PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T59	0-80	Sentence	denotes	EBV Latent Membrane Protein 1 Activates Akt, NFκB, and Stat3 in B Cell Lymphomas
T1	0-129	Sentence	denotes	EBV Latent Membrane Protein 1 Activates Akt, NFκB, and Stat3 in B Cell Lymphomas LMP1 Activates Akt, NFκB, and Stat3 in Lymphomas
T2	132-140	Sentence	denotes	Abstract
T60	141-227	Sentence	denotes	Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV).
T3	141-227	Sentence	denotes	Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV).
T61	228-309	Sentence	denotes	In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age.
T4	228-309	Sentence	denotes	In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age.
T62	310-439	Sentence	denotes	This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice.
T5	310-439	Sentence	denotes	This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice.
T63	440-623	Sentence	denotes	The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFκB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro.
T6	440-623	Sentence	denotes	The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFκB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro.
T64	624-760	Sentence	denotes	Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling.
T7	624-760	Sentence	denotes	Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling.
T65	761-881	Sentence	denotes	These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition.
T8	761-881	Sentence	denotes	These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition.
T66	882-1022	Sentence	denotes	These results suggest that Akt, NFκB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.
T9	882-1022	Sentence	denotes	These results suggest that Akt, NFκB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.
T10	1024-1038	Sentence	denotes	Author Summary
T11	1040-1197	Sentence	denotes	Epstein-Barr virus (EBV) is linked to the development of multiple cancers, including post-transplant lymphoma, Hodgkin disease, and nasopharyngeal carcinoma.
T12	1198-1379	Sentence	denotes	Latent membrane protein 1 (LMP1) is expressed in many EBV-associated cancers and is responsible for most of the altered cellular growth properties that are induced by EBV infection.
T13	1380-1511	Sentence	denotes	This study reveals that LMP1 induces lymphomas in B-1a lymphocytes, a cell type that is susceptible to transformation in aged mice.
T14	1512-1598	Sentence	denotes	The lymphomas require Akt, NFκB, and Stat3 signaling for enhanced growth and survival.
T15	1599-1734	Sentence	denotes	The activation of the Stat3, Akt, and NFκB signaling pathways likely underlies the ability of LMP1 to promote malignant transformation.
T16	1737-1749	Sentence	denotes	Introduction
T780	1750-1923	Sentence	denotes	Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus that infects humans predominantly at an early age with greater than 90% of the adult population infected with EBV [1].
T17	1750-1923	Sentence	denotes	Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus that infects humans predominantly at an early age with greater than 90% of the adult population infected with EBV [1].
T781	1924-2210	Sentence	denotes	EBV is linked to the development of both B lymphocyte and epithelial cell malignancies, including Burkitt lymphoma, Hodgkin disease (HD), and nasopharyngeal carcinoma (NPC), and cancers linked to immunosuppression, including post-transplant lymphoma and AIDS-associated lymphomas [2,3].
T18	1924-2210	Sentence	denotes	EBV is linked to the development of both B lymphocyte and epithelial cell malignancies, including Burkitt lymphoma, Hodgkin disease (HD), and nasopharyngeal carcinoma (NPC), and cancers linked to immunosuppression, including post-transplant lymphoma and AIDS-associated lymphomas [2,3].
T782	2211-2395	Sentence	denotes	In vitro infection of B lymphocytes with EBV induces permanent growth transformation, and this ability to affect cell growth regulation likely contributes to the development of cancer.
T19	2211-2395	Sentence	denotes	In vitro infection of B lymphocytes with EBV induces permanent growth transformation, and this ability to affect cell growth regulation likely contributes to the development of cancer.
T783	2396-2640	Sentence	denotes	Many of the viral proteins expressed in transformed cells, including the EBV nuclear antigens and latent membrane proteins, have profound effects on cell growth regulation and are required for EBV latent infection and B cell transformation [1].
T20	2396-2640	Sentence	denotes	Many of the viral proteins expressed in transformed cells, including the EBV nuclear antigens and latent membrane proteins, have profound effects on cell growth regulation and are required for EBV latent infection and B cell transformation [1].
T784	2641-2860	Sentence	denotes	Latent membrane protein 1 (LMP1) is considered the major oncoprotein of EBV, as it transforms rodent fibroblasts to tumorigenicity in nude mice and is expressed in HD, NPC, and immunosuppression-associated tumors [4–8].
T21	2641-2860	Sentence	denotes	Latent membrane protein 1 (LMP1) is considered the major oncoprotein of EBV, as it transforms rodent fibroblasts to tumorigenicity in nude mice and is expressed in HD, NPC, and immunosuppression-associated tumors [4–8].
T785	2861-2990	Sentence	denotes	In B lymphocytes, LMP1 mimics CD40 signaling, and both LMP1 and CD40 are essential for EBV-mediated B cell transformation [9–11].
T22	2861-2990	Sentence	denotes	In B lymphocytes, LMP1 mimics CD40 signaling, and both LMP1 and CD40 are essential for EBV-mediated B cell transformation [9–11].
T786	2991-3187	Sentence	denotes	While CD40 interacts with CD40 ligand expressed on activated T cells to induce B cell activation and differentiation, LMP1 acts as a constitutive signal through ligand-independent oligomerization.
T23	2991-3187	Sentence	denotes	While CD40 interacts with CD40 ligand expressed on activated T cells to induce B cell activation and differentiation, LMP1 acts as a constitutive signal through ligand-independent oligomerization.
T787	3188-3385	Sentence	denotes	LMP1 and CD40 interact with the same tumor necrosis factor receptor–associated factors (TRAFs) leading to activation of NFκB, c-Jun N terminal kinase (JNK), and p38 MAPK signaling pathways [12–16].
T24	3188-3385	Sentence	denotes	LMP1 and CD40 interact with the same tumor necrosis factor receptor–associated factors (TRAFs) leading to activation of NFκB, c-Jun N terminal kinase (JNK), and p38 MAPK signaling pathways [12–16].
T788	3386-3512	Sentence	denotes	Activation of NFκB is required for EBV-induced B cell transformation and its inhibition rapidly results in cell death [17,18].
T25	3386-3512	Sentence	denotes	Activation of NFκB is required for EBV-induced B cell transformation and its inhibition rapidly results in cell death [17,18].
T789	3513-3712	Sentence	denotes	Recent studies indicate that LMP1 also activates phosphatidylinositol 3 kinase (PI3K)/Akt signaling and that this activation is required for LMP1-mediated transformation of rodent fibroblasts [5,19].
T26	3513-3712	Sentence	denotes	Recent studies indicate that LMP1 also activates phosphatidylinositol 3 kinase (PI3K)/Akt signaling and that this activation is required for LMP1-mediated transformation of rodent fibroblasts [5,19].
T790	3713-3808	Sentence	denotes	In vitro, primary B cells can be maintained by CD40 ligation in combination with IL4 treatment.
T27	3713-3808	Sentence	denotes	In vitro, primary B cells can be maintained by CD40 ligation in combination with IL4 treatment.
T791	3809-3996	Sentence	denotes	In vivo, CD40 signaling is necessary for germinal center (GC) formation such that mice deficient for CD40 or CD40L are unable to form GCs in response to T cell–dependent antigens [20,21].
T28	3809-3996	Sentence	denotes	In vivo, CD40 signaling is necessary for germinal center (GC) formation such that mice deficient for CD40 or CD40L are unable to form GCs in response to T cell–dependent antigens [20,21].
T792	3997-4272	Sentence	denotes	Both the membrane proximal and distal cytoplasmic regions of CD40 that bind TRAF6 and TRAFs2/3/5, respectively, are necessary for GC formation, but either region is sufficient to induce extrafollicular B cell differentiation and restore low affinity antibody production [22].
T29	3997-4272	Sentence	denotes	Both the membrane proximal and distal cytoplasmic regions of CD40 that bind TRAF6 and TRAFs2/3/5, respectively, are necessary for GC formation, but either region is sufficient to induce extrafollicular B cell differentiation and restore low affinity antibody production [22].
T793	4273-4527	Sentence	denotes	Functionally, LMP1 can rescue CD40-deficient mice and restore immunoglobulin (Ig) class switching, most likely because LMP1 recruits similar TRAF molecules, TRAFs 1/2/3/5 and TRAF6, through the C-terminal activation regions 1 and 2 domains, respectively.
T30	4273-4527	Sentence	denotes	Functionally, LMP1 can rescue CD40-deficient mice and restore immunoglobulin (Ig) class switching, most likely because LMP1 recruits similar TRAF molecules, TRAFs 1/2/3/5 and TRAF6, through the C-terminal activation regions 1 and 2 domains, respectively.
T794	4528-4605	Sentence	denotes	However, LMP1 is unable to restore affinity maturation and GC formation [23].
T31	4528-4605	Sentence	denotes	However, LMP1 is unable to restore affinity maturation and GC formation [23].
T795	4606-4816	Sentence	denotes	Several EBV transforming proteins have been studied in transgenic mouse models, however, only LMP1 induces tumor development when expressed under the control of the Ig heavy chain promoter and enhancer [24–26].
T32	4606-4816	Sentence	denotes	Several EBV transforming proteins have been studied in transgenic mouse models, however, only LMP1 induces tumor development when expressed under the control of the Ig heavy chain promoter and enhancer [24–26].
T796	4817-5066	Sentence	denotes	The LMP1 transgenic mice (IgLMP1) express LMP1 in B lymphocytes, and in mice older than 12 mo, lymphoma develops with increased incidence (40%–50%) compared to wild-type control mice (11%), suggesting that LMP1 contributes to tumor development [26].
T33	4817-5066	Sentence	denotes	The LMP1 transgenic mice (IgLMP1) express LMP1 in B lymphocytes, and in mice older than 12 mo, lymphoma develops with increased incidence (40%–50%) compared to wild-type control mice (11%), suggesting that LMP1 contributes to tumor development [26].
T797	5067-5216	Sentence	denotes	The LMP1 lymphomas have rearranged Ig genes and have activated Akt, JNK, p38, and NFκB, with specific activation of the NFκB family member cRel [27].
T34	5067-5216	Sentence	denotes	The LMP1 lymphomas have rearranged Ig genes and have activated Akt, JNK, p38, and NFκB, with specific activation of the NFκB family member cRel [27].
T798	5217-5358	Sentence	denotes	In this study, the LMP1 transgenic lymphocytes and lymphomas were further characterized and their growth properties in vitro were determined.
T35	5217-5358	Sentence	denotes	In this study, the LMP1 transgenic lymphocytes and lymphomas were further characterized and their growth properties in vitro were determined.
T799	5359-5533	Sentence	denotes	To obtain pure populations of malignant lymphocytes and to enable more detailed biochemical analyses, examples of primary lymphomas were inoculated and passaged in SCID mice.
T36	5359-5533	Sentence	denotes	To obtain pure populations of malignant lymphocytes and to enable more detailed biochemical analyses, examples of primary lymphomas were inoculated and passaged in SCID mice.
T800	5534-5683	Sentence	denotes	Interestingly, lymphoma development was restricted to B-1a lymphocytes, a self-replenishing population of cells that are prone to malignancy [28,29].
T37	5534-5683	Sentence	denotes	Interestingly, lymphoma development was restricted to B-1a lymphocytes, a self-replenishing population of cells that are prone to malignancy [28,29].
T801	5684-5796	Sentence	denotes	LMP1 transgenic lymphocytes had increased viability in vitro and viability was increased by the addition of IL4.
T38	5684-5796	Sentence	denotes	LMP1 transgenic lymphocytes had increased viability in vitro and viability was increased by the addition of IL4.
T802	5797-5996	Sentence	denotes	In contrast, both LMP1-positive and -negative lymphoma cells were independent of IL4 co-stimulation for survival and proliferation in vitro with a complete absence of activated Stat6, the IL4 target.
T39	5797-5996	Sentence	denotes	In contrast, both LMP1-positive and -negative lymphoma cells were independent of IL4 co-stimulation for survival and proliferation in vitro with a complete absence of activated Stat6, the IL4 target.
T803	5997-6117	Sentence	denotes	The lymphomas were also distinguished by constitutive activation of Stat3 and deregulation of the Rb cell cycle pathway.
T40	5997-6117	Sentence	denotes	The lymphomas were also distinguished by constitutive activation of Stat3 and deregulation of the Rb cell cycle pathway.
T804	6118-6334	Sentence	denotes	Inhibition of the PI3K/Akt, NFκB, and Stat3 signaling pathways blocked the enhanced growth of both LMP1 transgenic and malignant lymphocytes, suggesting that these pathways are required for their growth and survival.
T41	6118-6334	Sentence	denotes	Inhibition of the PI3K/Akt, NFκB, and Stat3 signaling pathways blocked the enhanced growth of both LMP1 transgenic and malignant lymphocytes, suggesting that these pathways are required for their growth and survival.
T805	6335-6472	Sentence	denotes	These appear to be the same targets that are deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition.
T42	6335-6472	Sentence	denotes	These appear to be the same targets that are deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition.
T806	6473-6673	Sentence	denotes	This study reveals that LMP1 promotes malignancy in cells with the inherent ability to proliferate and that the Akt, NFκB, and Stat3 signaling pathways are required for its growth stimulatory effects.
T43	6473-6673	Sentence	denotes	This study reveals that LMP1 promotes malignancy in cells with the inherent ability to proliferate and that the Akt, NFκB, and Stat3 signaling pathways are required for its growth stimulatory effects.
T44	6675-6682	Sentence	denotes	Results
T3205	6684-6758	Sentence	denotes	High Levels of LMP1 Expression Correlates with the Development of Lymphoma
T45	6684-6758	Sentence	denotes	High Levels of LMP1 Expression Correlates with the Development of Lymphoma
T3206	6759-6876	Sentence	denotes	LMP1 expression in IgLMP1 mice was directed to B cells under the control of the Ig heavy chain promoter and enhancer.
T46	6759-6876	Sentence	denotes	LMP1 expression in IgLMP1 mice was directed to B cells under the control of the Ig heavy chain promoter and enhancer.
T3207	6877-7044	Sentence	denotes	It has previously been shown that in these transgenic mice, LMP1 expression was restricted to B220+ B cells with lymphoma detected in greatly enlarged spleens [23,26].
T47	6877-7044	Sentence	denotes	It has previously been shown that in these transgenic mice, LMP1 expression was restricted to B220+ B cells with lymphoma detected in greatly enlarged spleens [23,26].
T3208	7045-7285	Sentence	denotes	To investigate whether LMP1 expression contributes to lymphoma development, B cells were purified from splenocytes by positive selection using anti-CD19 MACS magnetic beads, and equivalent amounts of B cells were analyzed by immunoblotting.
T48	7045-7285	Sentence	denotes	To investigate whether LMP1 expression contributes to lymphoma development, B cells were purified from splenocytes by positive selection using anti-CD19 MACS magnetic beads, and equivalent amounts of B cells were analyzed by immunoblotting.
T3209	7286-7487	Sentence	denotes	LMP1 was detectable in LMP1 transgenic B cells, but upon development of lymphoma, LMP1 expression was stronger in 5/7 lymphomas analyzed with concomitant appearance of degradation products (Figure 1A).
T49	7286-7487	Sentence	denotes	LMP1 was detectable in LMP1 transgenic B cells, but upon development of lymphoma, LMP1 expression was stronger in 5/7 lymphomas analyzed with concomitant appearance of degradation products (Figure 1A).
T3210	7488-7680	Sentence	denotes	To determine whether the higher level of LMP1 detected was due to an expansion of malignant lymphocytes, expression of LMP1 in the spleen was further evaluated by immunohistochemical staining.
T50	7488-7680	Sentence	denotes	To determine whether the higher level of LMP1 detected was due to an expansion of malignant lymphocytes, expression of LMP1 in the spleen was further evaluated by immunohistochemical staining.
T3211	7681-7866	Sentence	denotes	Immunohistochemistry analysis of spleen sections detected LMP1 in the plasma membrane of cells in both the follicular white pulp and circulating lymphocytes in the red pulp (Figure 1B).
T51	7681-7866	Sentence	denotes	Immunohistochemistry analysis of spleen sections detected LMP1 in the plasma membrane of cells in both the follicular white pulp and circulating lymphocytes in the red pulp (Figure 1B).
T3212	7867-7995	Sentence	denotes	LMP1 expression was heterogeneous with strong LMP1 staining interspersed amongst a background of cells staining weakly for LMP1.
T52	7867-7995	Sentence	denotes	LMP1 expression was heterogeneous with strong LMP1 staining interspersed amongst a background of cells staining weakly for LMP1.
T3213	7996-8115	Sentence	denotes	Upon development to lymphoma, LMP1 expression was more abundantly detected with multiple foci of intense LMP1 staining.
T53	7996-8115	Sentence	denotes	Upon development to lymphoma, LMP1 expression was more abundantly detected with multiple foci of intense LMP1 staining.
T3214	8116-8312	Sentence	denotes	This demonstrates that the increased LMP1 detected by immunoblotting upon malignant progression reflects an increase in LMP1 expression and an accumulation of cells expressing high levels of LMP1.
T54	8116-8312	Sentence	denotes	This demonstrates that the increased LMP1 detected by immunoblotting upon malignant progression reflects an increase in LMP1 expression and an accumulation of cells expressing high levels of LMP1.
T3215	8313-8467	Sentence	denotes	This correlation between high LMP1 expression and the development of lymphoma suggests that progression to lymphoma results from increased levels of LMP1.
T55	8313-8467	Sentence	denotes	This correlation between high LMP1 expression and the development of lymphoma suggests that progression to lymphoma results from increased levels of LMP1.
T56	8468-8542	Sentence	denotes	Figure 1 High LMP1 Expression Correlates with the Development of Lymphoma
T21805	8478-8542	Sentence	denotes	High LMP1 Expression Correlates with the Development of Lymphoma
T21806	8543-8718	Sentence	denotes	LMP1 expression is shown by (A) immunoblotting of purified B cells (CD19+) and (B) immunohistochemistry staining of spleen tissue from wild-type (WT) and LMP1 transgenic mice.
T57	8543-8718	Sentence	denotes	LMP1 expression is shown by (A) immunoblotting of purified B cells (CD19+) and (B) immunohistochemistry staining of spleen tissue from wild-type (WT) and LMP1 transgenic mice.
T6392	8678-17061	Sentence	denotes	wild-type (WT) and LMP1 transgenic mice. (A) Lymphomas are identified with a number (1–7). Arrows indicate the LMP1-specific band and its degradation products as well as a non-specific band. Actin was used as a loading control. (B) White and red pulps are shown, but this architecture is lost upon development of lymphoma. Scale bar, 20 μm. LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion To determine if LMP1 signaling affects B cell differentiation and to immunophenotype the lymphomas that arise from LMP1 expression, surface Ig expression of heavy chains (IgM, IgG, IgD) and light chains (κ, λ) were analyzed by flow cytometry. Similar numbers of naïve (IgM+IgD+IgG−) splenic B cells with a strong bias towards κ light chain were detected from wild-type or LMP1 transgenic mice, indicating that LMP1 signaling does not affect B cell maturation (unpublished data). Flow cytometry analysis of the SCID-passaged wild-type and LMP1 transgenic lymphomas revealed an IgMhighIgDlow phenotype (Figure 2A), indicative of marginal zone, B-1, or memory B cells. B-1 cells are further separated into CD5+ (B-1a) and CD5− (B-1b) subsets. To differentiate between these cell types, lymphoma cells were further analyzed for the B-1a marker CD5. All (5/5) of the tested LMP1 transgenic lymphomas displayed an IgMhighIgDlowCD5+ phenotype (Figure 2A), an expression pattern that distinguishes B-1a cells. Interestingly, a spontaneous wild-type lymphoma also developed in B-1a cells (Figure 2A). These cells are an interesting population that is self replenishing with an increased likelihood to become malignant in aged mice [30]. Analysis of LMP1 transgenic mice before the development of lymphoma showed similar numbers of splenic B-1a (CD19+CD5+) and B-1b or B-2 populations (CD19+CD5−), indicating that LMP1 does not affect B cell differentiation (Figure 2B). Figure 2 LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion (A) Flow cytometry analysis of splenocytes from a WT or LMP1 transgenic lymphoma for the pan–B cell (CD19), B-1a cell (CD5), and Ig heavy chain (IgM and IgD) and light chain (κ and λ) markers. Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 4. This analysis was repeated on four other LMP1 transgenic lymphomas (1, 2, 3, and 6) showing a similar B-1a phenotype, of which lymphomas 2 and 4 were also doubly positive for κ and λ light chains. (B) Flow cytometry analysis of WT or LMP1 transgenic splenocytes for B-1a (CD19+CD5+) and B-1b or B2 subsets (CD19+CD5−). Percentages of B-1a and B-1b or B2 subsets are shown in each quadrant. This analysis was repeated on three other WT and two other LMP1 transgenic mice with similar results. (C) Immunoblot analysis for κ and λ light chains of B cells (CD19+) purified from WT and LMP1 transgenic mice. Actin was used as a loading control. Due to allelic exclusion, mature B cells that have been exposed to antigen will typically express only one heavy chain isotype (IgG, IgE, or IgA) and either a κ or λ light chain. Interestingly, 2/5 LMP1 transgenic lymphomas analyzed (lymphomas 2 and 4) were doubly positive for low levels of both κ and λ light chains (Figure 2A). Previous characterization of the LMP1 lymphomas had revealed that the lymphomas were clonal as determined by Ig heavy chain rearrangement [26], and analysis of κ chain rearrangement (Figure S1) of the samples analyzed in this study confirmed clonality. To further assess light chain expression, the passaged samples were tested by immunoblotting for κ and λ light chains (Figure 2C). Interestingly, very low levels of expression of both light chains were detected by flow cytometry. The low levels of expression may reflect a limitation of the total number of light chains that can be expressed on the surface of a B cell. In agreement with the flow cytometry analysis, LMP1 transgenic lymphomas 2 and 4 were also positive for κ and λ light chains by immunoblot analysis (Figure 2C), confirming that these lymphomas express both light chains. A previous study of mice that developed leukemia due to an expansion of self-reactive B-1a cells determined that the B-1a leukemias were also doubly positive for κ and λ light chains [31]. These findings indicate that expression of LMP1 in B-1a cells promotes the development of malignancy and can result in the aberrant escape from allelic exclusion. LMP1 Promotes B Cell Survival and Proliferation In Vitro Primary B cell cultures can be maintained through CD40 ligation and supplementation with IL4 [32]. To investigate whether LMP1 affects primary B cell survival and proliferation, splenocytes were cultured in the presence or absence of IL4 and analyzed by MTS as a metabolic marker, by ethidium monoazide (EMA) exclusion for viability, and by 5-bromo-2′-deoxy-uridine (BrdU) incorporation for proliferation. In the MTS assay, as expected, splenocytes from wild-type mice did not survive even with the addition of IL4 due to a lack of CD40 ligation (Figure 3A). In contrast, LMP1 splenocytes had increased metabolism even in the absence of IL4, which was further enhanced upon addition of IL4. Wild-type and LMP1 transgenic lymphoma cells had high levels of MTS activity even in the absence of IL4 (Figure 3A). The LMP1 transgenic lymphoma cells had approximately 4-fold higher MTS activity than the normal transgenic lymphocytes and were at least 2-fold higher than the control lymphoma. As previously published, lymphoma usually develops in mice over 12 mo of age and all mice are sacrificed by 18–20 mo. The ages of the transgenic mice with or without lymphoma ranged between 6 and 20 mo old. There was no correlation between age and MTS activity. Figure 3 LMP1 Promotes B Cell Survival and Proliferation In Vitro (A) MTS assay of splenocytes from WT and LMP1 transgenic mice. Splenocytes were cultured in the presence (grey bars) or absence (black bars) of IL4 for 3 d. The results are the mean ± SEM of triplicate samples averaged from multiple mice where “n” the number of mice analyzed is as follows: n = 2 for WT lymphocytes and WT lymphomas, n = 11 for LMP1 transgenic lymphocytes, and n = 13 for LMP1 transgenic lymphomas. (B) EMA exclusion of CD19+ gated splenocytes from WT and LMP1 transgenic mice showing percentage of viable B cells cultured with (white bars) or without (black bars) IL4 for 2 d. (C) Flow cytometry analysis for incorporated BrdU in WT and LMP1 transgenic lymphoma cells cultured with or without IL4 for 2 d. Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 2. Percentages of cells in each quadrant are shown. EMA exclusion of CD19+ gated B cells prepared from two wild-type and two LMP1 transgenic mice indicated a 2-fold increase in viability in the LMP1 transgenic lymphocytes compared to wild-type lymphocytes. Two examples of LMP1 transgenic lymphoma cells had greatly increased viability that was not increased by IL4 treatment, indicating that the lymphoma cells are independent of IL4 co-stimulation (Figure 3B). Enhancement in MTS activity was observed in LMP1 transgenic lymphoma cells by the addition of IL4; however, EMA exclusion did not reveal a similar increase. This could reflect a difference for IL4 requirement in the metabolic activity versus the viability of LMP1 transgenic lymphoma cells. Although expression of LMP1 could enhance survival of non-malignant primary lymphocytes, BrdU incorporation revealed that LMP1 expression alone was not sufficient to induce proliferation in culture (unpublished data). Only lymphoma cells had detectable levels of BrdU incorporation detected by flow cytometry (Figure 3C). Interestingly, LMP1 lymphoma cells had significantly higher levels of proliferation in comparison to the spontaneous lymphoma that developed in an LMP1-negative littermate (25% versus 4%). This higher level of proliferation was observed in lymphomas that express both high (Table 1, LMP1-L2 and LMP1-L3) and low (Table 1, LMP1-L5) levels of LMP1, suggesting that even small amounts of LMP1 is sufficient to induce dramatic effects in proliferation. The level of proliferation was not enhanced upon IL4 addition, confirming the IL4 independence observed in the viability studies (Figure 3C; Table1). Table 1 Summary of Analysis Performed on Wild-Type and LMP1 Transgenic Lymphomas Wild-Type and LMP1 Transgenic Lymphoma Cells Do Not Require IL4 and Stat6 Signaling
T21807	8719-8768	Sentence	denotes	(A) Lymphomas are identified with a number (1–7).
T58	8719-8768	Sentence	denotes	(A) Lymphomas are identified with a number (1–7).
T21808	8769-8868	Sentence	denotes	Arrows indicate the LMP1-specific band and its degradation products as well as a non-specific band.
T59	8769-8868	Sentence	denotes	Arrows indicate the LMP1-specific band and its degradation products as well as a non-specific band.
T21809	8869-8905	Sentence	denotes	Actin was used as a loading control.
T60	8869-8905	Sentence	denotes	Actin was used as a loading control.
T21810	8906-9000	Sentence	denotes	(B) White and red pulps are shown, but this architecture is lost upon development of lymphoma.
T61	8906-9000	Sentence	denotes	(B) White and red pulps are shown, but this architecture is lost upon development of lymphoma.
T21811	9001-9018	Sentence	denotes	Scale bar, 20 μm.
T62	9001-9018	Sentence	denotes	Scale bar, 20 μm.
T4090	9020-9082	Sentence	denotes	LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion
T63	9020-9082	Sentence	denotes	LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion
T4091	9083-9325	Sentence	denotes	To determine if LMP1 signaling affects B cell differentiation and to immunophenotype the lymphomas that arise from LMP1 expression, surface Ig expression of heavy chains (IgM, IgG, IgD) and light chains (κ, λ) were analyzed by flow cytometry.
T64	9083-9325	Sentence	denotes	To determine if LMP1 signaling affects B cell differentiation and to immunophenotype the lymphomas that arise from LMP1 expression, surface Ig expression of heavy chains (IgM, IgG, IgD) and light chains (κ, λ) were analyzed by flow cytometry.
T4092	9326-9561	Sentence	denotes	Similar numbers of naïve (IgM+IgD+IgG−) splenic B cells with a strong bias towards κ light chain were detected from wild-type or LMP1 transgenic mice, indicating that LMP1 signaling does not affect B cell maturation (unpublished data).
T65	9326-9561	Sentence	denotes	Similar numbers of naïve (IgM+IgD+IgG−) splenic B cells with a strong bias towards κ light chain were detected from wild-type or LMP1 transgenic mice, indicating that LMP1 signaling does not affect B cell maturation (unpublished data).
T4093	9562-9748	Sentence	denotes	Flow cytometry analysis of the SCID-passaged wild-type and LMP1 transgenic lymphomas revealed an IgMhighIgDlow phenotype (Figure 2A), indicative of marginal zone, B-1, or memory B cells.
T66	9562-9748	Sentence	denotes	Flow cytometry analysis of the SCID-passaged wild-type and LMP1 transgenic lymphomas revealed an IgMhighIgDlow phenotype (Figure 2A), indicative of marginal zone, B-1, or memory B cells.
T4094	9749-9822	Sentence	denotes	B-1 cells are further separated into CD5+ (B-1a) and CD5− (B-1b) subsets.
T67	9749-9822	Sentence	denotes	B-1 cells are further separated into CD5+ (B-1a) and CD5− (B-1b) subsets.
T4095	9823-9927	Sentence	denotes	To differentiate between these cell types, lymphoma cells were further analyzed for the B-1a marker CD5.
T68	9823-9927	Sentence	denotes	To differentiate between these cell types, lymphoma cells were further analyzed for the B-1a marker CD5.
T4096	9928-10084	Sentence	denotes	All (5/5) of the tested LMP1 transgenic lymphomas displayed an IgMhighIgDlowCD5+ phenotype (Figure 2A), an expression pattern that distinguishes B-1a cells.
T69	9928-10084	Sentence	denotes	All (5/5) of the tested LMP1 transgenic lymphomas displayed an IgMhighIgDlowCD5+ phenotype (Figure 2A), an expression pattern that distinguishes B-1a cells.
T4097	10085-10174	Sentence	denotes	Interestingly, a spontaneous wild-type lymphoma also developed in B-1a cells (Figure 2A).
T70	10085-10174	Sentence	denotes	Interestingly, a spontaneous wild-type lymphoma also developed in B-1a cells (Figure 2A).
T4098	10175-10310	Sentence	denotes	These cells are an interesting population that is self replenishing with an increased likelihood to become malignant in aged mice [30].
T71	10175-10310	Sentence	denotes	These cells are an interesting population that is self replenishing with an increased likelihood to become malignant in aged mice [30].
T4099	10311-10543	Sentence	denotes	Analysis of LMP1 transgenic mice before the development of lymphoma showed similar numbers of splenic B-1a (CD19+CD5+) and B-1b or B-2 populations (CD19+CD5−), indicating that LMP1 does not affect B cell differentiation (Figure 2B).
T72	10311-10543	Sentence	denotes	Analysis of LMP1 transgenic mice before the development of lymphoma showed similar numbers of splenic B-1a (CD19+CD5+) and B-1b or B-2 populations (CD19+CD5−), indicating that LMP1 does not affect B cell differentiation (Figure 2B).
T73	10544-10616	Sentence	denotes	Figure 2 LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion
T22113	10554-10616	Sentence	denotes	LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion
T22114	10617-10809	Sentence	denotes	(A) Flow cytometry analysis of splenocytes from a WT or LMP1 transgenic lymphoma for the pan–B cell (CD19), B-1a cell (CD5), and Ig heavy chain (IgM and IgD) and light chain (κ and λ) markers.
T74	10617-10809	Sentence	denotes	(A) Flow cytometry analysis of splenocytes from a WT or LMP1 transgenic lymphoma for the pan–B cell (CD19), B-1a cell (CD5), and Ig heavy chain (IgM and IgD) and light chain (κ and λ) markers.
T22115	10810-10882	Sentence	denotes	Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 4.
T75	10810-10882	Sentence	denotes	Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 4.
T22116	10883-11079	Sentence	denotes	This analysis was repeated on four other LMP1 transgenic lymphomas (1, 2, 3, and 6) showing a similar B-1a phenotype, of which lymphomas 2 and 4 were also doubly positive for κ and λ light chains.
T76	10883-11079	Sentence	denotes	This analysis was repeated on four other LMP1 transgenic lymphomas (1, 2, 3, and 6) showing a similar B-1a phenotype, of which lymphomas 2 and 4 were also doubly positive for κ and λ light chains.
T22117	11080-11201	Sentence	denotes	(B) Flow cytometry analysis of WT or LMP1 transgenic splenocytes for B-1a (CD19+CD5+) and B-1b or B2 subsets (CD19+CD5−).
T77	11080-11201	Sentence	denotes	(B) Flow cytometry analysis of WT or LMP1 transgenic splenocytes for B-1a (CD19+CD5+) and B-1b or B2 subsets (CD19+CD5−).
T22118	11202-11272	Sentence	denotes	Percentages of B-1a and B-1b or B2 subsets are shown in each quadrant.
T78	11202-11272	Sentence	denotes	Percentages of B-1a and B-1b or B2 subsets are shown in each quadrant.
T22119	11273-11374	Sentence	denotes	This analysis was repeated on three other WT and two other LMP1 transgenic mice with similar results.
T79	11273-11374	Sentence	denotes	This analysis was repeated on three other WT and two other LMP1 transgenic mice with similar results.
T22120	11375-11485	Sentence	denotes	(C) Immunoblot analysis for κ and λ light chains of B cells (CD19+) purified from WT and LMP1 transgenic mice.
T80	11375-11485	Sentence	denotes	(C) Immunoblot analysis for κ and λ light chains of B cells (CD19+) purified from WT and LMP1 transgenic mice.
T22121	11486-11522	Sentence	denotes	Actin was used as a loading control.
T81	11486-11522	Sentence	denotes	Actin was used as a loading control.
T4100	11525-11703	Sentence	denotes	Due to allelic exclusion, mature B cells that have been exposed to antigen will typically express only one heavy chain isotype (IgG, IgE, or IgA) and either a κ or λ light chain.
T82	11525-11703	Sentence	denotes	Due to allelic exclusion, mature B cells that have been exposed to antigen will typically express only one heavy chain isotype (IgG, IgE, or IgA) and either a κ or λ light chain.
T4101	11704-11855	Sentence	denotes	Interestingly, 2/5 LMP1 transgenic lymphomas analyzed (lymphomas 2 and 4) were doubly positive for low levels of both κ and λ light chains (Figure 2A).
T83	11704-11855	Sentence	denotes	Interestingly, 2/5 LMP1 transgenic lymphomas analyzed (lymphomas 2 and 4) were doubly positive for low levels of both κ and λ light chains (Figure 2A).
T4102	11856-12108	Sentence	denotes	Previous characterization of the LMP1 lymphomas had revealed that the lymphomas were clonal as determined by Ig heavy chain rearrangement [26], and analysis of κ chain rearrangement (Figure S1) of the samples analyzed in this study confirmed clonality.
T84	11856-12108	Sentence	denotes	Previous characterization of the LMP1 lymphomas had revealed that the lymphomas were clonal as determined by Ig heavy chain rearrangement [26], and analysis of κ chain rearrangement (Figure S1) of the samples analyzed in this study confirmed clonality.
T4103	12109-12239	Sentence	denotes	To further assess light chain expression, the passaged samples were tested by immunoblotting for κ and λ light chains (Figure 2C).
T85	12109-12239	Sentence	denotes	To further assess light chain expression, the passaged samples were tested by immunoblotting for κ and λ light chains (Figure 2C).
T4104	12240-12338	Sentence	denotes	Interestingly, very low levels of expression of both light chains were detected by flow cytometry.
T86	12240-12338	Sentence	denotes	Interestingly, very low levels of expression of both light chains were detected by flow cytometry.
T4105	12339-12478	Sentence	denotes	The low levels of expression may reflect a limitation of the total number of light chains that can be expressed on the surface of a B cell.
T87	12339-12478	Sentence	denotes	The low levels of expression may reflect a limitation of the total number of light chains that can be expressed on the surface of a B cell.
T4106	12479-12698	Sentence	denotes	In agreement with the flow cytometry analysis, LMP1 transgenic lymphomas 2 and 4 were also positive for κ and λ light chains by immunoblot analysis (Figure 2C), confirming that these lymphomas express both light chains.
T88	12479-12698	Sentence	denotes	In agreement with the flow cytometry analysis, LMP1 transgenic lymphomas 2 and 4 were also positive for κ and λ light chains by immunoblot analysis (Figure 2C), confirming that these lymphomas express both light chains.
T4107	12699-12887	Sentence	denotes	A previous study of mice that developed leukemia due to an expansion of self-reactive B-1a cells determined that the B-1a leukemias were also doubly positive for κ and λ light chains [31].
T89	12699-12887	Sentence	denotes	A previous study of mice that developed leukemia due to an expansion of self-reactive B-1a cells determined that the B-1a leukemias were also doubly positive for κ and λ light chains [31].
T4108	12888-13050	Sentence	denotes	These findings indicate that expression of LMP1 in B-1a cells promotes the development of malignancy and can result in the aberrant escape from allelic exclusion.
T90	12888-13050	Sentence	denotes	These findings indicate that expression of LMP1 in B-1a cells promotes the development of malignancy and can result in the aberrant escape from allelic exclusion.
T5525	13052-13108	Sentence	denotes	LMP1 Promotes B Cell Survival and Proliferation In Vitro
T91	13052-13108	Sentence	denotes	LMP1 Promotes B Cell Survival and Proliferation In Vitro
T5526	13109-13207	Sentence	denotes	Primary B cell cultures can be maintained through CD40 ligation and supplementation with IL4 [32].
T92	13109-13207	Sentence	denotes	Primary B cell cultures can be maintained through CD40 ligation and supplementation with IL4 [32].
T5527	13208-13514	Sentence	denotes	To investigate whether LMP1 affects primary B cell survival and proliferation, splenocytes were cultured in the presence or absence of IL4 and analyzed by MTS as a metabolic marker, by ethidium monoazide (EMA) exclusion for viability, and by 5-bromo-2′-deoxy-uridine (BrdU) incorporation for proliferation.
T93	13208-13514	Sentence	denotes	To investigate whether LMP1 affects primary B cell survival and proliferation, splenocytes were cultured in the presence or absence of IL4 and analyzed by MTS as a metabolic marker, by ethidium monoazide (EMA) exclusion for viability, and by 5-bromo-2′-deoxy-uridine (BrdU) incorporation for proliferation.
T5528	13515-13667	Sentence	denotes	In the MTS assay, as expected, splenocytes from wild-type mice did not survive even with the addition of IL4 due to a lack of CD40 ligation (Figure 3A).
T94	13515-13667	Sentence	denotes	In the MTS assay, as expected, splenocytes from wild-type mice did not survive even with the addition of IL4 due to a lack of CD40 ligation (Figure 3A).
T5529	13668-13799	Sentence	denotes	In contrast, LMP1 splenocytes had increased metabolism even in the absence of IL4, which was further enhanced upon addition of IL4.
T95	13668-13799	Sentence	denotes	In contrast, LMP1 splenocytes had increased metabolism even in the absence of IL4, which was further enhanced upon addition of IL4.
T5530	13800-13916	Sentence	denotes	Wild-type and LMP1 transgenic lymphoma cells had high levels of MTS activity even in the absence of IL4 (Figure 3A).
T96	13800-13916	Sentence	denotes	Wild-type and LMP1 transgenic lymphoma cells had high levels of MTS activity even in the absence of IL4 (Figure 3A).
T5531	13917-14094	Sentence	denotes	The LMP1 transgenic lymphoma cells had approximately 4-fold higher MTS activity than the normal transgenic lymphocytes and were at least 2-fold higher than the control lymphoma.
T97	13917-14094	Sentence	denotes	The LMP1 transgenic lymphoma cells had approximately 4-fold higher MTS activity than the normal transgenic lymphocytes and were at least 2-fold higher than the control lymphoma.
T5532	14095-14212	Sentence	denotes	As previously published, lymphoma usually develops in mice over 12 mo of age and all mice are sacrificed by 18–20 mo.
T98	14095-14212	Sentence	denotes	As previously published, lymphoma usually develops in mice over 12 mo of age and all mice are sacrificed by 18–20 mo.
T5533	14213-14301	Sentence	denotes	The ages of the transgenic mice with or without lymphoma ranged between 6 and 20 mo old.
T99	14213-14301	Sentence	denotes	The ages of the transgenic mice with or without lymphoma ranged between 6 and 20 mo old.
T5534	14302-14356	Sentence	denotes	There was no correlation between age and MTS activity.
T100	14302-14356	Sentence	denotes	There was no correlation between age and MTS activity.
T101	14357-14423	Sentence	denotes	Figure 3 LMP1 Promotes B Cell Survival and Proliferation In Vitro
T22656	14367-14423	Sentence	denotes	LMP1 Promotes B Cell Survival and Proliferation In Vitro
T22657	14424-14486	Sentence	denotes	(A) MTS assay of splenocytes from WT and LMP1 transgenic mice.
T102	14424-14486	Sentence	denotes	(A) MTS assay of splenocytes from WT and LMP1 transgenic mice.
T22658	14487-14580	Sentence	denotes	Splenocytes were cultured in the presence (grey bars) or absence (black bars) of IL4 for 3 d.
T103	14487-14580	Sentence	denotes	Splenocytes were cultured in the presence (grey bars) or absence (black bars) of IL4 for 3 d.
T22659	14581-14839	Sentence	denotes	The results are the mean ± SEM of triplicate samples averaged from multiple mice where “n” the number of mice analyzed is as follows: n = 2 for WT lymphocytes and WT lymphomas, n = 11 for LMP1 transgenic lymphocytes, and n = 13 for LMP1 transgenic lymphomas.
T104	14581-14839	Sentence	denotes	The results are the mean ± SEM of triplicate samples averaged from multiple mice where “n” the number of mice analyzed is as follows: n = 2 for WT lymphocytes and WT lymphomas, n = 11 for LMP1 transgenic lymphocytes, and n = 13 for LMP1 transgenic lymphomas.
T22660	14840-15018	Sentence	denotes	(B) EMA exclusion of CD19+ gated splenocytes from WT and LMP1 transgenic mice showing percentage of viable B cells cultured with (white bars) or without (black bars) IL4 for 2 d.
T105	14840-15018	Sentence	denotes	(B) EMA exclusion of CD19+ gated splenocytes from WT and LMP1 transgenic mice showing percentage of viable B cells cultured with (white bars) or without (black bars) IL4 for 2 d.
T22661	15019-15147	Sentence	denotes	(C) Flow cytometry analysis for incorporated BrdU in WT and LMP1 transgenic lymphoma cells cultured with or without IL4 for 2 d.
T106	15019-15147	Sentence	denotes	(C) Flow cytometry analysis for incorporated BrdU in WT and LMP1 transgenic lymphoma cells cultured with or without IL4 for 2 d.
T22662	15148-15220	Sentence	denotes	Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 2.
T107	15148-15220	Sentence	denotes	Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 2.
T22663	15221-15269	Sentence	denotes	Percentages of cells in each quadrant are shown.
T108	15221-15269	Sentence	denotes	Percentages of cells in each quadrant are shown.
T5535	15272-15476	Sentence	denotes	EMA exclusion of CD19+ gated B cells prepared from two wild-type and two LMP1 transgenic mice indicated a 2-fold increase in viability in the LMP1 transgenic lymphocytes compared to wild-type lymphocytes.
T109	15272-15476	Sentence	denotes	EMA exclusion of CD19+ gated B cells prepared from two wild-type and two LMP1 transgenic mice indicated a 2-fold increase in viability in the LMP1 transgenic lymphocytes compared to wild-type lymphocytes.
T5536	15477-15682	Sentence	denotes	Two examples of LMP1 transgenic lymphoma cells had greatly increased viability that was not increased by IL4 treatment, indicating that the lymphoma cells are independent of IL4 co-stimulation (Figure 3B).
T110	15477-15682	Sentence	denotes	Two examples of LMP1 transgenic lymphoma cells had greatly increased viability that was not increased by IL4 treatment, indicating that the lymphoma cells are independent of IL4 co-stimulation (Figure 3B).
T5537	15683-15839	Sentence	denotes	Enhancement in MTS activity was observed in LMP1 transgenic lymphoma cells by the addition of IL4; however, EMA exclusion did not reveal a similar increase.
T111	15683-15839	Sentence	denotes	Enhancement in MTS activity was observed in LMP1 transgenic lymphoma cells by the addition of IL4; however, EMA exclusion did not reveal a similar increase.
T5538	15840-15973	Sentence	denotes	This could reflect a difference for IL4 requirement in the metabolic activity versus the viability of LMP1 transgenic lymphoma cells.
T112	15840-15973	Sentence	denotes	This could reflect a difference for IL4 requirement in the metabolic activity versus the viability of LMP1 transgenic lymphoma cells.
T5539	15974-16191	Sentence	denotes	Although expression of LMP1 could enhance survival of non-malignant primary lymphocytes, BrdU incorporation revealed that LMP1 expression alone was not sufficient to induce proliferation in culture (unpublished data).
T113	15974-16191	Sentence	denotes	Although expression of LMP1 could enhance survival of non-malignant primary lymphocytes, BrdU incorporation revealed that LMP1 expression alone was not sufficient to induce proliferation in culture (unpublished data).
T5540	16192-16295	Sentence	denotes	Only lymphoma cells had detectable levels of BrdU incorporation detected by flow cytometry (Figure 3C).
T114	16192-16295	Sentence	denotes	Only lymphoma cells had detectable levels of BrdU incorporation detected by flow cytometry (Figure 3C).
T5541	16296-16484	Sentence	denotes	Interestingly, LMP1 lymphoma cells had significantly higher levels of proliferation in comparison to the spontaneous lymphoma that developed in an LMP1-negative littermate (25% versus 4%).
T115	16296-16484	Sentence	denotes	Interestingly, LMP1 lymphoma cells had significantly higher levels of proliferation in comparison to the spontaneous lymphoma that developed in an LMP1-negative littermate (25% versus 4%).
T5542	16485-16744	Sentence	denotes	This higher level of proliferation was observed in lymphomas that express both high (Table 1, LMP1-L2 and LMP1-L3) and low (Table 1, LMP1-L5) levels of LMP1, suggesting that even small amounts of LMP1 is sufficient to induce dramatic effects in proliferation.
T116	16485-16744	Sentence	denotes	This higher level of proliferation was observed in lymphomas that express both high (Table 1, LMP1-L2 and LMP1-L3) and low (Table 1, LMP1-L5) levels of LMP1, suggesting that even small amounts of LMP1 is sufficient to induce dramatic effects in proliferation.
T5543	16745-16894	Sentence	denotes	The level of proliferation was not enhanced upon IL4 addition, confirming the IL4 independence observed in the viability studies (Figure 3C; Table1).
T117	16745-16894	Sentence	denotes	The level of proliferation was not enhanced upon IL4 addition, confirming the IL4 independence observed in the viability studies (Figure 3C; Table1).
T118	16895-16976	Sentence	denotes	Table 1 Summary of Analysis Performed on Wild-Type and LMP1 Transgenic Lymphomas
T25509	16904-16976	Sentence	denotes	Summary of Analysis Performed on Wild-Type and LMP1 Transgenic Lymphomas
T119	16978-17061	Sentence	denotes	Wild-Type and LMP1 Transgenic Lymphoma Cells Do Not Require IL4 and Stat6 Signaling
T6393	17062-17206	Sentence	denotes	To investigate whether IL4 independence was due to endogenous IL4 expression, IL4 transcription was assessed by an Rnase protection assay (RPA).
T120	17062-17206	Sentence	denotes	To investigate whether IL4 independence was due to endogenous IL4 expression, IL4 transcription was assessed by an Rnase protection assay (RPA).
T6394	17207-17318	Sentence	denotes	IL4 transcription was detectable with control RNA and faintly in the mouse lymphoma cell line K46μ (Figure 4A).
T121	17207-17318	Sentence	denotes	IL4 transcription was detectable with control RNA and faintly in the mouse lymphoma cell line K46μ (Figure 4A).
T6395	17319-17566	Sentence	denotes	However, IL4 transcription was not detectable in CD19+ MACS-purified B cells from wild-type lymphocytes (unpublished data), LMP1 transgenic lymphocytes, or lymphoma cells, although the GAPDH and L32 controls were effectively protected (Figure 4A).
T122	17319-17566	Sentence	denotes	However, IL4 transcription was not detectable in CD19+ MACS-purified B cells from wild-type lymphocytes (unpublished data), LMP1 transgenic lymphocytes, or lymphoma cells, although the GAPDH and L32 controls were effectively protected (Figure 4A).
T6396	17567-17705	Sentence	denotes	Activated Stat6 (pStat6), a target of the IL4 receptor pathway, was detected in the wild-type and LMP1 transgenic lymphocytes (Figure 4B).
T123	17567-17705	Sentence	denotes	Activated Stat6 (pStat6), a target of the IL4 receptor pathway, was detected in the wild-type and LMP1 transgenic lymphocytes (Figure 4B).
T6397	17706-17804	Sentence	denotes	In contrast, pStat6 was barely detected in either the wild-type or LMP1 transgenic lymphoma cells.
T124	17706-17804	Sentence	denotes	In contrast, pStat6 was barely detected in either the wild-type or LMP1 transgenic lymphoma cells.
T6398	17805-17930	Sentence	denotes	However, the pathway was not disabled, as treatment of the lymphoma cells with IL4 induced Stat6 phosphorylation (Figure 4C).
T125	17805-17930	Sentence	denotes	However, the pathway was not disabled, as treatment of the lymphoma cells with IL4 induced Stat6 phosphorylation (Figure 4C).
T126	17931-18041	Sentence	denotes	Figure 4 Wild-Type and LMP1 Transgenic Lymphoma Cells Survive Independently of IL4/Stat6 Signaling in Culture
T23143	17941-18041	Sentence	denotes	Wild-Type and LMP1 Transgenic Lymphoma Cells Survive Independently of IL4/Stat6 Signaling in Culture
T23144	18042-18152	Sentence	denotes	(A) Rnase protection assay for IL4 mRNA from purified B cells (CD19+) from WT and LMP1 transgenic splenocytes.
T127	18042-18152	Sentence	denotes	(A) Rnase protection assay for IL4 mRNA from purified B cells (CD19+) from WT and LMP1 transgenic splenocytes.
T23145	18153-18221	Sentence	denotes	The L32 and GAPDH housekeeping genes were used as a loading control.
T128	18153-18221	Sentence	denotes	The L32 and GAPDH housekeeping genes were used as a loading control.
T23146	18222-18274	Sentence	denotes	Arrow indicates the position of the protected probe.
T129	18222-18274	Sentence	denotes	Arrow indicates the position of the protected probe.
T23147	18275-18585	Sentence	denotes	(B and C) Immunoblot analysis of WT and LMP1 transgenic mice for activated pStat6 in (B) purified B cells (CD19+) at the time of harvest or in (C) whole splenocytes cultured with or without IL4. (B) Actin was used as a loading control, and the white line indicates that intervening lanes have been spliced out.
T130	18275-18585	Sentence	denotes	(B and C) Immunoblot analysis of WT and LMP1 transgenic mice for activated pStat6 in (B) purified B cells (CD19+) at the time of harvest or in (C) whole splenocytes cultured with or without IL4. (B) Actin was used as a loading control, and the white line indicates that intervening lanes have been spliced out.
T23148	18586-18779	Sentence	denotes	(D and E) MTS assay of (D) WT lymphocytes and (E) LMP1 transgenic lymphoma cells cultured with IL4, a neutralizing antibody to IL4, or a rat IgG isotype control at the indicated concentrations.
T131	18586-18779	Sentence	denotes	(D and E) MTS assay of (D) WT lymphocytes and (E) LMP1 transgenic lymphoma cells cultured with IL4, a neutralizing antibody to IL4, or a rat IgG isotype control at the indicated concentrations.
T23149	18780-18834	Sentence	denotes	Shown are the results from LMP1 transgenic lymphoma 3.
T132	18780-18834	Sentence	denotes	Shown are the results from LMP1 transgenic lymphoma 3.
T23150	18835-18973	Sentence	denotes	The results are the mean ± SEM of triplicate samples from a single representative experiment that was repeated twice with similar results.
T133	18835-18973	Sentence	denotes	The results are the mean ± SEM of triplicate samples from a single representative experiment that was repeated twice with similar results.
T6399	18976-19244	Sentence	denotes	Although wild-type lymphocytes cannot be maintained in culture with IL4 supplementation alone (Figure 3A), slight enhancement in MTS activity could be detected if the cells were analyzed at an earlier time point, at 1 d (Figure 4D) versus 3 d (Figure 3A) post-harvest.
T134	18976-19244	Sentence	denotes	Although wild-type lymphocytes cannot be maintained in culture with IL4 supplementation alone (Figure 3A), slight enhancement in MTS activity could be detected if the cells were analyzed at an earlier time point, at 1 d (Figure 4D) versus 3 d (Figure 3A) post-harvest.
T6400	19245-19382	Sentence	denotes	The enhancement of MTS activity induced by IL4 in wild-type lymphocytes could be neutralized by the addition of IL4 antibody (Figure 4D).
T135	19245-19382	Sentence	denotes	The enhancement of MTS activity induced by IL4 in wild-type lymphocytes could be neutralized by the addition of IL4 antibody (Figure 4D).
T6401	19383-19501	Sentence	denotes	However, neutralizing antibodies to IL4 did not affect the MTS activity of LMP1 transgenic lymphoma cells (Figure 4E).
T136	19383-19501	Sentence	denotes	However, neutralizing antibodies to IL4 did not affect the MTS activity of LMP1 transgenic lymphoma cells (Figure 4E).
T6402	19502-19635	Sentence	denotes	In summary, the wild-type and LMP1 transgenic lymphoma cells grew independently of IL4 treatment and did not require Stat6 signaling.
T137	19502-19635	Sentence	denotes	In summary, the wild-type and LMP1 transgenic lymphoma cells grew independently of IL4 treatment and did not require Stat6 signaling.
T7551	19637-19693	Sentence	denotes	LMP1 Upregulates IL10 and Constitutively Activates Stat3
T138	19637-19693	Sentence	denotes	LMP1 Upregulates IL10 and Constitutively Activates Stat3
T7552	19694-19958	Sentence	denotes	To identify cytokines that may contribute to the increased survival and growth of lymphomas, the expression levels of a panel of cytokines were screened on CD19+ MACS-purified B cells, using an RPA probe set for IL4, IL5, IL10, IL13, IL15, IL9, IL2, IL6, and IFNγ.
T139	19694-19958	Sentence	denotes	To identify cytokines that may contribute to the increased survival and growth of lymphomas, the expression levels of a panel of cytokines were screened on CD19+ MACS-purified B cells, using an RPA probe set for IL4, IL5, IL10, IL13, IL15, IL9, IL2, IL6, and IFNγ.
T7553	19959-20069	Sentence	denotes	Expression levels were quantified with a phosphorimager and normalized to the ribosomal housekeeping gene L32.
T140	19959-20069	Sentence	denotes	Expression levels were quantified with a phosphorimager and normalized to the ribosomal housekeeping gene L32.
T7554	20070-20221	Sentence	denotes	None of the tested cytokines were detected in wild-type lymphocytes, therefore cytokine:L32 ratios were set to 1 in the mouse B cell lymphoma line 967.
T141	20070-20221	Sentence	denotes	None of the tested cytokines were detected in wild-type lymphocytes, therefore cytokine:L32 ratios were set to 1 in the mouse B cell lymphoma line 967.
T7555	20222-20416	Sentence	denotes	Transcription of IL10, IL15, and IFNγ were reproducibly detected in LMP1 transgenic lymphocytes and lymphoma cells and was higher than in the B cell lymphoma cell lines 967 and K46μ (Figure 5A).
T142	20222-20416	Sentence	denotes	Transcription of IL10, IL15, and IFNγ were reproducibly detected in LMP1 transgenic lymphocytes and lymphoma cells and was higher than in the B cell lymphoma cell lines 967 and K46μ (Figure 5A).
T7556	20417-20697	Sentence	denotes	There was no significant difference in the expression of IL15 and IFNγ between LMP1 transgenic lymphocytes and lymphoma cells, suggesting that upregulation of IL15 and IFNγ is induced by LMP1 expression in healthy lymphocytes but is not a unique property of malignant lymphocytes.
T143	20417-20697	Sentence	denotes	There was no significant difference in the expression of IL15 and IFNγ between LMP1 transgenic lymphocytes and lymphoma cells, suggesting that upregulation of IL15 and IFNγ is induced by LMP1 expression in healthy lymphocytes but is not a unique property of malignant lymphocytes.
T7557	20698-20886	Sentence	denotes	Strikingly, IL10, a B lymphocyte stimulatory cytokine, was increased 1.5- to 5-fold in the wild-type and LMP1 transgenic lymphoma cells compared to LMP1 transgenic lymphocytes (Figure 5A).
T144	20698-20886	Sentence	denotes	Strikingly, IL10, a B lymphocyte stimulatory cytokine, was increased 1.5- to 5-fold in the wild-type and LMP1 transgenic lymphoma cells compared to LMP1 transgenic lymphocytes (Figure 5A).
T7558	20887-21026	Sentence	denotes	Production of IL15 and IFNγ has been associated with induction of cytotoxic effector responses in cells latently infected with EBV [33,34].
T145	20887-21026	Sentence	denotes	Production of IL15 and IFNγ has been associated with induction of cytotoxic effector responses in cells latently infected with EBV [33,34].
T7559	21027-21251	Sentence	denotes	However, transformation and growth properties induced by EBV are associated with the upregulation of IL10 [35–38]; hence, the effects of IL10 upregulation on the growth properties of the lymphoma cells were further examined.
T146	21027-21251	Sentence	denotes	However, transformation and growth properties induced by EBV are associated with the upregulation of IL10 [35–38]; hence, the effects of IL10 upregulation on the growth properties of the lymphoma cells were further examined.
T7560	21252-21496	Sentence	denotes	Immunoblot analysis indicated that LMP1 transgenic lymphocytes and wild-type and LMP1 transgenic lymphoma cells had corresponding increased levels of phosphorylated α and β isoforms of activated Stat3, a target of the IL10 receptor (Figure 5B).
T147	21252-21496	Sentence	denotes	Immunoblot analysis indicated that LMP1 transgenic lymphocytes and wild-type and LMP1 transgenic lymphoma cells had corresponding increased levels of phosphorylated α and β isoforms of activated Stat3, a target of the IL10 receptor (Figure 5B).
T7561	21497-21638	Sentence	denotes	However, when comparing the same lymphomas, there was no correlation between the levels of IL10 induction and the levels of Stat3 activation.
T148	21497-21638	Sentence	denotes	However, when comparing the same lymphomas, there was no correlation between the levels of IL10 induction and the levels of Stat3 activation.
T7562	21639-21757	Sentence	denotes	This suggests that the activation of Stat3 is not solely induced by IL10 or that Stat3 activation may be constitutive.
T149	21639-21757	Sentence	denotes	This suggests that the activation of Stat3 is not solely induced by IL10 or that Stat3 activation may be constitutive.
T7563	21758-21888	Sentence	denotes	Additionally, there was no correlation between the levels of LMP1 expression and the levels of IL10 induction (Figures 1A and 5A).
T150	21758-21888	Sentence	denotes	Additionally, there was no correlation between the levels of LMP1 expression and the levels of IL10 induction (Figures 1A and 5A).
T7564	21889-22031	Sentence	denotes	This indicates that the induction of IL10 is a general property associated with enhanced survival and may only be indirectly affected by LMP1.
T151	21889-22031	Sentence	denotes	This indicates that the induction of IL10 is a general property associated with enhanced survival and may only be indirectly affected by LMP1.
T7565	22032-22205	Sentence	denotes	Neutralizing antibodies to IL10 did not affect the survival of lymphoma cells as determined by the MTS assay (unpublished data), suggesting constitutive activation of Stat3.
T152	22032-22205	Sentence	denotes	Neutralizing antibodies to IL10 did not affect the survival of lymphoma cells as determined by the MTS assay (unpublished data), suggesting constitutive activation of Stat3.
T7566	22206-22429	Sentence	denotes	This was confirmed by immunoblot analysis such that in the presence of anti-IL10 neutralizing antibodies, pStat3 levels remained activated in lymphoma cells isolated from wild-type and LMP1 transgenic lymphomas (Figure 5C).
T153	22206-22429	Sentence	denotes	This was confirmed by immunoblot analysis such that in the presence of anti-IL10 neutralizing antibodies, pStat3 levels remained activated in lymphoma cells isolated from wild-type and LMP1 transgenic lymphomas (Figure 5C).
T7567	22430-22587	Sentence	denotes	Exogenous addition of IL10 enhanced pStat3 activation above constitutive levels, indicating that lymphoma cells are responsive to IL10 treatment (Figure 5C).
T154	22430-22587	Sentence	denotes	Exogenous addition of IL10 enhanced pStat3 activation above constitutive levels, indicating that lymphoma cells are responsive to IL10 treatment (Figure 5C).
T7568	22588-22713	Sentence	denotes	This means that although the lymphoma cells have constitutive Stat3 activation, it may be further enhanced by IL10 induction.
T155	22588-22713	Sentence	denotes	This means that although the lymphoma cells have constitutive Stat3 activation, it may be further enhanced by IL10 induction.
T7569	22714-22879	Sentence	denotes	The neutralizing effect of the anti-IL10 antibody was confirmed by pre-incubation of IL10 with anti-IL10 antibody compared to a rat IgG1 isotype control (Figure 5C).
T156	22714-22879	Sentence	denotes	The neutralizing effect of the anti-IL10 antibody was confirmed by pre-incubation of IL10 with anti-IL10 antibody compared to a rat IgG1 isotype control (Figure 5C).
T157	22880-22957	Sentence	denotes	Figure 5 LMP1 Upregulates IL10 Expression and Constitutively Activates Stat3
T23731	22890-22957	Sentence	denotes	LMP1 Upregulates IL10 Expression and Constitutively Activates Stat3
T23732	22958-23097	Sentence	denotes	(A) Relative expression of IL10, IL15, and IFNγ mRNA in WT and LMP1 transgenic B cells (CD19+), as detected with an Rnase protection assay.
T158	22958-23097	Sentence	denotes	(A) Relative expression of IL10, IL15, and IFNγ mRNA in WT and LMP1 transgenic B cells (CD19+), as detected with an Rnase protection assay.
T23733	23098-23159	Sentence	denotes	Mouse lymphoma cell lines 967 and K46μ were used as controls.
T159	23098-23159	Sentence	denotes	Mouse lymphoma cell lines 967 and K46μ were used as controls.
T23734	23160-23282	Sentence	denotes	Expression levels were quantified with a phosphorimager and values were normalized to the ribosomal housekeeping gene L32.
T160	23160-23282	Sentence	denotes	Expression levels were quantified with a phosphorimager and values were normalized to the ribosomal housekeeping gene L32.
T23735	23283-23357	Sentence	denotes	The cytokine:L32 ratio was set to 1 in the mouse B cell lymphoma line 967.
T161	23283-23357	Sentence	denotes	The cytokine:L32 ratio was set to 1 in the mouse B cell lymphoma line 967.
T23736	23358-23684	Sentence	denotes	(B and C) Immunoblot analysis of activated pStat3 in purified B cells (CD19+) from WT and LMP1 transgenic mice (B) at the time of harvest, and (C) 4 h after culture with or without IL10, a neutralizing antibody to IL10, or a rat IgG1 isotype control. (C) Shown are the results for WT lymphoma 1 and LMP1 transgenic lymphoma 1.
T162	23358-23684	Sentence	denotes	(B and C) Immunoblot analysis of activated pStat3 in purified B cells (CD19+) from WT and LMP1 transgenic mice (B) at the time of harvest, and (C) 4 h after culture with or without IL10, a neutralizing antibody to IL10, or a rat IgG1 isotype control. (C) Shown are the results for WT lymphoma 1 and LMP1 transgenic lymphoma 1.
T23737	23685-23748	Sentence	denotes	Arrows indicate the positions of the α and β isoforms of Stat3.
T163	23685-23748	Sentence	denotes	Arrows indicate the positions of the α and β isoforms of Stat3.
T23738	23749-23785	Sentence	denotes	Actin was used as a loading control.
T164	23749-23785	Sentence	denotes	Actin was used as a loading control.
T23739	23786-23895	Sentence	denotes	(D) Immunohistochemistry detection of activated nuclear pStat3 in the spleens of WT and LMP1 transgenic mice.
T165	23786-23895	Sentence	denotes	(D) Immunohistochemistry detection of activated nuclear pStat3 in the spleens of WT and LMP1 transgenic mice.
T23740	23896-23913	Sentence	denotes	Scale bar, 20 μm.
T166	23896-23913	Sentence	denotes	Scale bar, 20 μm.
T7570	23916-24086	Sentence	denotes	Nuclear translocation of pStat3 is a consequence of activation, and nuclear pStat3 was not detected by immunohistochemistry staining of spleen sections from control mice.
T167	23916-24086	Sentence	denotes	Nuclear translocation of pStat3 is a consequence of activation, and nuclear pStat3 was not detected by immunohistochemistry staining of spleen sections from control mice.
T7571	24087-24251	Sentence	denotes	However, nuclear pStat3 was detectable in LMP1 transgenic mice and wild-type lymphomas and was detected more homogeneously in LMP1 transgenic lymphomas (Figure 5D).
T168	24087-24251	Sentence	denotes	However, nuclear pStat3 was detectable in LMP1 transgenic mice and wild-type lymphomas and was detected more homogeneously in LMP1 transgenic lymphomas (Figure 5D).
T7572	24252-24397	Sentence	denotes	The constitutive activation of pStat3 and abundant nuclear Stat3 suggests that Stat3 signaling contributes to LMP1-mediated lymphoma development.
T169	24252-24397	Sentence	denotes	The constitutive activation of pStat3 and abundant nuclear Stat3 suggests that Stat3 signaling contributes to LMP1-mediated lymphoma development.
T9769	24399-24469	Sentence	denotes	LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway
T170	24399-24469	Sentence	denotes	LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway
T9770	24470-24552	Sentence	denotes	LMP1 transformation of rodent fibroblasts requires activation of PI3K and Akt [5].
T171	24470-24552	Sentence	denotes	LMP1 transformation of rodent fibroblasts requires activation of PI3K and Akt [5].
T9771	24553-24676	Sentence	denotes	Additionally, activated pAkt is frequently detected in NPC and the neoplastic Reed-Sternberg cells of classical HD [39,40].
T172	24553-24676	Sentence	denotes	Additionally, activated pAkt is frequently detected in NPC and the neoplastic Reed-Sternberg cells of classical HD [39,40].
T9772	24677-24848	Sentence	denotes	To determine if Akt signaling is activated in LMP1 transgenic mice, pAkt and several of its targets were assessed by immunoblotting of splenic CD19+ MACS-purified B cells.
T173	24677-24848	Sentence	denotes	To determine if Akt signaling is activated in LMP1 transgenic mice, pAkt and several of its targets were assessed by immunoblotting of splenic CD19+ MACS-purified B cells.
T9773	24849-25058	Sentence	denotes	LMP1 transgenic B cells had increased levels of pAkt compared to wild-type lymphocytes; however, progression to lymphoma in both LMP1-positive and -negative lymphoma cells did not further increase pAkt levels.
T174	24849-25058	Sentence	denotes	LMP1 transgenic B cells had increased levels of pAkt compared to wild-type lymphocytes; however, progression to lymphoma in both LMP1-positive and -negative lymphoma cells did not further increase pAkt levels.
T9774	25059-25287	Sentence	denotes	The Akt target glycogen synthase kinase 3 (GSK3) is inactivated by phosphorylation; however, increased phosphorylated GSK3 was not detected in the transgenic lymphocytes and was almost absent in the lymphoma samples (Figure 6A).
T175	25059-25287	Sentence	denotes	The Akt target glycogen synthase kinase 3 (GSK3) is inactivated by phosphorylation; however, increased phosphorylated GSK3 was not detected in the transgenic lymphocytes and was almost absent in the lymphoma samples (Figure 6A).
T9775	25288-25408	Sentence	denotes	This finding indicates that GSK3 is not a target of activated Akt in the LMP1 transgenic lymphocytes and lymphoma cells.
T176	25288-25408	Sentence	denotes	This finding indicates that GSK3 is not a target of activated Akt in the LMP1 transgenic lymphocytes and lymphoma cells.
T9776	25409-25520	Sentence	denotes	Similarly, activation of Akt without phosphorylation of GSK3 has been previously shown in EBV-positive HD [40].
T177	25409-25520	Sentence	denotes	Similarly, activation of Akt without phosphorylation of GSK3 has been previously shown in EBV-positive HD [40].
T9777	25521-25625	Sentence	denotes	In contrast, the wild-type lymphocytes lacked activated Akt but did have detectable phosphorylated GSK3.
T178	25521-25625	Sentence	denotes	In contrast, the wild-type lymphocytes lacked activated Akt but did have detectable phosphorylated GSK3.
T9778	25626-25712	Sentence	denotes	This further suggests that additional pathways are involved in the regulation of GSK3.
T179	25626-25712	Sentence	denotes	This further suggests that additional pathways are involved in the regulation of GSK3.
T180	25713-25793	Sentence	denotes	Figure 6 LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway
T24366	25723-25793	Sentence	denotes	LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway
T24367	25794-26058	Sentence	denotes	(A and B) Immunoblot analysis of purified B cells (CD19+) from the spleens of WT and LMP1 transgenic mice for Akt signaling, probing for (A) activated pAkt and downstream targets, including inactivated pGSK3α/β, and (B) activated p-mTOR, and total levels of FoxO1.
T181	25794-26058	Sentence	denotes	(A and B) Immunoblot analysis of purified B cells (CD19+) from the spleens of WT and LMP1 transgenic mice for Akt signaling, probing for (A) activated pAkt and downstream targets, including inactivated pGSK3α/β, and (B) activated p-mTOR, and total levels of FoxO1.
T24368	26059-26117	Sentence	denotes	Arrows indicate the positions of α and β isoforms of GSK3.
T182	26059-26117	Sentence	denotes	Arrows indicate the positions of α and β isoforms of GSK3.
T24369	26118-26188	Sentence	denotes	The white line indicates that intervening lanes have been spliced out.
T183	26118-26188	Sentence	denotes	The white line indicates that intervening lanes have been spliced out.
T24370	26189-26342	Sentence	denotes	(C) Immunoblot analysis for cell cycle proteins regulating the Rb pathway, probing for activated pRb, and total levels of Cdk2 and the Cdk inhibitor p27.
T184	26189-26342	Sentence	denotes	(C) Immunoblot analysis for cell cycle proteins regulating the Rb pathway, probing for activated pRb, and total levels of Cdk2 and the Cdk inhibitor p27.
T24371	26343-26379	Sentence	denotes	Actin was used as a loading control.
T185	26343-26379	Sentence	denotes	Actin was used as a loading control.
T9779	26382-26468	Sentence	denotes	To identify other potential Akt targets, immunoblot analysis for p-mTOR was performed.
T186	26382-26468	Sentence	denotes	To identify other potential Akt targets, immunoblot analysis for p-mTOR was performed.
T9780	26469-26690	Sentence	denotes	Activated p-mTOR was not increased in LMP1 transgenic lymphocytes or lymphoma cells, indicating that this pathway is not affected by LMP1-induced Akt activation and does not contribute to lymphoma development (Figure 6B).
T187	26469-26690	Sentence	denotes	Activated p-mTOR was not increased in LMP1 transgenic lymphocytes or lymphoma cells, indicating that this pathway is not affected by LMP1-induced Akt activation and does not contribute to lymphoma development (Figure 6B).
T9781	26691-26892	Sentence	denotes	Akt is also known to phosphorylate and induce the degradation of the pro-apoptotic Forkhead family of transcription factors, leading to cell cycle progression and survival in some human tumors [41,42].
T188	26691-26892	Sentence	denotes	Akt is also known to phosphorylate and induce the degradation of the pro-apoptotic Forkhead family of transcription factors, leading to cell cycle progression and survival in some human tumors [41,42].
T9782	26893-27020	Sentence	denotes	Immunoblot analysis of splenic B cells did not consistently detect p-FoxO1 levels, a signal that targets FoxO1 for degradation.
T189	26893-27020	Sentence	denotes	Immunoblot analysis of splenic B cells did not consistently detect p-FoxO1 levels, a signal that targets FoxO1 for degradation.
T9783	27021-27097	Sentence	denotes	Hence, degradation of FoxO1 was assessed by detection of total FoxO1 levels.
T190	27021-27097	Sentence	denotes	Hence, degradation of FoxO1 was assessed by detection of total FoxO1 levels.
T9784	27098-27346	Sentence	denotes	Immunoblot analysis indicated that total FoxO1 levels were greatly decreased in wild-type and LMP1 transgenic lymphomas (Figure 6B), suggesting that inhibition of the Forkhead signaling pathway is an important target of Akt in lymphoma development.
T191	27098-27346	Sentence	denotes	Immunoblot analysis indicated that total FoxO1 levels were greatly decreased in wild-type and LMP1 transgenic lymphomas (Figure 6B), suggesting that inhibition of the Forkhead signaling pathway is an important target of Akt in lymphoma development.
T9785	27347-27577	Sentence	denotes	However, considering that Akt activation did not induce FoxO1 degradation in LMP1 transgenic B cells, Akt may not be the sole regulator of FoxO1, and it may be that progression to lymphoma requires modulation of multiple pathways.
T192	27347-27577	Sentence	denotes	However, considering that Akt activation did not induce FoxO1 degradation in LMP1 transgenic B cells, Akt may not be the sole regulator of FoxO1, and it may be that progression to lymphoma requires modulation of multiple pathways.
T9786	27578-27690	Sentence	denotes	The Forkhead family of transcription factors is known to induce the expression of the Cdk inhibitor p27 [43,44].
T193	27578-27690	Sentence	denotes	The Forkhead family of transcription factors is known to induce the expression of the Cdk inhibitor p27 [43,44].
T9787	27691-27868	Sentence	denotes	LMP1-transformed rodent fibroblasts have decreased expression of p27, upregulation of Cdk2, and subsequent phosphorylation and inactivation of the tumor suppressor gene Rb [45].
T194	27691-27868	Sentence	denotes	LMP1-transformed rodent fibroblasts have decreased expression of p27, upregulation of Cdk2, and subsequent phosphorylation and inactivation of the tumor suppressor gene Rb [45].
T9788	27869-28060	Sentence	denotes	To investigate whether LMP1 affected cell cycle regulation through the Rb pathway in B cells, immunoblot analyses for pRb, Cdk2, and p27 were performed on splenic CD19+ MACS-purified B cells.
T195	27869-28060	Sentence	denotes	To investigate whether LMP1 affected cell cycle regulation through the Rb pathway in B cells, immunoblot analyses for pRb, Cdk2, and p27 were performed on splenic CD19+ MACS-purified B cells.
T9789	28061-28219	Sentence	denotes	LMP1 transgenic B cells had enhanced levels of pRb with concomitant stabilization of total Rb levels and Cdk2 compared to wild-type B lymphocytes (Figure 6C).
T196	28061-28219	Sentence	denotes	LMP1 transgenic B cells had enhanced levels of pRb with concomitant stabilization of total Rb levels and Cdk2 compared to wild-type B lymphocytes (Figure 6C).
T9790	28220-28405	Sentence	denotes	Progression to lymphoma in both wild-type and LMP1 transgenic lymphoma cells led to increased levels of Rb, correspondingly high levels of Cdk2, and decreased levels of p27 (Figure 6C).
T197	28220-28405	Sentence	denotes	Progression to lymphoma in both wild-type and LMP1 transgenic lymphoma cells led to increased levels of Rb, correspondingly high levels of Cdk2, and decreased levels of p27 (Figure 6C).
T9791	28406-28569	Sentence	denotes	These data indicate that the Rb pathway is deregulated in LMP1 transgenic lymphocytes and that lymphoma cells are distinguished by loss of FoxO1 and decreased p27.
T198	28406-28569	Sentence	denotes	These data indicate that the Rb pathway is deregulated in LMP1 transgenic lymphocytes and that lymphoma cells are distinguished by loss of FoxO1 and decreased p27.
T11843	28571-28662	Sentence	denotes	LMP1 Promotes Tumor Growth and Survival through Activation of Akt, NFκB, and Stat3 Pathways
T199	28571-28662	Sentence	denotes	LMP1 Promotes Tumor Growth and Survival through Activation of Akt, NFκB, and Stat3 Pathways
T11844	28663-28952	Sentence	denotes	To explore which pathways were required for the enhanced growth and survival of LMP1-induced lymphomas, splenocytes from wild-type and LMP1 transgenic mice were cultured in the presence of inhibitors for Akt, NFκB, Stat3, mTOR, or MAPK and assayed for growth and survival by the MTS assay.
T200	28663-28952	Sentence	denotes	To explore which pathways were required for the enhanced growth and survival of LMP1-induced lymphomas, splenocytes from wild-type and LMP1 transgenic mice were cultured in the presence of inhibitors for Akt, NFκB, Stat3, mTOR, or MAPK and assayed for growth and survival by the MTS assay.
T11845	28953-29067	Sentence	denotes	As previously shown, wild-type lymphocytes were not viable in culture and could not be tested with the inhibitors.
T201	28953-29067	Sentence	denotes	As previously shown, wild-type lymphocytes were not viable in culture and could not be tested with the inhibitors.
T11846	29068-29306	Sentence	denotes	However, the enhanced viability of LMP1 transgenic lymphocytes was effectively blocked by treatment with triciribine, BAY11–7085, cucurbitacin I, and slightly with SB203580, but not by treatment with rapamycin, U0126, or AG490 (Figure 7).
T202	29068-29306	Sentence	denotes	However, the enhanced viability of LMP1 transgenic lymphocytes was effectively blocked by treatment with triciribine, BAY11–7085, cucurbitacin I, and slightly with SB203580, but not by treatment with rapamycin, U0126, or AG490 (Figure 7).
T11847	29307-29450	Sentence	denotes	Triciribine inhibits the activation of Akt and at 20 μM has been shown to induce growth arrest in cancer cells with aberrant Akt activity [46].
T203	29307-29450	Sentence	denotes	Triciribine inhibits the activation of Akt and at 20 μM has been shown to induce growth arrest in cancer cells with aberrant Akt activity [46].
T11848	29451-29702	Sentence	denotes	The effects of triciribine on cell growth of the transgenic lymphocytes and lymphomas were apparent as low as 1 μM, suggesting that activation of Akt is required for the survival and growth of LMP1 transgenic lymphocytes and lymphoma cells (Figure 7).
T204	29451-29702	Sentence	denotes	The effects of triciribine on cell growth of the transgenic lymphocytes and lymphomas were apparent as low as 1 μM, suggesting that activation of Akt is required for the survival and growth of LMP1 transgenic lymphocytes and lymphoma cells (Figure 7).
T11849	29703-29825	Sentence	denotes	The effects of the inhibitors were assessed by identifying phosphorylated Akt, Stat3, and total levels of IκBα (Figure 8).
T205	29703-29825	Sentence	denotes	The effects of the inhibitors were assessed by identifying phosphorylated Akt, Stat3, and total levels of IκBα (Figure 8).
T11850	29826-29953	Sentence	denotes	Treatment with triciribine effectively blocked phosphorylation of Akt, and phosphorylated Akt was no longer detected past 5 μM.
T206	29826-29953	Sentence	denotes	Treatment with triciribine effectively blocked phosphorylation of Akt, and phosphorylated Akt was no longer detected past 5 μM.
T11851	29954-30012	Sentence	denotes	Phosphorylated Stat3 and IκBα were still present at 25 μM.
T207	29954-30012	Sentence	denotes	Phosphorylated Stat3 and IκBα were still present at 25 μM.
T11852	30013-30190	Sentence	denotes	These findings suggest that triciribine specifically targets Akt and that Akt activation is required for the enhanced viability of the transgenic lymphocytes and lymphoma cells.
T208	30013-30190	Sentence	denotes	These findings suggest that triciribine specifically targets Akt and that Akt activation is required for the enhanced viability of the transgenic lymphocytes and lymphoma cells.
T209	30191-30290	Sentence	denotes	Figure 7 Akt, NFκB, and Stat3 Signaling Are Required for the Growth and Survival of Lymphoma Cells
T24794	30201-30290	Sentence	denotes	Akt, NFκB, and Stat3 Signaling Are Required for the Growth and Survival of Lymphoma Cells
T24795	30291-30415	Sentence	denotes	MTS assay of splenocytes from (A and B) WT or (C and D) LMP1 transgenic lymphomas and (E and F) LMP1 transgenic lymphoctyes.
T210	30291-30415	Sentence	denotes	MTS assay of splenocytes from (A and B) WT or (C and D) LMP1 transgenic lymphomas and (E and F) LMP1 transgenic lymphoctyes.
T24796	30416-30623	Sentence	denotes	Splenocytes were cultured with or without inhibitors of NFκB (BAY11), mTOR (rapamycin), Akt (triciribine), MEK1/2 (U0126), p38 (SB202190), or Stat3 (cucurbitacin I and AG490) at the indicated concentrations.
T211	30416-30623	Sentence	denotes	Splenocytes were cultured with or without inhibitors of NFκB (BAY11), mTOR (rapamycin), Akt (triciribine), MEK1/2 (U0126), p38 (SB202190), or Stat3 (cucurbitacin I and AG490) at the indicated concentrations.
T24797	30624-30677	Sentence	denotes	The results are the mean ± SEM of triplicate samples.
T212	30624-30677	Sentence	denotes	The results are the mean ± SEM of triplicate samples.
T24798	30678-30826	Sentence	denotes	Shown are the results for (A and B) WT lymphoma 1, (C and D) LMP1 transgenic lymphoma 2, and (E and F) one out of two LMP1 transgenic mice analyzed.
T213	30678-30826	Sentence	denotes	Shown are the results for (A and B) WT lymphoma 1, (C and D) LMP1 transgenic lymphoma 2, and (E and F) one out of two LMP1 transgenic mice analyzed.
T24799	30827-30911	Sentence	denotes	This analysis was repeated with LMP1 transgenic lymphoma 4 yielding similar results.
T214	30827-30911	Sentence	denotes	This analysis was repeated with LMP1 transgenic lymphoma 4 yielding similar results.
T215	30912-31024	Sentence	denotes	Figure 8 Analysis of Akt, NFκB, and Stat3 Pathways in Contribution to the Growth and Survival of Lymphoma Cells
T25238	30922-31024	Sentence	denotes	Analysis of Akt, NFκB, and Stat3 Pathways in Contribution to the Growth and Survival of Lymphoma Cells
T25239	31025-31311	Sentence	denotes	(A–D) Immunoblot analysis of wild-type and LMP1 transgenic lymphomas for Akt, NFκB, and Stat3 signaling after treatment with (A) an Akt inhibitor, triciribine, (B) an NFκB inhibitor, BAY11–7085, and the Stat3 inhibitors (C) cucurbitacin I and (D) AG490, at the indicated concentrations.
T216	31025-31311	Sentence	denotes	(A–D) Immunoblot analysis of wild-type and LMP1 transgenic lymphomas for Akt, NFκB, and Stat3 signaling after treatment with (A) an Akt inhibitor, triciribine, (B) an NFκB inhibitor, BAY11–7085, and the Stat3 inhibitors (C) cucurbitacin I and (D) AG490, at the indicated concentrations.
T25240	31312-31371	Sentence	denotes	Arrows indicate the positions of α and β isoforms of Stat3.
T217	31312-31371	Sentence	denotes	Arrows indicate the positions of α and β isoforms of Stat3.
T25241	31372-31408	Sentence	denotes	Actin was used as a loading control.
T218	31372-31408	Sentence	denotes	Actin was used as a loading control.
T11853	31411-31506	Sentence	denotes	Inhibition of NFκB signaling rapidly induces cell death of EBV-transformed lymphocytes [17,18].
T219	31411-31506	Sentence	denotes	Inhibition of NFκB signaling rapidly induces cell death of EBV-transformed lymphocytes [17,18].
T11854	31507-31726	Sentence	denotes	BAY11–7085, an inhibitor of NFκB signaling, also greatly decreased the viability of the LMP1 transgenic lymphocytes and lymphoma cells at doses as low as 1 μM, and at 5 μM the cells were completely nonviable (Figure 7).
T220	31507-31726	Sentence	denotes	BAY11–7085, an inhibitor of NFκB signaling, also greatly decreased the viability of the LMP1 transgenic lymphocytes and lymphoma cells at doses as low as 1 μM, and at 5 μM the cells were completely nonviable (Figure 7).
T11855	31727-31774	Sentence	denotes	This is well within the reported IC50 of 10 μM.
T221	31727-31774	Sentence	denotes	This is well within the reported IC50 of 10 μM.
T11856	31775-31907	Sentence	denotes	Phosphorylated Akt, Stat3, and total IκBα were still present up to treatment with 15 μM and then were no longer detected (Figure 8).
T222	31775-31907	Sentence	denotes	Phosphorylated Akt, Stat3, and total IκBα were still present up to treatment with 15 μM and then were no longer detected (Figure 8).
T11857	31908-32084	Sentence	denotes	This finding suggests that inhibition of NFκB can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells without significant effects on activation of Akt or Stat3.
T223	31908-32084	Sentence	denotes	This finding suggests that inhibition of NFκB can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells without significant effects on activation of Akt or Stat3.
T11858	32085-32178	Sentence	denotes	Cucurbitacin I inhibits activation of Stat3 by suppressing the activation of its kinase JAK2.
T224	32085-32178	Sentence	denotes	Cucurbitacin I inhibits activation of Stat3 by suppressing the activation of its kinase JAK2.
T11859	32179-32282	Sentence	denotes	It has been shown to selectively inhibit the growth of tumors with constitutively activated Stat3 [47].
T225	32179-32282	Sentence	denotes	It has been shown to selectively inhibit the growth of tumors with constitutively activated Stat3 [47].
T11860	32283-32485	Sentence	denotes	Similarly, LMP1 transgenic lymphocytes and lymphoma cells were susceptible to cucurbitacin I treatment starting at 0.1 μM, a dose that corresponds closely to the reported IC50 of 500 nM (Figure 7) [47].
T226	32283-32485	Sentence	denotes	Similarly, LMP1 transgenic lymphocytes and lymphoma cells were susceptible to cucurbitacin I treatment starting at 0.1 μM, a dose that corresponds closely to the reported IC50 of 500 nM (Figure 7) [47].
T11861	32486-32674	Sentence	denotes	Phosphorylated Akt, Stat3, and total IκBα were not detectable past 1 μM and at higher doses all protein levels were greatly decreased, indicative of the total loss of viability (Figure 8).
T227	32486-32674	Sentence	denotes	Phosphorylated Akt, Stat3, and total IκBα were not detectable past 1 μM and at higher doses all protein levels were greatly decreased, indicative of the total loss of viability (Figure 8).
T11862	32675-32838	Sentence	denotes	A second reported inhibitor of Stat3, AG490, had no effect on growth (Figure 7), but activation of Akt, Stat3, or levels of IκBα were also not affected (Figure 8).
T228	32675-32838	Sentence	denotes	A second reported inhibitor of Stat3, AG490, had no effect on growth (Figure 7), but activation of Akt, Stat3, or levels of IκBα were also not affected (Figure 8).
T11863	32839-33047	Sentence	denotes	These findings suggest that inhibition of Stat3 can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells, but Stat3 inhibition also has considerable crossover effects on Akt and NFκB signaling.
T229	32839-33047	Sentence	denotes	These findings suggest that inhibition of Stat3 can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells, but Stat3 inhibition also has considerable crossover effects on Akt and NFκB signaling.
T11864	33048-33283	Sentence	denotes	LMP1 has also been shown to activate JNK and p38 MAPK pathways [13,48], and LMP1 transgenic lymphocytes were mildly susceptible to growth inhibition by SB202190, an inhibitor of p38 MAPK, but not U0126, an inhibitor of MEK1/2 activity.
T230	33048-33283	Sentence	denotes	LMP1 has also been shown to activate JNK and p38 MAPK pathways [13,48], and LMP1 transgenic lymphocytes were mildly susceptible to growth inhibition by SB202190, an inhibitor of p38 MAPK, but not U0126, an inhibitor of MEK1/2 activity.
T11865	33284-33548	Sentence	denotes	However, effects of SB202190 were only apparent at high doses (>10 μM), much higher than the reported IC50 of 0.35 μM, suggesting that p38 MAPK does not significantly contribute to the enhanced viability in LMP1 transgenic lymphocytes or lymphoma cells (Figure 7).
T231	33284-33548	Sentence	denotes	However, effects of SB202190 were only apparent at high doses (>10 μM), much higher than the reported IC50 of 0.35 μM, suggesting that p38 MAPK does not significantly contribute to the enhanced viability in LMP1 transgenic lymphocytes or lymphoma cells (Figure 7).
T11866	33549-34122	Sentence	denotes	Interestingly, both wild-type and LMP1 transgenic lymphomas were similarly susceptible to triciribine, BAY11–7085, and cucurbitacin I treatments, but not SB202190, AG490, or U0126 treatment, suggesting that activation of Akt, NFκB, and Stat3 but not MAPK pathways are characteristics associated with malignant transformation (Figure 7). rapamycin, an inhibitor of mTOR, did not affect the viability of the transgenic lymphocytes or lymphoma cells, confirming that mTOR is not targeted by Akt activation in LMP1 transgenic lymphocytes or malignant lymphoma cells (Figure 7).
T232	33549-34122	Sentence	denotes	Interestingly, both wild-type and LMP1 transgenic lymphomas were similarly susceptible to triciribine, BAY11–7085, and cucurbitacin I treatments, but not SB202190, AG490, or U0126 treatment, suggesting that activation of Akt, NFκB, and Stat3 but not MAPK pathways are characteristics associated with malignant transformation (Figure 7). rapamycin, an inhibitor of mTOR, did not affect the viability of the transgenic lymphocytes or lymphoma cells, confirming that mTOR is not targeted by Akt activation in LMP1 transgenic lymphocytes or malignant lymphoma cells (Figure 7).
T233	34124-34134	Sentence	denotes	Discussion
T14502	34135-34223	Sentence	denotes	This study defines the oncogenic properties of LMP1 in promoting B cell lymphomagenesis.
T234	34135-34223	Sentence	denotes	This study defines the oncogenic properties of LMP1 in promoting B cell lymphomagenesis.
T14503	34224-34450	Sentence	denotes	LMP1 transgenic mice have a higher incidence of lymphoma [26] and the progression to lymphoma correlates with higher expression levels of LMP1 (Figure 1A and 1B), suggesting that LMP1 is directly involved in tumor development.
T235	34224-34450	Sentence	denotes	LMP1 transgenic mice have a higher incidence of lymphoma [26] and the progression to lymphoma correlates with higher expression levels of LMP1 (Figure 1A and 1B), suggesting that LMP1 is directly involved in tumor development.
T14504	34451-34574	Sentence	denotes	Table 1 summarizes the biological and molecular properties that were identified in wild-type and LMP1 transgenic lymphomas.
T236	34451-34574	Sentence	denotes	Table 1 summarizes the biological and molecular properties that were identified in wild-type and LMP1 transgenic lymphomas.
T14505	34575-34838	Sentence	denotes	Although many of the molecular properties studied were similar between wild-type and LMP1 transgenic lymphomas, there were distinguishing biological properties, namely the ability of LMP1 transgenic lymphomas to induce higher levels of survival and proliferation.
T237	34575-34838	Sentence	denotes	Although many of the molecular properties studied were similar between wild-type and LMP1 transgenic lymphomas, there were distinguishing biological properties, namely the ability of LMP1 transgenic lymphomas to induce higher levels of survival and proliferation.
T14506	34839-35069	Sentence	denotes	Interestingly, although LMP1 transgenic mice develop lymphomas in the same B-1a cell type as spontaneous wild-type lymphomas (Figure 2), some signaling effects induced by LMP1 may explain the enhanced promotion to lymphomagenesis.
T238	34839-35069	Sentence	denotes	Interestingly, although LMP1 transgenic mice develop lymphomas in the same B-1a cell type as spontaneous wild-type lymphomas (Figure 2), some signaling effects induced by LMP1 may explain the enhanced promotion to lymphomagenesis.
T14507	35070-35310	Sentence	denotes	Since CD40-deficient mice have decreased numbers of IgMhighIgDlow cells, a phenotype associated with B-1, marginal zone, and memory B cells, the mimicry of CD40 signaling by LMP1 could possibly contribute to the expansion of B-1 cells [20].
T239	35070-35310	Sentence	denotes	Since CD40-deficient mice have decreased numbers of IgMhighIgDlow cells, a phenotype associated with B-1, marginal zone, and memory B cells, the mimicry of CD40 signaling by LMP1 could possibly contribute to the expansion of B-1 cells [20].
T14508	35311-35500	Sentence	denotes	It is noteworthy that expression of LMP1 in transgenic mice has been shown to inhibit the formation of GCs [23,49], preventing typical B-2 cells from antigen-driven selection and expansion.
T240	35311-35500	Sentence	denotes	It is noteworthy that expression of LMP1 in transgenic mice has been shown to inhibit the formation of GCs [23,49], preventing typical B-2 cells from antigen-driven selection and expansion.
T14509	35501-35604	Sentence	denotes	The lack of GC reactions may contribute to the bias of LMP1 transgenic mice towards B-1 cell lymphomas.
T241	35501-35604	Sentence	denotes	The lack of GC reactions may contribute to the bias of LMP1 transgenic mice towards B-1 cell lymphomas.
T14510	35605-35722	Sentence	denotes	Interestingly, LMP2 signaling also favors development of B-1 cells, but this occurs in the absence of transformation.
T242	35605-35722	Sentence	denotes	Interestingly, LMP2 signaling also favors development of B-1 cells, but this occurs in the absence of transformation.
T14511	35723-35867	Sentence	denotes	These results suggest that the mimicry of B cell receptor signaling by LMP2 promotes B-1 cell differentiation but not transformation [24,50,51].
T243	35723-35867	Sentence	denotes	These results suggest that the mimicry of B cell receptor signaling by LMP2 promotes B-1 cell differentiation but not transformation [24,50,51].
T14512	35868-36005	Sentence	denotes	This promotion of B-1 differentiation may account for the ability of LMP2 to exacerbate autoimmunity and bypass anergy induction [52,53].
T244	35868-36005	Sentence	denotes	This promotion of B-1 differentiation may account for the ability of LMP2 to exacerbate autoimmunity and bypass anergy induction [52,53].
T14513	36006-36248	Sentence	denotes	In contrast, the preponderance of tumors of B-1a origin does not reflect effects of LMP1 signaling on B cell differentiation, as splenic B cells from healthy LMP1 transgenic mice contain similar numbers of B-1 and B-2 cells as wild-type mice.
T245	36006-36248	Sentence	denotes	In contrast, the preponderance of tumors of B-1a origin does not reflect effects of LMP1 signaling on B cell differentiation, as splenic B cells from healthy LMP1 transgenic mice contain similar numbers of B-1 and B-2 cells as wild-type mice.
T14514	36249-36380	Sentence	denotes	In support of this lack of effect, the differentiation of B-1 versus B-2 cells is thought to be independent of CD40 signaling [54].
T246	36249-36380	Sentence	denotes	In support of this lack of effect, the differentiation of B-1 versus B-2 cells is thought to be independent of CD40 signaling [54].
T14515	36381-36571	Sentence	denotes	B-1 cells constitute the predominant lymphocyte population in the peritoneal and pleuropericardial cavities, while B-2 cells are mainly found in the spleen, lymph node, and peripheral blood.
T247	36381-36571	Sentence	denotes	B-1 cells constitute the predominant lymphocyte population in the peritoneal and pleuropericardial cavities, while B-2 cells are mainly found in the spleen, lymph node, and peripheral blood.
T14516	36572-36722	Sentence	denotes	B-1 cells produce the main source of IgM and IgA antibodies in serum, which are involved in T cell–independent responses to common microbial antigens.
T248	36572-36722	Sentence	denotes	B-1 cells produce the main source of IgM and IgA antibodies in serum, which are involved in T cell–independent responses to common microbial antigens.
T14517	36723-36840	Sentence	denotes	Importantly, B-1 cells have the unique capacity to self replenish and are also predisposed to transformation [28,29].
T249	36723-36840	Sentence	denotes	Importantly, B-1 cells have the unique capacity to self replenish and are also predisposed to transformation [28,29].
T14518	36841-37019	Sentence	denotes	Clonal expansion of B-1 cells can be detected in aging mice above 18 mo of age, and B-1 cells are thought to be the murine progenitor of B cell chronic lymphocytic leukemia [30].
T250	36841-37019	Sentence	denotes	Clonal expansion of B-1 cells can be detected in aging mice above 18 mo of age, and B-1 cells are thought to be the murine progenitor of B cell chronic lymphocytic leukemia [30].
T14519	37020-37194	Sentence	denotes	The data presented in this study indicate that although LMP1 is expressed in all B lymphocytes in the transgenic mice, malignancy develops in this specific subset of B cells.
T251	37020-37194	Sentence	denotes	The data presented in this study indicate that although LMP1 is expressed in all B lymphocytes in the transgenic mice, malignancy develops in this specific subset of B cells.
T14520	37195-37316	Sentence	denotes	The elevated expression of LMP1 in B-1a cells and the activation of specific pathways apparently induce malignant growth.
T252	37195-37316	Sentence	denotes	The elevated expression of LMP1 in B-1a cells and the activation of specific pathways apparently induce malignant growth.
T14521	37317-37429	Sentence	denotes	These same pathways can also become sporadically activated in aged mice and also result in lymphoma development.
T253	37317-37429	Sentence	denotes	These same pathways can also become sporadically activated in aged mice and also result in lymphoma development.
T14522	37430-37609	Sentence	denotes	This is similar to EBV-associated cancers in vivo, where pathways that are activated by LMP1 are also activated in the less prevalent EBV-negative forms of the cancers [40,55–58].
T254	37430-37609	Sentence	denotes	This is similar to EBV-associated cancers in vivo, where pathways that are activated by LMP1 are also activated in the less prevalent EBV-negative forms of the cancers [40,55–58].
T14523	37610-37798	Sentence	denotes	Thus, the contribution of EBV and LMP1 to tumor development is apparently the continuous activation of pathways that can also be sporadically activated and contribute to tumor development.
T255	37610-37798	Sentence	denotes	Thus, the contribution of EBV and LMP1 to tumor development is apparently the continuous activation of pathways that can also be sporadically activated and contribute to tumor development.

T59

0-80

Sentence

denotes

EBV Latent Membrane Protein 1 Activates Akt, NFκB, and Stat3 in B Cell Lymphomas

T1

0-129

Sentence

denotes

EBV Latent Membrane Protein 1 Activates Akt, NFκB, and Stat3 in B Cell Lymphomas LMP1 Activates Akt, NFκB, and Stat3 in Lymphomas

T2

132-140

Sentence

denotes

Abstract

T60

141-227

Sentence

denotes

Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV).

T3

141-227

Sentence

denotes

Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV).

T61

228-309

Sentence

denotes

In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age.

T4

228-309

Sentence

denotes

In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age.

T62

310-439

Sentence

denotes

This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice.

T5

310-439

Sentence

denotes

This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice.

T63

440-623

Sentence

denotes

The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFκB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro.

T6

440-623

Sentence

denotes

The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFκB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro.

T64

624-760

Sentence

denotes

Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling.

T7

624-760

Sentence

denotes

Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling.

T65

761-881

Sentence

denotes

These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition.

T8

761-881

Sentence

denotes

These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition.

T66

882-1022

Sentence

denotes

These results suggest that Akt, NFκB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.

T9

882-1022

Sentence

denotes

These results suggest that Akt, NFκB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.

T10

1024-1038

Sentence

denotes

Author Summary

T11

1040-1197

Sentence

denotes

Epstein-Barr virus (EBV) is linked to the development of multiple cancers, including post-transplant lymphoma, Hodgkin disease, and nasopharyngeal carcinoma.

T12

1198-1379

Sentence

denotes

Latent membrane protein 1 (LMP1) is expressed in many EBV-associated cancers and is responsible for most of the altered cellular growth properties that are induced by EBV infection.

T13

1380-1511

Sentence

denotes

This study reveals that LMP1 induces lymphomas in B-1a lymphocytes, a cell type that is susceptible to transformation in aged mice.

T14

1512-1598

Sentence

denotes

The lymphomas require Akt, NFκB, and Stat3 signaling for enhanced growth and survival.

T15

1599-1734

Sentence

denotes

The activation of the Stat3, Akt, and NFκB signaling pathways likely underlies the ability of LMP1 to promote malignant transformation.

T16

1737-1749

Sentence

denotes

Introduction

T780

1750-1923

Sentence

denotes

Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus that infects humans predominantly at an early age with greater than 90% of the adult population infected with EBV [1].

T17

1750-1923

Sentence

denotes

Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus that infects humans predominantly at an early age with greater than 90% of the adult population infected with EBV [1].

T781

1924-2210

Sentence

denotes

EBV is linked to the development of both B lymphocyte and epithelial cell malignancies, including Burkitt lymphoma, Hodgkin disease (HD), and nasopharyngeal carcinoma (NPC), and cancers linked to immunosuppression, including post-transplant lymphoma and AIDS-associated lymphomas [2,3].

T18

1924-2210

Sentence

denotes

EBV is linked to the development of both B lymphocyte and epithelial cell malignancies, including Burkitt lymphoma, Hodgkin disease (HD), and nasopharyngeal carcinoma (NPC), and cancers linked to immunosuppression, including post-transplant lymphoma and AIDS-associated lymphomas [2,3].

T782

2211-2395

Sentence

denotes

In vitro infection of B lymphocytes with EBV induces permanent growth transformation, and this ability to affect cell growth regulation likely contributes to the development of cancer.

T19

2211-2395

Sentence

denotes

In vitro infection of B lymphocytes with EBV induces permanent growth transformation, and this ability to affect cell growth regulation likely contributes to the development of cancer.

T783

2396-2640

Sentence

denotes

Many of the viral proteins expressed in transformed cells, including the EBV nuclear antigens and latent membrane proteins, have profound effects on cell growth regulation and are required for EBV latent infection and B cell transformation [1].

T20

2396-2640

Sentence

denotes

Many of the viral proteins expressed in transformed cells, including the EBV nuclear antigens and latent membrane proteins, have profound effects on cell growth regulation and are required for EBV latent infection and B cell transformation [1].

T784

2641-2860

Sentence

denotes

Latent membrane protein 1 (LMP1) is considered the major oncoprotein of EBV, as it transforms rodent fibroblasts to tumorigenicity in nude mice and is expressed in HD, NPC, and immunosuppression-associated tumors [4–8].

T21

2641-2860

Sentence

denotes

Latent membrane protein 1 (LMP1) is considered the major oncoprotein of EBV, as it transforms rodent fibroblasts to tumorigenicity in nude mice and is expressed in HD, NPC, and immunosuppression-associated tumors [4–8].

T785

2861-2990

Sentence

denotes

In B lymphocytes, LMP1 mimics CD40 signaling, and both LMP1 and CD40 are essential for EBV-mediated B cell transformation [9–11].

T22

2861-2990

Sentence

denotes

In B lymphocytes, LMP1 mimics CD40 signaling, and both LMP1 and CD40 are essential for EBV-mediated B cell transformation [9–11].

T786

2991-3187

Sentence

denotes

While CD40 interacts with CD40 ligand expressed on activated T cells to induce B cell activation and differentiation, LMP1 acts as a constitutive signal through ligand-independent oligomerization.

T23

2991-3187

Sentence

denotes

While CD40 interacts with CD40 ligand expressed on activated T cells to induce B cell activation and differentiation, LMP1 acts as a constitutive signal through ligand-independent oligomerization.

T787

3188-3385

Sentence

denotes

LMP1 and CD40 interact with the same tumor necrosis factor receptor–associated factors (TRAFs) leading to activation of NFκB, c-Jun N terminal kinase (JNK), and p38 MAPK signaling pathways [12–16].

T24

3188-3385

Sentence

denotes

LMP1 and CD40 interact with the same tumor necrosis factor receptor–associated factors (TRAFs) leading to activation of NFκB, c-Jun N terminal kinase (JNK), and p38 MAPK signaling pathways [12–16].

T788

3386-3512

Sentence

denotes

Activation of NFκB is required for EBV-induced B cell transformation and its inhibition rapidly results in cell death [17,18].

T25

3386-3512

Sentence

denotes

Activation of NFκB is required for EBV-induced B cell transformation and its inhibition rapidly results in cell death [17,18].

T789

3513-3712

Sentence

denotes

Recent studies indicate that LMP1 also activates phosphatidylinositol 3 kinase (PI3K)/Akt signaling and that this activation is required for LMP1-mediated transformation of rodent fibroblasts [5,19].

T26

3513-3712

Sentence

denotes

Recent studies indicate that LMP1 also activates phosphatidylinositol 3 kinase (PI3K)/Akt signaling and that this activation is required for LMP1-mediated transformation of rodent fibroblasts [5,19].

T790

3713-3808

Sentence

denotes

In vitro, primary B cells can be maintained by CD40 ligation in combination with IL4 treatment.

T27

3713-3808

Sentence

denotes

In vitro, primary B cells can be maintained by CD40 ligation in combination with IL4 treatment.

T791

3809-3996

Sentence

denotes

In vivo, CD40 signaling is necessary for germinal center (GC) formation such that mice deficient for CD40 or CD40L are unable to form GCs in response to T cell–dependent antigens [20,21].

T28

3809-3996

Sentence

denotes

In vivo, CD40 signaling is necessary for germinal center (GC) formation such that mice deficient for CD40 or CD40L are unable to form GCs in response to T cell–dependent antigens [20,21].

T792

3997-4272

Sentence

denotes

Both the membrane proximal and distal cytoplasmic regions of CD40 that bind TRAF6 and TRAFs2/3/5, respectively, are necessary for GC formation, but either region is sufficient to induce extrafollicular B cell differentiation and restore low affinity antibody production [22].

T29

3997-4272

Sentence

denotes

Both the membrane proximal and distal cytoplasmic regions of CD40 that bind TRAF6 and TRAFs2/3/5, respectively, are necessary for GC formation, but either region is sufficient to induce extrafollicular B cell differentiation and restore low affinity antibody production [22].

T793

4273-4527

Sentence

denotes

Functionally, LMP1 can rescue CD40-deficient mice and restore immunoglobulin (Ig) class switching, most likely because LMP1 recruits similar TRAF molecules, TRAFs 1/2/3/5 and TRAF6, through the C-terminal activation regions 1 and 2 domains, respectively.

T30

4273-4527

Sentence

denotes

Functionally, LMP1 can rescue CD40-deficient mice and restore immunoglobulin (Ig) class switching, most likely because LMP1 recruits similar TRAF molecules, TRAFs 1/2/3/5 and TRAF6, through the C-terminal activation regions 1 and 2 domains, respectively.

T794

4528-4605

Sentence

denotes

However, LMP1 is unable to restore affinity maturation and GC formation [23].

T31

4528-4605

Sentence

denotes

However, LMP1 is unable to restore affinity maturation and GC formation [23].

T795

4606-4816

Sentence

denotes

Several EBV transforming proteins have been studied in transgenic mouse models, however, only LMP1 induces tumor development when expressed under the control of the Ig heavy chain promoter and enhancer [24–26].

T32

4606-4816

Sentence

denotes

Several EBV transforming proteins have been studied in transgenic mouse models, however, only LMP1 induces tumor development when expressed under the control of the Ig heavy chain promoter and enhancer [24–26].

T796

4817-5066

Sentence

denotes

The LMP1 transgenic mice (IgLMP1) express LMP1 in B lymphocytes, and in mice older than 12 mo, lymphoma develops with increased incidence (40%–50%) compared to wild-type control mice (11%), suggesting that LMP1 contributes to tumor development [26].

T33

4817-5066

Sentence

denotes

The LMP1 transgenic mice (IgLMP1) express LMP1 in B lymphocytes, and in mice older than 12 mo, lymphoma develops with increased incidence (40%–50%) compared to wild-type control mice (11%), suggesting that LMP1 contributes to tumor development [26].

T797

5067-5216

Sentence

denotes

The LMP1 lymphomas have rearranged Ig genes and have activated Akt, JNK, p38, and NFκB, with specific activation of the NFκB family member cRel [27].

T34

5067-5216

Sentence

denotes

The LMP1 lymphomas have rearranged Ig genes and have activated Akt, JNK, p38, and NFκB, with specific activation of the NFκB family member cRel [27].

T798

5217-5358

Sentence

denotes

In this study, the LMP1 transgenic lymphocytes and lymphomas were further characterized and their growth properties in vitro were determined.

T35

5217-5358

Sentence

denotes

In this study, the LMP1 transgenic lymphocytes and lymphomas were further characterized and their growth properties in vitro were determined.

T799

5359-5533

Sentence

denotes

To obtain pure populations of malignant lymphocytes and to enable more detailed biochemical analyses, examples of primary lymphomas were inoculated and passaged in SCID mice.

T36

5359-5533

Sentence

denotes

To obtain pure populations of malignant lymphocytes and to enable more detailed biochemical analyses, examples of primary lymphomas were inoculated and passaged in SCID mice.

T800

5534-5683

Sentence

denotes

Interestingly, lymphoma development was restricted to B-1a lymphocytes, a self-replenishing population of cells that are prone to malignancy [28,29].

T37

5534-5683

Sentence

denotes

Interestingly, lymphoma development was restricted to B-1a lymphocytes, a self-replenishing population of cells that are prone to malignancy [28,29].

T801

5684-5796

Sentence

denotes

LMP1 transgenic lymphocytes had increased viability in vitro and viability was increased by the addition of IL4.

T38

5684-5796

Sentence

denotes

LMP1 transgenic lymphocytes had increased viability in vitro and viability was increased by the addition of IL4.

T802

5797-5996

Sentence

denotes

In contrast, both LMP1-positive and -negative lymphoma cells were independent of IL4 co-stimulation for survival and proliferation in vitro with a complete absence of activated Stat6, the IL4 target.

T39

5797-5996

Sentence

denotes

In contrast, both LMP1-positive and -negative lymphoma cells were independent of IL4 co-stimulation for survival and proliferation in vitro with a complete absence of activated Stat6, the IL4 target.

T803

5997-6117

Sentence

denotes

The lymphomas were also distinguished by constitutive activation of Stat3 and deregulation of the Rb cell cycle pathway.

T40

5997-6117

Sentence

denotes

The lymphomas were also distinguished by constitutive activation of Stat3 and deregulation of the Rb cell cycle pathway.

T804

6118-6334

Sentence

denotes

Inhibition of the PI3K/Akt, NFκB, and Stat3 signaling pathways blocked the enhanced growth of both LMP1 transgenic and malignant lymphocytes, suggesting that these pathways are required for their growth and survival.

T41

6118-6334

Sentence

denotes

Inhibition of the PI3K/Akt, NFκB, and Stat3 signaling pathways blocked the enhanced growth of both LMP1 transgenic and malignant lymphocytes, suggesting that these pathways are required for their growth and survival.

T805

6335-6472

Sentence

denotes

These appear to be the same targets that are deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition.

T42

6335-6472

Sentence

denotes

These appear to be the same targets that are deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition.

T806

6473-6673

Sentence

denotes

This study reveals that LMP1 promotes malignancy in cells with the inherent ability to proliferate and that the Akt, NFκB, and Stat3 signaling pathways are required for its growth stimulatory effects.

T43

6473-6673

Sentence

denotes

This study reveals that LMP1 promotes malignancy in cells with the inherent ability to proliferate and that the Akt, NFκB, and Stat3 signaling pathways are required for its growth stimulatory effects.

T44

6675-6682

Sentence

denotes

Results

T3205

6684-6758

Sentence

denotes

High Levels of LMP1 Expression Correlates with the Development of Lymphoma

T45

6684-6758

Sentence

denotes

High Levels of LMP1 Expression Correlates with the Development of Lymphoma

T3206

6759-6876

Sentence

denotes

LMP1 expression in IgLMP1 mice was directed to B cells under the control of the Ig heavy chain promoter and enhancer.

T46

6759-6876

Sentence

denotes

LMP1 expression in IgLMP1 mice was directed to B cells under the control of the Ig heavy chain promoter and enhancer.

T3207

6877-7044

Sentence

denotes

It has previously been shown that in these transgenic mice, LMP1 expression was restricted to B220+ B cells with lymphoma detected in greatly enlarged spleens [23,26].

T47

6877-7044

Sentence

denotes

It has previously been shown that in these transgenic mice, LMP1 expression was restricted to B220+ B cells with lymphoma detected in greatly enlarged spleens [23,26].

T3208

7045-7285

Sentence

denotes

To investigate whether LMP1 expression contributes to lymphoma development, B cells were purified from splenocytes by positive selection using anti-CD19 MACS magnetic beads, and equivalent amounts of B cells were analyzed by immunoblotting.

T48

7045-7285

Sentence

denotes

To investigate whether LMP1 expression contributes to lymphoma development, B cells were purified from splenocytes by positive selection using anti-CD19 MACS magnetic beads, and equivalent amounts of B cells were analyzed by immunoblotting.

T3209

7286-7487

Sentence

denotes

LMP1 was detectable in LMP1 transgenic B cells, but upon development of lymphoma, LMP1 expression was stronger in 5/7 lymphomas analyzed with concomitant appearance of degradation products (Figure 1A).

T49

7286-7487

Sentence

denotes

LMP1 was detectable in LMP1 transgenic B cells, but upon development of lymphoma, LMP1 expression was stronger in 5/7 lymphomas analyzed with concomitant appearance of degradation products (Figure 1A).

T3210

7488-7680

Sentence

denotes

To determine whether the higher level of LMP1 detected was due to an expansion of malignant lymphocytes, expression of LMP1 in the spleen was further evaluated by immunohistochemical staining.

T50

7488-7680

Sentence

denotes

To determine whether the higher level of LMP1 detected was due to an expansion of malignant lymphocytes, expression of LMP1 in the spleen was further evaluated by immunohistochemical staining.

T3211

7681-7866

Sentence

denotes

Immunohistochemistry analysis of spleen sections detected LMP1 in the plasma membrane of cells in both the follicular white pulp and circulating lymphocytes in the red pulp (Figure 1B).

T51

7681-7866

Sentence

denotes

Immunohistochemistry analysis of spleen sections detected LMP1 in the plasma membrane of cells in both the follicular white pulp and circulating lymphocytes in the red pulp (Figure 1B).

T3212

7867-7995

Sentence

denotes

LMP1 expression was heterogeneous with strong LMP1 staining interspersed amongst a background of cells staining weakly for LMP1.

T52

7867-7995

Sentence

denotes

LMP1 expression was heterogeneous with strong LMP1 staining interspersed amongst a background of cells staining weakly for LMP1.

T3213

7996-8115

Sentence

denotes

Upon development to lymphoma, LMP1 expression was more abundantly detected with multiple foci of intense LMP1 staining.

T53

7996-8115

Sentence

denotes

Upon development to lymphoma, LMP1 expression was more abundantly detected with multiple foci of intense LMP1 staining.

T3214

8116-8312

Sentence

denotes

This demonstrates that the increased LMP1 detected by immunoblotting upon malignant progression reflects an increase in LMP1 expression and an accumulation of cells expressing high levels of LMP1.

T54

8116-8312

Sentence

denotes

This demonstrates that the increased LMP1 detected by immunoblotting upon malignant progression reflects an increase in LMP1 expression and an accumulation of cells expressing high levels of LMP1.

T3215

8313-8467

Sentence

denotes

This correlation between high LMP1 expression and the development of lymphoma suggests that progression to lymphoma results from increased levels of LMP1.

T55

8313-8467

Sentence

denotes

This correlation between high LMP1 expression and the development of lymphoma suggests that progression to lymphoma results from increased levels of LMP1.

T56

8468-8542

Sentence

denotes

Figure 1 High LMP1 Expression Correlates with the Development of Lymphoma

T21805

8478-8542

Sentence

denotes

High LMP1 Expression Correlates with the Development of Lymphoma

T21806

8543-8718

Sentence

denotes

LMP1 expression is shown by (A) immunoblotting of purified B cells (CD19+) and (B) immunohistochemistry staining of spleen tissue from wild-type (WT) and LMP1 transgenic mice.

T57

8543-8718

Sentence

denotes

LMP1 expression is shown by (A) immunoblotting of purified B cells (CD19+) and (B) immunohistochemistry staining of spleen tissue from wild-type (WT) and LMP1 transgenic mice.

T6392

8678-17061

Sentence

denotes

wild-type (WT) and LMP1 transgenic mice. (A) Lymphomas are identified with a number (1–7). Arrows indicate the LMP1-specific band and its degradation products as well as a non-specific band. Actin was used as a loading control. (B) White and red pulps are shown, but this architecture is lost upon development of lymphoma. Scale bar, 20 μm. LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion To determine if LMP1 signaling affects B cell differentiation and to immunophenotype the lymphomas that arise from LMP1 expression, surface Ig expression of heavy chains (IgM, IgG, IgD) and light chains (κ, λ) were analyzed by flow cytometry. Similar numbers of naïve (IgM+IgD+IgG−) splenic B cells with a strong bias towards κ light chain were detected from wild-type or LMP1 transgenic mice, indicating that LMP1 signaling does not affect B cell maturation (unpublished data). Flow cytometry analysis of the SCID-passaged wild-type and LMP1 transgenic lymphomas revealed an IgMhighIgDlow phenotype (Figure 2A), indicative of marginal zone, B-1, or memory B cells. B-1 cells are further separated into CD5+ (B-1a) and CD5− (B-1b) subsets. To differentiate between these cell types, lymphoma cells were further analyzed for the B-1a marker CD5. All (5/5) of the tested LMP1 transgenic lymphomas displayed an IgMhighIgDlowCD5+ phenotype (Figure 2A), an expression pattern that distinguishes B-1a cells. Interestingly, a spontaneous wild-type lymphoma also developed in B-1a cells (Figure 2A). These cells are an interesting population that is self replenishing with an increased likelihood to become malignant in aged mice [30]. Analysis of LMP1 transgenic mice before the development of lymphoma showed similar numbers of splenic B-1a (CD19+CD5+) and B-1b or B-2 populations (CD19+CD5−), indicating that LMP1 does not affect B cell differentiation (Figure 2B). Figure 2 LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion (A) Flow cytometry analysis of splenocytes from a WT or LMP1 transgenic lymphoma for the pan–B cell (CD19), B-1a cell (CD5), and Ig heavy chain (IgM and IgD) and light chain (κ and λ) markers. Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 4. This analysis was repeated on four other LMP1 transgenic lymphomas (1, 2, 3, and 6) showing a similar B-1a phenotype, of which lymphomas 2 and 4 were also doubly positive for κ and λ light chains. (B) Flow cytometry analysis of WT or LMP1 transgenic splenocytes for B-1a (CD19+CD5+) and B-1b or B2 subsets (CD19+CD5−). Percentages of B-1a and B-1b or B2 subsets are shown in each quadrant. This analysis was repeated on three other WT and two other LMP1 transgenic mice with similar results. (C) Immunoblot analysis for κ and λ light chains of B cells (CD19+) purified from WT and LMP1 transgenic mice. Actin was used as a loading control. Due to allelic exclusion, mature B cells that have been exposed to antigen will typically express only one heavy chain isotype (IgG, IgE, or IgA) and either a κ or λ light chain. Interestingly, 2/5 LMP1 transgenic lymphomas analyzed (lymphomas 2 and 4) were doubly positive for low levels of both κ and λ light chains (Figure 2A). Previous characterization of the LMP1 lymphomas had revealed that the lymphomas were clonal as determined by Ig heavy chain rearrangement [26], and analysis of κ chain rearrangement (Figure S1) of the samples analyzed in this study confirmed clonality. To further assess light chain expression, the passaged samples were tested by immunoblotting for κ and λ light chains (Figure 2C). Interestingly, very low levels of expression of both light chains were detected by flow cytometry. The low levels of expression may reflect a limitation of the total number of light chains that can be expressed on the surface of a B cell. In agreement with the flow cytometry analysis, LMP1 transgenic lymphomas 2 and 4 were also positive for κ and λ light chains by immunoblot analysis (Figure 2C), confirming that these lymphomas express both light chains. A previous study of mice that developed leukemia due to an expansion of self-reactive B-1a cells determined that the B-1a leukemias were also doubly positive for κ and λ light chains [31]. These findings indicate that expression of LMP1 in B-1a cells promotes the development of malignancy and can result in the aberrant escape from allelic exclusion. LMP1 Promotes B Cell Survival and Proliferation In Vitro Primary B cell cultures can be maintained through CD40 ligation and supplementation with IL4 [32]. To investigate whether LMP1 affects primary B cell survival and proliferation, splenocytes were cultured in the presence or absence of IL4 and analyzed by MTS as a metabolic marker, by ethidium monoazide (EMA) exclusion for viability, and by 5-bromo-2′-deoxy-uridine (BrdU) incorporation for proliferation. In the MTS assay, as expected, splenocytes from wild-type mice did not survive even with the addition of IL4 due to a lack of CD40 ligation (Figure 3A). In contrast, LMP1 splenocytes had increased metabolism even in the absence of IL4, which was further enhanced upon addition of IL4. Wild-type and LMP1 transgenic lymphoma cells had high levels of MTS activity even in the absence of IL4 (Figure 3A). The LMP1 transgenic lymphoma cells had approximately 4-fold higher MTS activity than the normal transgenic lymphocytes and were at least 2-fold higher than the control lymphoma. As previously published, lymphoma usually develops in mice over 12 mo of age and all mice are sacrificed by 18–20 mo. The ages of the transgenic mice with or without lymphoma ranged between 6 and 20 mo old. There was no correlation between age and MTS activity. Figure 3 LMP1 Promotes B Cell Survival and Proliferation In Vitro (A) MTS assay of splenocytes from WT and LMP1 transgenic mice. Splenocytes were cultured in the presence (grey bars) or absence (black bars) of IL4 for 3 d. The results are the mean ± SEM of triplicate samples averaged from multiple mice where “n” the number of mice analyzed is as follows: n = 2 for WT lymphocytes and WT lymphomas, n = 11 for LMP1 transgenic lymphocytes, and n = 13 for LMP1 transgenic lymphomas. (B) EMA exclusion of CD19+ gated splenocytes from WT and LMP1 transgenic mice showing percentage of viable B cells cultured with (white bars) or without (black bars) IL4 for 2 d. (C) Flow cytometry analysis for incorporated BrdU in WT and LMP1 transgenic lymphoma cells cultured with or without IL4 for 2 d. Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 2. Percentages of cells in each quadrant are shown. EMA exclusion of CD19+ gated B cells prepared from two wild-type and two LMP1 transgenic mice indicated a 2-fold increase in viability in the LMP1 transgenic lymphocytes compared to wild-type lymphocytes. Two examples of LMP1 transgenic lymphoma cells had greatly increased viability that was not increased by IL4 treatment, indicating that the lymphoma cells are independent of IL4 co-stimulation (Figure 3B). Enhancement in MTS activity was observed in LMP1 transgenic lymphoma cells by the addition of IL4; however, EMA exclusion did not reveal a similar increase. This could reflect a difference for IL4 requirement in the metabolic activity versus the viability of LMP1 transgenic lymphoma cells. Although expression of LMP1 could enhance survival of non-malignant primary lymphocytes, BrdU incorporation revealed that LMP1 expression alone was not sufficient to induce proliferation in culture (unpublished data). Only lymphoma cells had detectable levels of BrdU incorporation detected by flow cytometry (Figure 3C). Interestingly, LMP1 lymphoma cells had significantly higher levels of proliferation in comparison to the spontaneous lymphoma that developed in an LMP1-negative littermate (25% versus 4%). This higher level of proliferation was observed in lymphomas that express both high (Table 1, LMP1-L2 and LMP1-L3) and low (Table 1, LMP1-L5) levels of LMP1, suggesting that even small amounts of LMP1 is sufficient to induce dramatic effects in proliferation. The level of proliferation was not enhanced upon IL4 addition, confirming the IL4 independence observed in the viability studies (Figure 3C; Table1). Table 1 Summary of Analysis Performed on Wild-Type and LMP1 Transgenic Lymphomas Wild-Type and LMP1 Transgenic Lymphoma Cells Do Not Require IL4 and Stat6 Signaling

T21807

8719-8768

Sentence

denotes

(A) Lymphomas are identified with a number (1–7).

T58

8719-8768

Sentence

denotes

(A) Lymphomas are identified with a number (1–7).

T21808

8769-8868

Sentence

denotes

Arrows indicate the LMP1-specific band and its degradation products as well as a non-specific band.

T59

8769-8868

Sentence

denotes

Arrows indicate the LMP1-specific band and its degradation products as well as a non-specific band.

T21809

8869-8905

Sentence

denotes

Actin was used as a loading control.

T60

8869-8905

Sentence

denotes

Actin was used as a loading control.

T21810

8906-9000

Sentence

denotes

(B) White and red pulps are shown, but this architecture is lost upon development of lymphoma.

T61

8906-9000

Sentence

denotes

(B) White and red pulps are shown, but this architecture is lost upon development of lymphoma.

T21811

9001-9018

Sentence

denotes

Scale bar, 20 μm.

T62

9001-9018

Sentence

denotes

Scale bar, 20 μm.

T4090

9020-9082

Sentence

denotes

LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion

T63

9020-9082

Sentence

denotes

LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion

T4091

9083-9325

Sentence

denotes

To determine if LMP1 signaling affects B cell differentiation and to immunophenotype the lymphomas that arise from LMP1 expression, surface Ig expression of heavy chains (IgM, IgG, IgD) and light chains (κ, λ) were analyzed by flow cytometry.

T64

9083-9325

Sentence

denotes

To determine if LMP1 signaling affects B cell differentiation and to immunophenotype the lymphomas that arise from LMP1 expression, surface Ig expression of heavy chains (IgM, IgG, IgD) and light chains (κ, λ) were analyzed by flow cytometry.

T4092

9326-9561

Sentence

denotes

Similar numbers of naïve (IgM+IgD+IgG−) splenic B cells with a strong bias towards κ light chain were detected from wild-type or LMP1 transgenic mice, indicating that LMP1 signaling does not affect B cell maturation (unpublished data).

T65

9326-9561

Sentence

denotes

Similar numbers of naïve (IgM+IgD+IgG−) splenic B cells with a strong bias towards κ light chain were detected from wild-type or LMP1 transgenic mice, indicating that LMP1 signaling does not affect B cell maturation (unpublished data).

T4093

9562-9748

Sentence

denotes

Flow cytometry analysis of the SCID-passaged wild-type and LMP1 transgenic lymphomas revealed an IgMhighIgDlow phenotype (Figure 2A), indicative of marginal zone, B-1, or memory B cells.

T66

9562-9748

Sentence

denotes

Flow cytometry analysis of the SCID-passaged wild-type and LMP1 transgenic lymphomas revealed an IgMhighIgDlow phenotype (Figure 2A), indicative of marginal zone, B-1, or memory B cells.

T4094

9749-9822

Sentence

denotes

B-1 cells are further separated into CD5+ (B-1a) and CD5− (B-1b) subsets.

T67

9749-9822

Sentence

denotes

B-1 cells are further separated into CD5+ (B-1a) and CD5− (B-1b) subsets.

T4095

9823-9927

Sentence

denotes

To differentiate between these cell types, lymphoma cells were further analyzed for the B-1a marker CD5.

T68

9823-9927

Sentence

denotes

To differentiate between these cell types, lymphoma cells were further analyzed for the B-1a marker CD5.

T4096

9928-10084

Sentence

denotes

All (5/5) of the tested LMP1 transgenic lymphomas displayed an IgMhighIgDlowCD5+ phenotype (Figure 2A), an expression pattern that distinguishes B-1a cells.

T69

9928-10084

Sentence

denotes

All (5/5) of the tested LMP1 transgenic lymphomas displayed an IgMhighIgDlowCD5+ phenotype (Figure 2A), an expression pattern that distinguishes B-1a cells.

T4097

10085-10174

Sentence

denotes

Interestingly, a spontaneous wild-type lymphoma also developed in B-1a cells (Figure 2A).

T70

10085-10174

Sentence

denotes

Interestingly, a spontaneous wild-type lymphoma also developed in B-1a cells (Figure 2A).

T4098

10175-10310

Sentence

denotes

These cells are an interesting population that is self replenishing with an increased likelihood to become malignant in aged mice [30].

T71

10175-10310

Sentence

denotes

These cells are an interesting population that is self replenishing with an increased likelihood to become malignant in aged mice [30].

T4099

10311-10543

Sentence

denotes

Analysis of LMP1 transgenic mice before the development of lymphoma showed similar numbers of splenic B-1a (CD19+CD5+) and B-1b or B-2 populations (CD19+CD5−), indicating that LMP1 does not affect B cell differentiation (Figure 2B).

T72

10311-10543

Sentence

denotes

Analysis of LMP1 transgenic mice before the development of lymphoma showed similar numbers of splenic B-1a (CD19+CD5+) and B-1b or B-2 populations (CD19+CD5−), indicating that LMP1 does not affect B cell differentiation (Figure 2B).

T73

10544-10616

Sentence

denotes

Figure 2 LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion

T22113

10554-10616

Sentence

denotes

LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion

T22114

10617-10809

Sentence

denotes

(A) Flow cytometry analysis of splenocytes from a WT or LMP1 transgenic lymphoma for the pan–B cell (CD19), B-1a cell (CD5), and Ig heavy chain (IgM and IgD) and light chain (κ and λ) markers.

T74

10617-10809

Sentence

denotes

(A) Flow cytometry analysis of splenocytes from a WT or LMP1 transgenic lymphoma for the pan–B cell (CD19), B-1a cell (CD5), and Ig heavy chain (IgM and IgD) and light chain (κ and λ) markers.

T22115

10810-10882

Sentence

denotes

Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 4.

T75

10810-10882

Sentence

denotes

Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 4.

T22116

10883-11079

Sentence

denotes

This analysis was repeated on four other LMP1 transgenic lymphomas (1, 2, 3, and 6) showing a similar B-1a phenotype, of which lymphomas 2 and 4 were also doubly positive for κ and λ light chains.

T76

10883-11079

Sentence

denotes

This analysis was repeated on four other LMP1 transgenic lymphomas (1, 2, 3, and 6) showing a similar B-1a phenotype, of which lymphomas 2 and 4 were also doubly positive for κ and λ light chains.

T22117

11080-11201

Sentence

denotes

(B) Flow cytometry analysis of WT or LMP1 transgenic splenocytes for B-1a (CD19+CD5+) and B-1b or B2 subsets (CD19+CD5−).

T77

11080-11201

Sentence

denotes

(B) Flow cytometry analysis of WT or LMP1 transgenic splenocytes for B-1a (CD19+CD5+) and B-1b or B2 subsets (CD19+CD5−).

T22118

11202-11272

Sentence

denotes

Percentages of B-1a and B-1b or B2 subsets are shown in each quadrant.

T78

11202-11272

Sentence

denotes

Percentages of B-1a and B-1b or B2 subsets are shown in each quadrant.

T22119

11273-11374

Sentence

denotes

This analysis was repeated on three other WT and two other LMP1 transgenic mice with similar results.

T79

11273-11374

Sentence

denotes

This analysis was repeated on three other WT and two other LMP1 transgenic mice with similar results.

T22120

11375-11485

Sentence

denotes

(C) Immunoblot analysis for κ and λ light chains of B cells (CD19+) purified from WT and LMP1 transgenic mice.

T80

11375-11485

Sentence

denotes

(C) Immunoblot analysis for κ and λ light chains of B cells (CD19+) purified from WT and LMP1 transgenic mice.

T22121

11486-11522

Sentence

denotes

Actin was used as a loading control.

T81

11486-11522

Sentence

denotes

Actin was used as a loading control.

T4100

11525-11703

Sentence

denotes

Due to allelic exclusion, mature B cells that have been exposed to antigen will typically express only one heavy chain isotype (IgG, IgE, or IgA) and either a κ or λ light chain.

T82

11525-11703

Sentence

denotes

Due to allelic exclusion, mature B cells that have been exposed to antigen will typically express only one heavy chain isotype (IgG, IgE, or IgA) and either a κ or λ light chain.

T4101

11704-11855

Sentence

denotes

Interestingly, 2/5 LMP1 transgenic lymphomas analyzed (lymphomas 2 and 4) were doubly positive for low levels of both κ and λ light chains (Figure 2A).

T83

11704-11855

Sentence

denotes

Interestingly, 2/5 LMP1 transgenic lymphomas analyzed (lymphomas 2 and 4) were doubly positive for low levels of both κ and λ light chains (Figure 2A).

T4102

11856-12108

Sentence

denotes

Previous characterization of the LMP1 lymphomas had revealed that the lymphomas were clonal as determined by Ig heavy chain rearrangement [26], and analysis of κ chain rearrangement (Figure S1) of the samples analyzed in this study confirmed clonality.

T84

11856-12108

Sentence

denotes

Previous characterization of the LMP1 lymphomas had revealed that the lymphomas were clonal as determined by Ig heavy chain rearrangement [26], and analysis of κ chain rearrangement (Figure S1) of the samples analyzed in this study confirmed clonality.

T4103

12109-12239

Sentence

denotes

To further assess light chain expression, the passaged samples were tested by immunoblotting for κ and λ light chains (Figure 2C).

T85

12109-12239

Sentence

denotes

To further assess light chain expression, the passaged samples were tested by immunoblotting for κ and λ light chains (Figure 2C).

T4104

12240-12338

Sentence

denotes

Interestingly, very low levels of expression of both light chains were detected by flow cytometry.

T86

12240-12338

Sentence

denotes

Interestingly, very low levels of expression of both light chains were detected by flow cytometry.

T4105

12339-12478

Sentence

denotes

The low levels of expression may reflect a limitation of the total number of light chains that can be expressed on the surface of a B cell.

T87

12339-12478

Sentence

denotes

The low levels of expression may reflect a limitation of the total number of light chains that can be expressed on the surface of a B cell.

T4106

12479-12698

Sentence

denotes

In agreement with the flow cytometry analysis, LMP1 transgenic lymphomas 2 and 4 were also positive for κ and λ light chains by immunoblot analysis (Figure 2C), confirming that these lymphomas express both light chains.

T88

12479-12698

Sentence

denotes

In agreement with the flow cytometry analysis, LMP1 transgenic lymphomas 2 and 4 were also positive for κ and λ light chains by immunoblot analysis (Figure 2C), confirming that these lymphomas express both light chains.

T4107

12699-12887

Sentence

denotes

A previous study of mice that developed leukemia due to an expansion of self-reactive B-1a cells determined that the B-1a leukemias were also doubly positive for κ and λ light chains [31].

T89

12699-12887

Sentence

denotes

A previous study of mice that developed leukemia due to an expansion of self-reactive B-1a cells determined that the B-1a leukemias were also doubly positive for κ and λ light chains [31].

T4108

12888-13050

Sentence

denotes

These findings indicate that expression of LMP1 in B-1a cells promotes the development of malignancy and can result in the aberrant escape from allelic exclusion.

T90

12888-13050

Sentence

denotes

These findings indicate that expression of LMP1 in B-1a cells promotes the development of malignancy and can result in the aberrant escape from allelic exclusion.

T5525

13052-13108

Sentence

denotes

LMP1 Promotes B Cell Survival and Proliferation In Vitro

T91

13052-13108

Sentence

denotes

LMP1 Promotes B Cell Survival and Proliferation In Vitro

T5526

13109-13207

Sentence

denotes

Primary B cell cultures can be maintained through CD40 ligation and supplementation with IL4 [32].

T92

13109-13207

Sentence

denotes

Primary B cell cultures can be maintained through CD40 ligation and supplementation with IL4 [32].

T5527

13208-13514

Sentence

denotes

To investigate whether LMP1 affects primary B cell survival and proliferation, splenocytes were cultured in the presence or absence of IL4 and analyzed by MTS as a metabolic marker, by ethidium monoazide (EMA) exclusion for viability, and by 5-bromo-2′-deoxy-uridine (BrdU) incorporation for proliferation.

T93

13208-13514

Sentence

denotes

To investigate whether LMP1 affects primary B cell survival and proliferation, splenocytes were cultured in the presence or absence of IL4 and analyzed by MTS as a metabolic marker, by ethidium monoazide (EMA) exclusion for viability, and by 5-bromo-2′-deoxy-uridine (BrdU) incorporation for proliferation.

T5528

13515-13667

Sentence

denotes

In the MTS assay, as expected, splenocytes from wild-type mice did not survive even with the addition of IL4 due to a lack of CD40 ligation (Figure 3A).

T94

13515-13667

Sentence

denotes

In the MTS assay, as expected, splenocytes from wild-type mice did not survive even with the addition of IL4 due to a lack of CD40 ligation (Figure 3A).

T5529

13668-13799

Sentence

denotes

In contrast, LMP1 splenocytes had increased metabolism even in the absence of IL4, which was further enhanced upon addition of IL4.

T95

13668-13799

Sentence

denotes

In contrast, LMP1 splenocytes had increased metabolism even in the absence of IL4, which was further enhanced upon addition of IL4.

T5530

13800-13916

Sentence

denotes

Wild-type and LMP1 transgenic lymphoma cells had high levels of MTS activity even in the absence of IL4 (Figure 3A).

T96

13800-13916

Sentence

denotes

Wild-type and LMP1 transgenic lymphoma cells had high levels of MTS activity even in the absence of IL4 (Figure 3A).

T5531

13917-14094

Sentence

denotes

The LMP1 transgenic lymphoma cells had approximately 4-fold higher MTS activity than the normal transgenic lymphocytes and were at least 2-fold higher than the control lymphoma.

T97

13917-14094

Sentence

denotes

The LMP1 transgenic lymphoma cells had approximately 4-fold higher MTS activity than the normal transgenic lymphocytes and were at least 2-fold higher than the control lymphoma.

T5532

14095-14212

Sentence

denotes

As previously published, lymphoma usually develops in mice over 12 mo of age and all mice are sacrificed by 18–20 mo.

T98

14095-14212

Sentence

denotes

As previously published, lymphoma usually develops in mice over 12 mo of age and all mice are sacrificed by 18–20 mo.

T5533

14213-14301

Sentence

denotes

The ages of the transgenic mice with or without lymphoma ranged between 6 and 20 mo old.

T99

14213-14301

Sentence

denotes

The ages of the transgenic mice with or without lymphoma ranged between 6 and 20 mo old.

T5534

14302-14356

Sentence

denotes

There was no correlation between age and MTS activity.

T100

14302-14356

Sentence

denotes

There was no correlation between age and MTS activity.

T101

14357-14423

Sentence

denotes

Figure 3 LMP1 Promotes B Cell Survival and Proliferation In Vitro

T22656

14367-14423

Sentence

denotes

LMP1 Promotes B Cell Survival and Proliferation In Vitro

T22657

14424-14486

Sentence

denotes

(A) MTS assay of splenocytes from WT and LMP1 transgenic mice.

T102

14424-14486

Sentence

denotes

(A) MTS assay of splenocytes from WT and LMP1 transgenic mice.

T22658

14487-14580

Sentence

denotes

Splenocytes were cultured in the presence (grey bars) or absence (black bars) of IL4 for 3 d.

T103

14487-14580

Sentence

denotes

Splenocytes were cultured in the presence (grey bars) or absence (black bars) of IL4 for 3 d.

T22659

14581-14839

Sentence

denotes

The results are the mean ± SEM of triplicate samples averaged from multiple mice where “n” the number of mice analyzed is as follows: n = 2 for WT lymphocytes and WT lymphomas, n = 11 for LMP1 transgenic lymphocytes, and n = 13 for LMP1 transgenic lymphomas.

T104

14581-14839

Sentence

denotes

The results are the mean ± SEM of triplicate samples averaged from multiple mice where “n” the number of mice analyzed is as follows: n = 2 for WT lymphocytes and WT lymphomas, n = 11 for LMP1 transgenic lymphocytes, and n = 13 for LMP1 transgenic lymphomas.

T22660

14840-15018

Sentence

denotes

(B) EMA exclusion of CD19+ gated splenocytes from WT and LMP1 transgenic mice showing percentage of viable B cells cultured with (white bars) or without (black bars) IL4 for 2 d.

T105

14840-15018

Sentence

denotes

(B) EMA exclusion of CD19+ gated splenocytes from WT and LMP1 transgenic mice showing percentage of viable B cells cultured with (white bars) or without (black bars) IL4 for 2 d.

T22661

15019-15147

Sentence

denotes

(C) Flow cytometry analysis for incorporated BrdU in WT and LMP1 transgenic lymphoma cells cultured with or without IL4 for 2 d.

T106

15019-15147

Sentence

denotes

(C) Flow cytometry analysis for incorporated BrdU in WT and LMP1 transgenic lymphoma cells cultured with or without IL4 for 2 d.

T22662

15148-15220

Sentence

denotes

Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 2.

T107

15148-15220

Sentence

denotes

Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 2.

T22663

15221-15269

Sentence

denotes

Percentages of cells in each quadrant are shown.

T108

15221-15269

Sentence

denotes

Percentages of cells in each quadrant are shown.

T5535

15272-15476

Sentence

denotes

EMA exclusion of CD19+ gated B cells prepared from two wild-type and two LMP1 transgenic mice indicated a 2-fold increase in viability in the LMP1 transgenic lymphocytes compared to wild-type lymphocytes.

T109

15272-15476

Sentence

denotes

EMA exclusion of CD19+ gated B cells prepared from two wild-type and two LMP1 transgenic mice indicated a 2-fold increase in viability in the LMP1 transgenic lymphocytes compared to wild-type lymphocytes.

T5536

15477-15682

Sentence

denotes

Two examples of LMP1 transgenic lymphoma cells had greatly increased viability that was not increased by IL4 treatment, indicating that the lymphoma cells are independent of IL4 co-stimulation (Figure 3B).

T110

15477-15682

Sentence

denotes

Two examples of LMP1 transgenic lymphoma cells had greatly increased viability that was not increased by IL4 treatment, indicating that the lymphoma cells are independent of IL4 co-stimulation (Figure 3B).

T5537

15683-15839

Sentence

denotes

Enhancement in MTS activity was observed in LMP1 transgenic lymphoma cells by the addition of IL4; however, EMA exclusion did not reveal a similar increase.

T111

15683-15839

Sentence

denotes

Enhancement in MTS activity was observed in LMP1 transgenic lymphoma cells by the addition of IL4; however, EMA exclusion did not reveal a similar increase.

T5538

15840-15973

Sentence

denotes

This could reflect a difference for IL4 requirement in the metabolic activity versus the viability of LMP1 transgenic lymphoma cells.

T112

15840-15973

Sentence

denotes

This could reflect a difference for IL4 requirement in the metabolic activity versus the viability of LMP1 transgenic lymphoma cells.

T5539

15974-16191

Sentence

denotes

Although expression of LMP1 could enhance survival of non-malignant primary lymphocytes, BrdU incorporation revealed that LMP1 expression alone was not sufficient to induce proliferation in culture (unpublished data).

T113

15974-16191

Sentence

denotes

Although expression of LMP1 could enhance survival of non-malignant primary lymphocytes, BrdU incorporation revealed that LMP1 expression alone was not sufficient to induce proliferation in culture (unpublished data).

T5540

16192-16295

Sentence

denotes

Only lymphoma cells had detectable levels of BrdU incorporation detected by flow cytometry (Figure 3C).

T114

16192-16295

Sentence

denotes

Only lymphoma cells had detectable levels of BrdU incorporation detected by flow cytometry (Figure 3C).

T5541

16296-16484

Sentence

denotes

Interestingly, LMP1 lymphoma cells had significantly higher levels of proliferation in comparison to the spontaneous lymphoma that developed in an LMP1-negative littermate (25% versus 4%).

T115

16296-16484

Sentence

denotes

Interestingly, LMP1 lymphoma cells had significantly higher levels of proliferation in comparison to the spontaneous lymphoma that developed in an LMP1-negative littermate (25% versus 4%).

T5542

16485-16744

Sentence

denotes

This higher level of proliferation was observed in lymphomas that express both high (Table 1, LMP1-L2 and LMP1-L3) and low (Table 1, LMP1-L5) levels of LMP1, suggesting that even small amounts of LMP1 is sufficient to induce dramatic effects in proliferation.

T116

16485-16744

Sentence

denotes

This higher level of proliferation was observed in lymphomas that express both high (Table 1, LMP1-L2 and LMP1-L3) and low (Table 1, LMP1-L5) levels of LMP1, suggesting that even small amounts of LMP1 is sufficient to induce dramatic effects in proliferation.

T5543

16745-16894

Sentence

denotes

The level of proliferation was not enhanced upon IL4 addition, confirming the IL4 independence observed in the viability studies (Figure 3C; Table1).

T117

16745-16894

Sentence

denotes

The level of proliferation was not enhanced upon IL4 addition, confirming the IL4 independence observed in the viability studies (Figure 3C; Table1).

T118

16895-16976

Sentence

denotes

Table 1 Summary of Analysis Performed on Wild-Type and LMP1 Transgenic Lymphomas

T25509

16904-16976

Sentence

denotes

Summary of Analysis Performed on Wild-Type and LMP1 Transgenic Lymphomas

T119

16978-17061

Sentence

denotes

Wild-Type and LMP1 Transgenic Lymphoma Cells Do Not Require IL4 and Stat6 Signaling

T6393

17062-17206

Sentence

denotes

To investigate whether IL4 independence was due to endogenous IL4 expression, IL4 transcription was assessed by an Rnase protection assay (RPA).

T120

17062-17206

Sentence

denotes

To investigate whether IL4 independence was due to endogenous IL4 expression, IL4 transcription was assessed by an Rnase protection assay (RPA).

T6394

17207-17318

Sentence

denotes

IL4 transcription was detectable with control RNA and faintly in the mouse lymphoma cell line K46μ (Figure 4A).

T121

17207-17318

Sentence

denotes

IL4 transcription was detectable with control RNA and faintly in the mouse lymphoma cell line K46μ (Figure 4A).

T6395

17319-17566

Sentence

denotes

However, IL4 transcription was not detectable in CD19+ MACS-purified B cells from wild-type lymphocytes (unpublished data), LMP1 transgenic lymphocytes, or lymphoma cells, although the GAPDH and L32 controls were effectively protected (Figure 4A).

T122

17319-17566

Sentence

denotes

However, IL4 transcription was not detectable in CD19+ MACS-purified B cells from wild-type lymphocytes (unpublished data), LMP1 transgenic lymphocytes, or lymphoma cells, although the GAPDH and L32 controls were effectively protected (Figure 4A).

T6396

17567-17705

Sentence

denotes

Activated Stat6 (pStat6), a target of the IL4 receptor pathway, was detected in the wild-type and LMP1 transgenic lymphocytes (Figure 4B).

T123

17567-17705

Sentence

denotes

Activated Stat6 (pStat6), a target of the IL4 receptor pathway, was detected in the wild-type and LMP1 transgenic lymphocytes (Figure 4B).

T6397

17706-17804

Sentence

denotes

In contrast, pStat6 was barely detected in either the wild-type or LMP1 transgenic lymphoma cells.

T124

17706-17804

Sentence

denotes

In contrast, pStat6 was barely detected in either the wild-type or LMP1 transgenic lymphoma cells.

T6398

17805-17930

Sentence

denotes

However, the pathway was not disabled, as treatment of the lymphoma cells with IL4 induced Stat6 phosphorylation (Figure 4C).

T125

17805-17930

Sentence

denotes

However, the pathway was not disabled, as treatment of the lymphoma cells with IL4 induced Stat6 phosphorylation (Figure 4C).

T126

17931-18041

Sentence

denotes

Figure 4 Wild-Type and LMP1 Transgenic Lymphoma Cells Survive Independently of IL4/Stat6 Signaling in Culture

T23143

17941-18041

Sentence

denotes

Wild-Type and LMP1 Transgenic Lymphoma Cells Survive Independently of IL4/Stat6 Signaling in Culture

T23144

18042-18152

Sentence

denotes

(A) Rnase protection assay for IL4 mRNA from purified B cells (CD19+) from WT and LMP1 transgenic splenocytes.

T127

18042-18152

Sentence

denotes

(A) Rnase protection assay for IL4 mRNA from purified B cells (CD19+) from WT and LMP1 transgenic splenocytes.

T23145

18153-18221

Sentence

denotes

The L32 and GAPDH housekeeping genes were used as a loading control.

T128

18153-18221

Sentence

denotes

The L32 and GAPDH housekeeping genes were used as a loading control.

T23146

18222-18274

Sentence

denotes

Arrow indicates the position of the protected probe.

T129

18222-18274

Sentence

denotes

Arrow indicates the position of the protected probe.

T23147

18275-18585

Sentence

denotes

(B and C) Immunoblot analysis of WT and LMP1 transgenic mice for activated pStat6 in (B) purified B cells (CD19+) at the time of harvest or in (C) whole splenocytes cultured with or without IL4. (B) Actin was used as a loading control, and the white line indicates that intervening lanes have been spliced out.

T130

18275-18585

Sentence

denotes

(B and C) Immunoblot analysis of WT and LMP1 transgenic mice for activated pStat6 in (B) purified B cells (CD19+) at the time of harvest or in (C) whole splenocytes cultured with or without IL4. (B) Actin was used as a loading control, and the white line indicates that intervening lanes have been spliced out.

T23148

18586-18779

Sentence

denotes

(D and E) MTS assay of (D) WT lymphocytes and (E) LMP1 transgenic lymphoma cells cultured with IL4, a neutralizing antibody to IL4, or a rat IgG isotype control at the indicated concentrations.

T131

18586-18779

Sentence

denotes

(D and E) MTS assay of (D) WT lymphocytes and (E) LMP1 transgenic lymphoma cells cultured with IL4, a neutralizing antibody to IL4, or a rat IgG isotype control at the indicated concentrations.

T23149

18780-18834

Sentence

denotes

Shown are the results from LMP1 transgenic lymphoma 3.

T132

18780-18834

Sentence

denotes

Shown are the results from LMP1 transgenic lymphoma 3.

T23150

18835-18973

Sentence

denotes

The results are the mean ± SEM of triplicate samples from a single representative experiment that was repeated twice with similar results.

T133

18835-18973

Sentence

denotes

The results are the mean ± SEM of triplicate samples from a single representative experiment that was repeated twice with similar results.

T6399

18976-19244

Sentence

denotes

Although wild-type lymphocytes cannot be maintained in culture with IL4 supplementation alone (Figure 3A), slight enhancement in MTS activity could be detected if the cells were analyzed at an earlier time point, at 1 d (Figure 4D) versus 3 d (Figure 3A) post-harvest.

T134

18976-19244

Sentence

denotes

Although wild-type lymphocytes cannot be maintained in culture with IL4 supplementation alone (Figure 3A), slight enhancement in MTS activity could be detected if the cells were analyzed at an earlier time point, at 1 d (Figure 4D) versus 3 d (Figure 3A) post-harvest.

T6400

19245-19382

Sentence

denotes

The enhancement of MTS activity induced by IL4 in wild-type lymphocytes could be neutralized by the addition of IL4 antibody (Figure 4D).

T135

19245-19382

Sentence

denotes

The enhancement of MTS activity induced by IL4 in wild-type lymphocytes could be neutralized by the addition of IL4 antibody (Figure 4D).

T6401

19383-19501

Sentence

denotes

However, neutralizing antibodies to IL4 did not affect the MTS activity of LMP1 transgenic lymphoma cells (Figure 4E).

T136

19383-19501

Sentence

denotes

However, neutralizing antibodies to IL4 did not affect the MTS activity of LMP1 transgenic lymphoma cells (Figure 4E).

T6402

19502-19635

Sentence

denotes

In summary, the wild-type and LMP1 transgenic lymphoma cells grew independently of IL4 treatment and did not require Stat6 signaling.

T137

19502-19635

Sentence

denotes

In summary, the wild-type and LMP1 transgenic lymphoma cells grew independently of IL4 treatment and did not require Stat6 signaling.

T7551

19637-19693

Sentence

denotes

LMP1 Upregulates IL10 and Constitutively Activates Stat3

T138

19637-19693

Sentence

denotes

LMP1 Upregulates IL10 and Constitutively Activates Stat3

T7552

19694-19958

Sentence

denotes

To identify cytokines that may contribute to the increased survival and growth of lymphomas, the expression levels of a panel of cytokines were screened on CD19+ MACS-purified B cells, using an RPA probe set for IL4, IL5, IL10, IL13, IL15, IL9, IL2, IL6, and IFNγ.

T139

19694-19958

Sentence

denotes

To identify cytokines that may contribute to the increased survival and growth of lymphomas, the expression levels of a panel of cytokines were screened on CD19+ MACS-purified B cells, using an RPA probe set for IL4, IL5, IL10, IL13, IL15, IL9, IL2, IL6, and IFNγ.

T7553

19959-20069

Sentence

denotes

Expression levels were quantified with a phosphorimager and normalized to the ribosomal housekeeping gene L32.

T140

19959-20069

Sentence

denotes

Expression levels were quantified with a phosphorimager and normalized to the ribosomal housekeeping gene L32.

T7554

20070-20221

Sentence

denotes

None of the tested cytokines were detected in wild-type lymphocytes, therefore cytokine:L32 ratios were set to 1 in the mouse B cell lymphoma line 967.

T141

20070-20221

Sentence

denotes

None of the tested cytokines were detected in wild-type lymphocytes, therefore cytokine:L32 ratios were set to 1 in the mouse B cell lymphoma line 967.

T7555

20222-20416

Sentence

denotes

Transcription of IL10, IL15, and IFNγ were reproducibly detected in LMP1 transgenic lymphocytes and lymphoma cells and was higher than in the B cell lymphoma cell lines 967 and K46μ (Figure 5A).

T142

20222-20416

Sentence

denotes

Transcription of IL10, IL15, and IFNγ were reproducibly detected in LMP1 transgenic lymphocytes and lymphoma cells and was higher than in the B cell lymphoma cell lines 967 and K46μ (Figure 5A).

T7556

20417-20697

Sentence

denotes

There was no significant difference in the expression of IL15 and IFNγ between LMP1 transgenic lymphocytes and lymphoma cells, suggesting that upregulation of IL15 and IFNγ is induced by LMP1 expression in healthy lymphocytes but is not a unique property of malignant lymphocytes.

T143

20417-20697

Sentence

denotes

There was no significant difference in the expression of IL15 and IFNγ between LMP1 transgenic lymphocytes and lymphoma cells, suggesting that upregulation of IL15 and IFNγ is induced by LMP1 expression in healthy lymphocytes but is not a unique property of malignant lymphocytes.

T7557

20698-20886

Sentence

denotes

Strikingly, IL10, a B lymphocyte stimulatory cytokine, was increased 1.5- to 5-fold in the wild-type and LMP1 transgenic lymphoma cells compared to LMP1 transgenic lymphocytes (Figure 5A).

T144

20698-20886

Sentence

denotes

Strikingly, IL10, a B lymphocyte stimulatory cytokine, was increased 1.5- to 5-fold in the wild-type and LMP1 transgenic lymphoma cells compared to LMP1 transgenic lymphocytes (Figure 5A).

T7558

20887-21026

Sentence

denotes

Production of IL15 and IFNγ has been associated with induction of cytotoxic effector responses in cells latently infected with EBV [33,34].

T145

20887-21026

Sentence

denotes

Production of IL15 and IFNγ has been associated with induction of cytotoxic effector responses in cells latently infected with EBV [33,34].

T7559

21027-21251

Sentence

denotes

However, transformation and growth properties induced by EBV are associated with the upregulation of IL10 [35–38]; hence, the effects of IL10 upregulation on the growth properties of the lymphoma cells were further examined.

T146

21027-21251

Sentence

denotes

However, transformation and growth properties induced by EBV are associated with the upregulation of IL10 [35–38]; hence, the effects of IL10 upregulation on the growth properties of the lymphoma cells were further examined.

T7560

21252-21496

Sentence

denotes

Immunoblot analysis indicated that LMP1 transgenic lymphocytes and wild-type and LMP1 transgenic lymphoma cells had corresponding increased levels of phosphorylated α and β isoforms of activated Stat3, a target of the IL10 receptor (Figure 5B).

T147

21252-21496

Sentence

denotes

Immunoblot analysis indicated that LMP1 transgenic lymphocytes and wild-type and LMP1 transgenic lymphoma cells had corresponding increased levels of phosphorylated α and β isoforms of activated Stat3, a target of the IL10 receptor (Figure 5B).

T7561

21497-21638

Sentence

denotes

However, when comparing the same lymphomas, there was no correlation between the levels of IL10 induction and the levels of Stat3 activation.

T148

21497-21638

Sentence

denotes

However, when comparing the same lymphomas, there was no correlation between the levels of IL10 induction and the levels of Stat3 activation.

T7562

21639-21757

Sentence

denotes

This suggests that the activation of Stat3 is not solely induced by IL10 or that Stat3 activation may be constitutive.

T149

21639-21757

Sentence

denotes

This suggests that the activation of Stat3 is not solely induced by IL10 or that Stat3 activation may be constitutive.

T7563

21758-21888

Sentence

denotes

Additionally, there was no correlation between the levels of LMP1 expression and the levels of IL10 induction (Figures 1A and 5A).

T150

21758-21888

Sentence

denotes

Additionally, there was no correlation between the levels of LMP1 expression and the levels of IL10 induction (Figures 1A and 5A).

T7564

21889-22031

Sentence

denotes

This indicates that the induction of IL10 is a general property associated with enhanced survival and may only be indirectly affected by LMP1.

T151

21889-22031

Sentence

denotes

This indicates that the induction of IL10 is a general property associated with enhanced survival and may only be indirectly affected by LMP1.

T7565

22032-22205

Sentence

denotes

Neutralizing antibodies to IL10 did not affect the survival of lymphoma cells as determined by the MTS assay (unpublished data), suggesting constitutive activation of Stat3.

T152

22032-22205

Sentence

denotes

Neutralizing antibodies to IL10 did not affect the survival of lymphoma cells as determined by the MTS assay (unpublished data), suggesting constitutive activation of Stat3.

T7566

22206-22429

Sentence

denotes

This was confirmed by immunoblot analysis such that in the presence of anti-IL10 neutralizing antibodies, pStat3 levels remained activated in lymphoma cells isolated from wild-type and LMP1 transgenic lymphomas (Figure 5C).

T153

22206-22429

Sentence

denotes

This was confirmed by immunoblot analysis such that in the presence of anti-IL10 neutralizing antibodies, pStat3 levels remained activated in lymphoma cells isolated from wild-type and LMP1 transgenic lymphomas (Figure 5C).

T7567

22430-22587

Sentence

denotes

Exogenous addition of IL10 enhanced pStat3 activation above constitutive levels, indicating that lymphoma cells are responsive to IL10 treatment (Figure 5C).

T154

22430-22587

Sentence

denotes

Exogenous addition of IL10 enhanced pStat3 activation above constitutive levels, indicating that lymphoma cells are responsive to IL10 treatment (Figure 5C).

T7568

22588-22713

Sentence

denotes

This means that although the lymphoma cells have constitutive Stat3 activation, it may be further enhanced by IL10 induction.

T155

22588-22713

Sentence

denotes

This means that although the lymphoma cells have constitutive Stat3 activation, it may be further enhanced by IL10 induction.

T7569

22714-22879

Sentence

denotes

The neutralizing effect of the anti-IL10 antibody was confirmed by pre-incubation of IL10 with anti-IL10 antibody compared to a rat IgG1 isotype control (Figure 5C).

T156

22714-22879

Sentence

denotes

The neutralizing effect of the anti-IL10 antibody was confirmed by pre-incubation of IL10 with anti-IL10 antibody compared to a rat IgG1 isotype control (Figure 5C).

T157

22880-22957

Sentence

denotes

Figure 5 LMP1 Upregulates IL10 Expression and Constitutively Activates Stat3

T23731

22890-22957

Sentence

denotes

LMP1 Upregulates IL10 Expression and Constitutively Activates Stat3

T23732

22958-23097

Sentence

denotes

(A) Relative expression of IL10, IL15, and IFNγ mRNA in WT and LMP1 transgenic B cells (CD19+), as detected with an Rnase protection assay.

T158

22958-23097

Sentence

denotes

(A) Relative expression of IL10, IL15, and IFNγ mRNA in WT and LMP1 transgenic B cells (CD19+), as detected with an Rnase protection assay.

T23733

23098-23159

Sentence

denotes

Mouse lymphoma cell lines 967 and K46μ were used as controls.

T159

23098-23159

Sentence

denotes

Mouse lymphoma cell lines 967 and K46μ were used as controls.

T23734

23160-23282

Sentence

denotes

Expression levels were quantified with a phosphorimager and values were normalized to the ribosomal housekeeping gene L32.

T160

23160-23282

Sentence

denotes

Expression levels were quantified with a phosphorimager and values were normalized to the ribosomal housekeeping gene L32.

T23735

23283-23357

Sentence

denotes

The cytokine:L32 ratio was set to 1 in the mouse B cell lymphoma line 967.

T161

23283-23357

Sentence

denotes

The cytokine:L32 ratio was set to 1 in the mouse B cell lymphoma line 967.

T23736

23358-23684

Sentence

denotes

(B and C) Immunoblot analysis of activated pStat3 in purified B cells (CD19+) from WT and LMP1 transgenic mice (B) at the time of harvest, and (C) 4 h after culture with or without IL10, a neutralizing antibody to IL10, or a rat IgG1 isotype control. (C) Shown are the results for WT lymphoma 1 and LMP1 transgenic lymphoma 1.

T162

23358-23684

Sentence

denotes

(B and C) Immunoblot analysis of activated pStat3 in purified B cells (CD19+) from WT and LMP1 transgenic mice (B) at the time of harvest, and (C) 4 h after culture with or without IL10, a neutralizing antibody to IL10, or a rat IgG1 isotype control. (C) Shown are the results for WT lymphoma 1 and LMP1 transgenic lymphoma 1.

T23737

23685-23748

Sentence

denotes

Arrows indicate the positions of the α and β isoforms of Stat3.

T163

23685-23748

Sentence

denotes

Arrows indicate the positions of the α and β isoforms of Stat3.

T23738

23749-23785

Sentence

denotes

Actin was used as a loading control.

T164

23749-23785

Sentence

denotes

Actin was used as a loading control.

T23739

23786-23895

Sentence

denotes

(D) Immunohistochemistry detection of activated nuclear pStat3 in the spleens of WT and LMP1 transgenic mice.

T165

23786-23895

Sentence

denotes

(D) Immunohistochemistry detection of activated nuclear pStat3 in the spleens of WT and LMP1 transgenic mice.

T23740

23896-23913

Sentence

denotes

Scale bar, 20 μm.

T166

23896-23913

Sentence

denotes

Scale bar, 20 μm.

T7570

23916-24086

Sentence

denotes

Nuclear translocation of pStat3 is a consequence of activation, and nuclear pStat3 was not detected by immunohistochemistry staining of spleen sections from control mice.

T167

23916-24086

Sentence

denotes

Nuclear translocation of pStat3 is a consequence of activation, and nuclear pStat3 was not detected by immunohistochemistry staining of spleen sections from control mice.

T7571

24087-24251

Sentence

denotes

However, nuclear pStat3 was detectable in LMP1 transgenic mice and wild-type lymphomas and was detected more homogeneously in LMP1 transgenic lymphomas (Figure 5D).

T168

24087-24251

Sentence

denotes

However, nuclear pStat3 was detectable in LMP1 transgenic mice and wild-type lymphomas and was detected more homogeneously in LMP1 transgenic lymphomas (Figure 5D).

T7572

24252-24397

Sentence

denotes

The constitutive activation of pStat3 and abundant nuclear Stat3 suggests that Stat3 signaling contributes to LMP1-mediated lymphoma development.

T169

24252-24397

Sentence

denotes

The constitutive activation of pStat3 and abundant nuclear Stat3 suggests that Stat3 signaling contributes to LMP1-mediated lymphoma development.

T9769

24399-24469

Sentence

denotes

LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway

T170

24399-24469

Sentence

denotes

LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway

T9770

24470-24552

Sentence

denotes

LMP1 transformation of rodent fibroblasts requires activation of PI3K and Akt [5].

T171

24470-24552

Sentence

denotes

LMP1 transformation of rodent fibroblasts requires activation of PI3K and Akt [5].

T9771

24553-24676

Sentence

denotes

Additionally, activated pAkt is frequently detected in NPC and the neoplastic Reed-Sternberg cells of classical HD [39,40].

T172

24553-24676

Sentence

denotes

Additionally, activated pAkt is frequently detected in NPC and the neoplastic Reed-Sternberg cells of classical HD [39,40].

T9772

24677-24848

Sentence

denotes

To determine if Akt signaling is activated in LMP1 transgenic mice, pAkt and several of its targets were assessed by immunoblotting of splenic CD19+ MACS-purified B cells.

T173

24677-24848

Sentence

denotes

To determine if Akt signaling is activated in LMP1 transgenic mice, pAkt and several of its targets were assessed by immunoblotting of splenic CD19+ MACS-purified B cells.

T9773

24849-25058

Sentence

denotes

LMP1 transgenic B cells had increased levels of pAkt compared to wild-type lymphocytes; however, progression to lymphoma in both LMP1-positive and -negative lymphoma cells did not further increase pAkt levels.

T174

24849-25058

Sentence

denotes

LMP1 transgenic B cells had increased levels of pAkt compared to wild-type lymphocytes; however, progression to lymphoma in both LMP1-positive and -negative lymphoma cells did not further increase pAkt levels.

T9774

25059-25287

Sentence

denotes

The Akt target glycogen synthase kinase 3 (GSK3) is inactivated by phosphorylation; however, increased phosphorylated GSK3 was not detected in the transgenic lymphocytes and was almost absent in the lymphoma samples (Figure 6A).

T175

25059-25287

Sentence

denotes

The Akt target glycogen synthase kinase 3 (GSK3) is inactivated by phosphorylation; however, increased phosphorylated GSK3 was not detected in the transgenic lymphocytes and was almost absent in the lymphoma samples (Figure 6A).

T9775

25288-25408

Sentence

denotes

This finding indicates that GSK3 is not a target of activated Akt in the LMP1 transgenic lymphocytes and lymphoma cells.

T176

25288-25408

Sentence

denotes

This finding indicates that GSK3 is not a target of activated Akt in the LMP1 transgenic lymphocytes and lymphoma cells.

T9776

25409-25520

Sentence

denotes

Similarly, activation of Akt without phosphorylation of GSK3 has been previously shown in EBV-positive HD [40].

T177

25409-25520

Sentence

denotes

Similarly, activation of Akt without phosphorylation of GSK3 has been previously shown in EBV-positive HD [40].

T9777

25521-25625

Sentence

denotes

In contrast, the wild-type lymphocytes lacked activated Akt but did have detectable phosphorylated GSK3.

T178

25521-25625

Sentence

denotes

In contrast, the wild-type lymphocytes lacked activated Akt but did have detectable phosphorylated GSK3.

T9778

25626-25712

Sentence

denotes

This further suggests that additional pathways are involved in the regulation of GSK3.

T179

25626-25712

Sentence

denotes

This further suggests that additional pathways are involved in the regulation of GSK3.

T180

25713-25793

Sentence

denotes

Figure 6 LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway

T24366

25723-25793

Sentence

denotes

LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway

T24367

25794-26058

Sentence

denotes

(A and B) Immunoblot analysis of purified B cells (CD19+) from the spleens of WT and LMP1 transgenic mice for Akt signaling, probing for (A) activated pAkt and downstream targets, including inactivated pGSK3α/β, and (B) activated p-mTOR, and total levels of FoxO1.

T181

25794-26058

Sentence

denotes

(A and B) Immunoblot analysis of purified B cells (CD19+) from the spleens of WT and LMP1 transgenic mice for Akt signaling, probing for (A) activated pAkt and downstream targets, including inactivated pGSK3α/β, and (B) activated p-mTOR, and total levels of FoxO1.

T24368

26059-26117

Sentence

denotes

Arrows indicate the positions of α and β isoforms of GSK3.

T182

26059-26117

Sentence

denotes

Arrows indicate the positions of α and β isoforms of GSK3.

T24369

26118-26188

Sentence

denotes

The white line indicates that intervening lanes have been spliced out.

T183

26118-26188

Sentence

denotes

The white line indicates that intervening lanes have been spliced out.

T24370

26189-26342

Sentence

denotes

(C) Immunoblot analysis for cell cycle proteins regulating the Rb pathway, probing for activated pRb, and total levels of Cdk2 and the Cdk inhibitor p27.

T184

26189-26342

Sentence

denotes

(C) Immunoblot analysis for cell cycle proteins regulating the Rb pathway, probing for activated pRb, and total levels of Cdk2 and the Cdk inhibitor p27.

T24371

26343-26379

Sentence

denotes

Actin was used as a loading control.

T185

26343-26379

Sentence

denotes

Actin was used as a loading control.

T9779

26382-26468

Sentence

denotes

To identify other potential Akt targets, immunoblot analysis for p-mTOR was performed.

T186

26382-26468

Sentence

denotes

To identify other potential Akt targets, immunoblot analysis for p-mTOR was performed.

T9780

26469-26690

Sentence

denotes

Activated p-mTOR was not increased in LMP1 transgenic lymphocytes or lymphoma cells, indicating that this pathway is not affected by LMP1-induced Akt activation and does not contribute to lymphoma development (Figure 6B).

T187

26469-26690

Sentence

denotes

Activated p-mTOR was not increased in LMP1 transgenic lymphocytes or lymphoma cells, indicating that this pathway is not affected by LMP1-induced Akt activation and does not contribute to lymphoma development (Figure 6B).

T9781

26691-26892

Sentence

denotes

Akt is also known to phosphorylate and induce the degradation of the pro-apoptotic Forkhead family of transcription factors, leading to cell cycle progression and survival in some human tumors [41,42].

T188

26691-26892

Sentence

denotes

Akt is also known to phosphorylate and induce the degradation of the pro-apoptotic Forkhead family of transcription factors, leading to cell cycle progression and survival in some human tumors [41,42].

T9782

26893-27020

Sentence

denotes

Immunoblot analysis of splenic B cells did not consistently detect p-FoxO1 levels, a signal that targets FoxO1 for degradation.

T189

26893-27020

Sentence

denotes

Immunoblot analysis of splenic B cells did not consistently detect p-FoxO1 levels, a signal that targets FoxO1 for degradation.

T9783

27021-27097

Sentence

denotes

Hence, degradation of FoxO1 was assessed by detection of total FoxO1 levels.

T190

27021-27097

Sentence

denotes

Hence, degradation of FoxO1 was assessed by detection of total FoxO1 levels.

T9784

27098-27346

Sentence

denotes

Immunoblot analysis indicated that total FoxO1 levels were greatly decreased in wild-type and LMP1 transgenic lymphomas (Figure 6B), suggesting that inhibition of the Forkhead signaling pathway is an important target of Akt in lymphoma development.

T191

27098-27346

Sentence

denotes

Immunoblot analysis indicated that total FoxO1 levels were greatly decreased in wild-type and LMP1 transgenic lymphomas (Figure 6B), suggesting that inhibition of the Forkhead signaling pathway is an important target of Akt in lymphoma development.

T9785

27347-27577

Sentence

denotes

However, considering that Akt activation did not induce FoxO1 degradation in LMP1 transgenic B cells, Akt may not be the sole regulator of FoxO1, and it may be that progression to lymphoma requires modulation of multiple pathways.

T192

27347-27577

Sentence

denotes

However, considering that Akt activation did not induce FoxO1 degradation in LMP1 transgenic B cells, Akt may not be the sole regulator of FoxO1, and it may be that progression to lymphoma requires modulation of multiple pathways.

T9786

27578-27690

Sentence

denotes

The Forkhead family of transcription factors is known to induce the expression of the Cdk inhibitor p27 [43,44].

T193

27578-27690

Sentence

denotes

The Forkhead family of transcription factors is known to induce the expression of the Cdk inhibitor p27 [43,44].

T9787

27691-27868

Sentence

denotes

LMP1-transformed rodent fibroblasts have decreased expression of p27, upregulation of Cdk2, and subsequent phosphorylation and inactivation of the tumor suppressor gene Rb [45].

T194

27691-27868

Sentence

denotes

LMP1-transformed rodent fibroblasts have decreased expression of p27, upregulation of Cdk2, and subsequent phosphorylation and inactivation of the tumor suppressor gene Rb [45].

T9788

27869-28060

Sentence

denotes

To investigate whether LMP1 affected cell cycle regulation through the Rb pathway in B cells, immunoblot analyses for pRb, Cdk2, and p27 were performed on splenic CD19+ MACS-purified B cells.

T195

27869-28060

Sentence

denotes

To investigate whether LMP1 affected cell cycle regulation through the Rb pathway in B cells, immunoblot analyses for pRb, Cdk2, and p27 were performed on splenic CD19+ MACS-purified B cells.

T9789

28061-28219

Sentence

denotes

LMP1 transgenic B cells had enhanced levels of pRb with concomitant stabilization of total Rb levels and Cdk2 compared to wild-type B lymphocytes (Figure 6C).

T196

28061-28219

Sentence

denotes

LMP1 transgenic B cells had enhanced levels of pRb with concomitant stabilization of total Rb levels and Cdk2 compared to wild-type B lymphocytes (Figure 6C).

T9790

28220-28405

Sentence

denotes

Progression to lymphoma in both wild-type and LMP1 transgenic lymphoma cells led to increased levels of Rb, correspondingly high levels of Cdk2, and decreased levels of p27 (Figure 6C).

T197

28220-28405

Sentence

denotes

Progression to lymphoma in both wild-type and LMP1 transgenic lymphoma cells led to increased levels of Rb, correspondingly high levels of Cdk2, and decreased levels of p27 (Figure 6C).

T9791

28406-28569

Sentence

denotes

These data indicate that the Rb pathway is deregulated in LMP1 transgenic lymphocytes and that lymphoma cells are distinguished by loss of FoxO1 and decreased p27.

T198

28406-28569

Sentence

denotes

These data indicate that the Rb pathway is deregulated in LMP1 transgenic lymphocytes and that lymphoma cells are distinguished by loss of FoxO1 and decreased p27.

T11843

28571-28662

Sentence

denotes

LMP1 Promotes Tumor Growth and Survival through Activation of Akt, NFκB, and Stat3 Pathways

T199

28571-28662

Sentence

denotes

LMP1 Promotes Tumor Growth and Survival through Activation of Akt, NFκB, and Stat3 Pathways

T11844

28663-28952

Sentence

denotes

To explore which pathways were required for the enhanced growth and survival of LMP1-induced lymphomas, splenocytes from wild-type and LMP1 transgenic mice were cultured in the presence of inhibitors for Akt, NFκB, Stat3, mTOR, or MAPK and assayed for growth and survival by the MTS assay.

T200

28663-28952

Sentence

denotes

To explore which pathways were required for the enhanced growth and survival of LMP1-induced lymphomas, splenocytes from wild-type and LMP1 transgenic mice were cultured in the presence of inhibitors for Akt, NFκB, Stat3, mTOR, or MAPK and assayed for growth and survival by the MTS assay.

T11845

28953-29067

Sentence

denotes

As previously shown, wild-type lymphocytes were not viable in culture and could not be tested with the inhibitors.

T201

28953-29067

Sentence

denotes

As previously shown, wild-type lymphocytes were not viable in culture and could not be tested with the inhibitors.

T11846

29068-29306

Sentence

denotes

However, the enhanced viability of LMP1 transgenic lymphocytes was effectively blocked by treatment with triciribine, BAY11–7085, cucurbitacin I, and slightly with SB203580, but not by treatment with rapamycin, U0126, or AG490 (Figure 7).

T202

29068-29306

Sentence

denotes

However, the enhanced viability of LMP1 transgenic lymphocytes was effectively blocked by treatment with triciribine, BAY11–7085, cucurbitacin I, and slightly with SB203580, but not by treatment with rapamycin, U0126, or AG490 (Figure 7).

T11847

29307-29450

Sentence

denotes

Triciribine inhibits the activation of Akt and at 20 μM has been shown to induce growth arrest in cancer cells with aberrant Akt activity [46].

T203

29307-29450

Sentence

denotes

Triciribine inhibits the activation of Akt and at 20 μM has been shown to induce growth arrest in cancer cells with aberrant Akt activity [46].

T11848

29451-29702

Sentence

denotes

The effects of triciribine on cell growth of the transgenic lymphocytes and lymphomas were apparent as low as 1 μM, suggesting that activation of Akt is required for the survival and growth of LMP1 transgenic lymphocytes and lymphoma cells (Figure 7).

T204

29451-29702

Sentence

denotes

The effects of triciribine on cell growth of the transgenic lymphocytes and lymphomas were apparent as low as 1 μM, suggesting that activation of Akt is required for the survival and growth of LMP1 transgenic lymphocytes and lymphoma cells (Figure 7).

T11849

29703-29825

Sentence

denotes

The effects of the inhibitors were assessed by identifying phosphorylated Akt, Stat3, and total levels of IκBα (Figure 8).

T205

29703-29825

Sentence

denotes

The effects of the inhibitors were assessed by identifying phosphorylated Akt, Stat3, and total levels of IκBα (Figure 8).

T11850

29826-29953

Sentence

denotes

Treatment with triciribine effectively blocked phosphorylation of Akt, and phosphorylated Akt was no longer detected past 5 μM.

T206

29826-29953

Sentence

denotes

Treatment with triciribine effectively blocked phosphorylation of Akt, and phosphorylated Akt was no longer detected past 5 μM.

T11851

29954-30012

Sentence

denotes

Phosphorylated Stat3 and IκBα were still present at 25 μM.

T207

29954-30012

Sentence

denotes

Phosphorylated Stat3 and IκBα were still present at 25 μM.

T11852

30013-30190

Sentence

denotes

These findings suggest that triciribine specifically targets Akt and that Akt activation is required for the enhanced viability of the transgenic lymphocytes and lymphoma cells.

T208

30013-30190

Sentence

denotes

These findings suggest that triciribine specifically targets Akt and that Akt activation is required for the enhanced viability of the transgenic lymphocytes and lymphoma cells.

T209

30191-30290

Sentence

denotes

Figure 7 Akt, NFκB, and Stat3 Signaling Are Required for the Growth and Survival of Lymphoma Cells

T24794

30201-30290

Sentence

denotes

Akt, NFκB, and Stat3 Signaling Are Required for the Growth and Survival of Lymphoma Cells

T24795

30291-30415

Sentence

denotes

MTS assay of splenocytes from (A and B) WT or (C and D) LMP1 transgenic lymphomas and (E and F) LMP1 transgenic lymphoctyes.

T210

30291-30415

Sentence

denotes

MTS assay of splenocytes from (A and B) WT or (C and D) LMP1 transgenic lymphomas and (E and F) LMP1 transgenic lymphoctyes.

T24796

30416-30623

Sentence

denotes

Splenocytes were cultured with or without inhibitors of NFκB (BAY11), mTOR (rapamycin), Akt (triciribine), MEK1/2 (U0126), p38 (SB202190), or Stat3 (cucurbitacin I and AG490) at the indicated concentrations.

T211

30416-30623

Sentence

denotes

Splenocytes were cultured with or without inhibitors of NFκB (BAY11), mTOR (rapamycin), Akt (triciribine), MEK1/2 (U0126), p38 (SB202190), or Stat3 (cucurbitacin I and AG490) at the indicated concentrations.

T24797

30624-30677

Sentence

denotes

The results are the mean ± SEM of triplicate samples.

T212

30624-30677

Sentence

denotes

The results are the mean ± SEM of triplicate samples.

T24798

30678-30826

Sentence

denotes

Shown are the results for (A and B) WT lymphoma 1, (C and D) LMP1 transgenic lymphoma 2, and (E and F) one out of two LMP1 transgenic mice analyzed.

T213

30678-30826

Sentence

denotes

Shown are the results for (A and B) WT lymphoma 1, (C and D) LMP1 transgenic lymphoma 2, and (E and F) one out of two LMP1 transgenic mice analyzed.

T24799

30827-30911

Sentence

denotes

This analysis was repeated with LMP1 transgenic lymphoma 4 yielding similar results.

T214

30827-30911

Sentence

denotes

This analysis was repeated with LMP1 transgenic lymphoma 4 yielding similar results.

T215

30912-31024

Sentence

denotes

Figure 8 Analysis of Akt, NFκB, and Stat3 Pathways in Contribution to the Growth and Survival of Lymphoma Cells

T25238

30922-31024

Sentence

denotes

Analysis of Akt, NFκB, and Stat3 Pathways in Contribution to the Growth and Survival of Lymphoma Cells

T25239

31025-31311

Sentence

denotes

(A–D) Immunoblot analysis of wild-type and LMP1 transgenic lymphomas for Akt, NFκB, and Stat3 signaling after treatment with (A) an Akt inhibitor, triciribine, (B) an NFκB inhibitor, BAY11–7085, and the Stat3 inhibitors (C) cucurbitacin I and (D) AG490, at the indicated concentrations.

T216

31025-31311

Sentence

denotes

(A–D) Immunoblot analysis of wild-type and LMP1 transgenic lymphomas for Akt, NFκB, and Stat3 signaling after treatment with (A) an Akt inhibitor, triciribine, (B) an NFκB inhibitor, BAY11–7085, and the Stat3 inhibitors (C) cucurbitacin I and (D) AG490, at the indicated concentrations.

T25240

31312-31371

Sentence

denotes

Arrows indicate the positions of α and β isoforms of Stat3.

T217

31312-31371

Sentence

denotes

Arrows indicate the positions of α and β isoforms of Stat3.

T25241

31372-31408

Sentence

denotes

Actin was used as a loading control.

T218

31372-31408

Sentence

denotes

Actin was used as a loading control.

T11853

31411-31506

Sentence

denotes

Inhibition of NFκB signaling rapidly induces cell death of EBV-transformed lymphocytes [17,18].

T219

31411-31506

Sentence

denotes

Inhibition of NFκB signaling rapidly induces cell death of EBV-transformed lymphocytes [17,18].

T11854

31507-31726

Sentence

denotes

BAY11–7085, an inhibitor of NFκB signaling, also greatly decreased the viability of the LMP1 transgenic lymphocytes and lymphoma cells at doses as low as 1 μM, and at 5 μM the cells were completely nonviable (Figure 7).

T220

31507-31726

Sentence

denotes

BAY11–7085, an inhibitor of NFκB signaling, also greatly decreased the viability of the LMP1 transgenic lymphocytes and lymphoma cells at doses as low as 1 μM, and at 5 μM the cells were completely nonviable (Figure 7).

T11855

31727-31774

Sentence

denotes

This is well within the reported IC50 of 10 μM.

T221

31727-31774

Sentence

denotes

This is well within the reported IC50 of 10 μM.

T11856

31775-31907

Sentence

denotes

Phosphorylated Akt, Stat3, and total IκBα were still present up to treatment with 15 μM and then were no longer detected (Figure 8).

T222

31775-31907

Sentence

denotes

Phosphorylated Akt, Stat3, and total IκBα were still present up to treatment with 15 μM and then were no longer detected (Figure 8).

T11857

31908-32084

Sentence

denotes

This finding suggests that inhibition of NFκB can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells without significant effects on activation of Akt or Stat3.

T223

31908-32084

Sentence

denotes

This finding suggests that inhibition of NFκB can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells without significant effects on activation of Akt or Stat3.

T11858

32085-32178

Sentence

denotes

Cucurbitacin I inhibits activation of Stat3 by suppressing the activation of its kinase JAK2.

T224

32085-32178

Sentence

denotes

Cucurbitacin I inhibits activation of Stat3 by suppressing the activation of its kinase JAK2.

T11859

32179-32282

Sentence

denotes

It has been shown to selectively inhibit the growth of tumors with constitutively activated Stat3 [47].

T225

32179-32282

Sentence

denotes

It has been shown to selectively inhibit the growth of tumors with constitutively activated Stat3 [47].

T11860

32283-32485

Sentence

denotes

Similarly, LMP1 transgenic lymphocytes and lymphoma cells were susceptible to cucurbitacin I treatment starting at 0.1 μM, a dose that corresponds closely to the reported IC50 of 500 nM (Figure 7) [47].

T226

32283-32485

Sentence

denotes

Similarly, LMP1 transgenic lymphocytes and lymphoma cells were susceptible to cucurbitacin I treatment starting at 0.1 μM, a dose that corresponds closely to the reported IC50 of 500 nM (Figure 7) [47].

T11861

32486-32674

Sentence

denotes

Phosphorylated Akt, Stat3, and total IκBα were not detectable past 1 μM and at higher doses all protein levels were greatly decreased, indicative of the total loss of viability (Figure 8).

T227

32486-32674

Sentence

denotes

Phosphorylated Akt, Stat3, and total IκBα were not detectable past 1 μM and at higher doses all protein levels were greatly decreased, indicative of the total loss of viability (Figure 8).

T11862

32675-32838

Sentence

denotes

A second reported inhibitor of Stat3, AG490, had no effect on growth (Figure 7), but activation of Akt, Stat3, or levels of IκBα were also not affected (Figure 8).

T228

32675-32838

Sentence

denotes

A second reported inhibitor of Stat3, AG490, had no effect on growth (Figure 7), but activation of Akt, Stat3, or levels of IκBα were also not affected (Figure 8).

T11863

32839-33047

Sentence

denotes

These findings suggest that inhibition of Stat3 can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells, but Stat3 inhibition also has considerable crossover effects on Akt and NFκB signaling.

T229

32839-33047

Sentence

denotes

These findings suggest that inhibition of Stat3 can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells, but Stat3 inhibition also has considerable crossover effects on Akt and NFκB signaling.

T11864

33048-33283

Sentence

denotes

LMP1 has also been shown to activate JNK and p38 MAPK pathways [13,48], and LMP1 transgenic lymphocytes were mildly susceptible to growth inhibition by SB202190, an inhibitor of p38 MAPK, but not U0126, an inhibitor of MEK1/2 activity.

T230

33048-33283

Sentence

denotes

LMP1 has also been shown to activate JNK and p38 MAPK pathways [13,48], and LMP1 transgenic lymphocytes were mildly susceptible to growth inhibition by SB202190, an inhibitor of p38 MAPK, but not U0126, an inhibitor of MEK1/2 activity.

T11865

33284-33548

Sentence

denotes

However, effects of SB202190 were only apparent at high doses (>10 μM), much higher than the reported IC50 of 0.35 μM, suggesting that p38 MAPK does not significantly contribute to the enhanced viability in LMP1 transgenic lymphocytes or lymphoma cells (Figure 7).

T231

33284-33548

Sentence

denotes

However, effects of SB202190 were only apparent at high doses (>10 μM), much higher than the reported IC50 of 0.35 μM, suggesting that p38 MAPK does not significantly contribute to the enhanced viability in LMP1 transgenic lymphocytes or lymphoma cells (Figure 7).

T11866

33549-34122

Sentence

denotes

Interestingly, both wild-type and LMP1 transgenic lymphomas were similarly susceptible to triciribine, BAY11–7085, and cucurbitacin I treatments, but not SB202190, AG490, or U0126 treatment, suggesting that activation of Akt, NFκB, and Stat3 but not MAPK pathways are characteristics associated with malignant transformation (Figure 7). rapamycin, an inhibitor of mTOR, did not affect the viability of the transgenic lymphocytes or lymphoma cells, confirming that mTOR is not targeted by Akt activation in LMP1 transgenic lymphocytes or malignant lymphoma cells (Figure 7).

T232

33549-34122

Sentence

denotes

Interestingly, both wild-type and LMP1 transgenic lymphomas were similarly susceptible to triciribine, BAY11–7085, and cucurbitacin I treatments, but not SB202190, AG490, or U0126 treatment, suggesting that activation of Akt, NFκB, and Stat3 but not MAPK pathways are characteristics associated with malignant transformation (Figure 7). rapamycin, an inhibitor of mTOR, did not affect the viability of the transgenic lymphocytes or lymphoma cells, confirming that mTOR is not targeted by Akt activation in LMP1 transgenic lymphocytes or malignant lymphoma cells (Figure 7).

T233

34124-34134

Sentence

denotes

Discussion

T14502

34135-34223

Sentence

denotes

This study defines the oncogenic properties of LMP1 in promoting B cell lymphomagenesis.

T234

34135-34223

Sentence

denotes

This study defines the oncogenic properties of LMP1 in promoting B cell lymphomagenesis.

T14503

34224-34450

Sentence

denotes

LMP1 transgenic mice have a higher incidence of lymphoma [26] and the progression to lymphoma correlates with higher expression levels of LMP1 (Figure 1A and 1B), suggesting that LMP1 is directly involved in tumor development.

T235

34224-34450

Sentence

denotes

LMP1 transgenic mice have a higher incidence of lymphoma [26] and the progression to lymphoma correlates with higher expression levels of LMP1 (Figure 1A and 1B), suggesting that LMP1 is directly involved in tumor development.

T14504

34451-34574

Sentence

denotes

Table 1 summarizes the biological and molecular properties that were identified in wild-type and LMP1 transgenic lymphomas.

T236

34451-34574

Sentence

denotes

Table 1 summarizes the biological and molecular properties that were identified in wild-type and LMP1 transgenic lymphomas.

T14505

34575-34838

Sentence

denotes

Although many of the molecular properties studied were similar between wild-type and LMP1 transgenic lymphomas, there were distinguishing biological properties, namely the ability of LMP1 transgenic lymphomas to induce higher levels of survival and proliferation.

T237

34575-34838

Sentence

denotes

Although many of the molecular properties studied were similar between wild-type and LMP1 transgenic lymphomas, there were distinguishing biological properties, namely the ability of LMP1 transgenic lymphomas to induce higher levels of survival and proliferation.

T14506

34839-35069

Sentence

denotes

Interestingly, although LMP1 transgenic mice develop lymphomas in the same B-1a cell type as spontaneous wild-type lymphomas (Figure 2), some signaling effects induced by LMP1 may explain the enhanced promotion to lymphomagenesis.

T238

34839-35069

Sentence

denotes

Interestingly, although LMP1 transgenic mice develop lymphomas in the same B-1a cell type as spontaneous wild-type lymphomas (Figure 2), some signaling effects induced by LMP1 may explain the enhanced promotion to lymphomagenesis.

T14507

35070-35310

Sentence

denotes

Since CD40-deficient mice have decreased numbers of IgMhighIgDlow cells, a phenotype associated with B-1, marginal zone, and memory B cells, the mimicry of CD40 signaling by LMP1 could possibly contribute to the expansion of B-1 cells [20].

T239

35070-35310

Sentence

denotes

Since CD40-deficient mice have decreased numbers of IgMhighIgDlow cells, a phenotype associated with B-1, marginal zone, and memory B cells, the mimicry of CD40 signaling by LMP1 could possibly contribute to the expansion of B-1 cells [20].

T14508

35311-35500

Sentence

denotes

It is noteworthy that expression of LMP1 in transgenic mice has been shown to inhibit the formation of GCs [23,49], preventing typical B-2 cells from antigen-driven selection and expansion.

T240

35311-35500

Sentence

denotes

It is noteworthy that expression of LMP1 in transgenic mice has been shown to inhibit the formation of GCs [23,49], preventing typical B-2 cells from antigen-driven selection and expansion.

T14509

35501-35604

Sentence

denotes

The lack of GC reactions may contribute to the bias of LMP1 transgenic mice towards B-1 cell lymphomas.

T241

35501-35604

Sentence

denotes

The lack of GC reactions may contribute to the bias of LMP1 transgenic mice towards B-1 cell lymphomas.

T14510

35605-35722

Sentence

denotes

Interestingly, LMP2 signaling also favors development of B-1 cells, but this occurs in the absence of transformation.

T242

35605-35722

Sentence

denotes

Interestingly, LMP2 signaling also favors development of B-1 cells, but this occurs in the absence of transformation.

T14511

35723-35867

Sentence

denotes

These results suggest that the mimicry of B cell receptor signaling by LMP2 promotes B-1 cell differentiation but not transformation [24,50,51].

T243

35723-35867

Sentence

denotes

These results suggest that the mimicry of B cell receptor signaling by LMP2 promotes B-1 cell differentiation but not transformation [24,50,51].

T14512

35868-36005

Sentence

denotes

This promotion of B-1 differentiation may account for the ability of LMP2 to exacerbate autoimmunity and bypass anergy induction [52,53].

T244

35868-36005

Sentence

denotes

This promotion of B-1 differentiation may account for the ability of LMP2 to exacerbate autoimmunity and bypass anergy induction [52,53].

T14513

36006-36248

Sentence

denotes

In contrast, the preponderance of tumors of B-1a origin does not reflect effects of LMP1 signaling on B cell differentiation, as splenic B cells from healthy LMP1 transgenic mice contain similar numbers of B-1 and B-2 cells as wild-type mice.

T245

36006-36248

Sentence

denotes

In contrast, the preponderance of tumors of B-1a origin does not reflect effects of LMP1 signaling on B cell differentiation, as splenic B cells from healthy LMP1 transgenic mice contain similar numbers of B-1 and B-2 cells as wild-type mice.

T14514

36249-36380

Sentence

denotes

In support of this lack of effect, the differentiation of B-1 versus B-2 cells is thought to be independent of CD40 signaling [54].

T246

36249-36380

Sentence

denotes

In support of this lack of effect, the differentiation of B-1 versus B-2 cells is thought to be independent of CD40 signaling [54].

T14515

36381-36571

Sentence

denotes

B-1 cells constitute the predominant lymphocyte population in the peritoneal and pleuropericardial cavities, while B-2 cells are mainly found in the spleen, lymph node, and peripheral blood.

T247

36381-36571

Sentence

denotes

B-1 cells constitute the predominant lymphocyte population in the peritoneal and pleuropericardial cavities, while B-2 cells are mainly found in the spleen, lymph node, and peripheral blood.

T14516

36572-36722

Sentence

denotes

B-1 cells produce the main source of IgM and IgA antibodies in serum, which are involved in T cell–independent responses to common microbial antigens.

T248

36572-36722

Sentence

denotes

B-1 cells produce the main source of IgM and IgA antibodies in serum, which are involved in T cell–independent responses to common microbial antigens.

T14517

36723-36840

Sentence

denotes

Importantly, B-1 cells have the unique capacity to self replenish and are also predisposed to transformation [28,29].

T249

36723-36840

Sentence

denotes

Importantly, B-1 cells have the unique capacity to self replenish and are also predisposed to transformation [28,29].

T14518

36841-37019

Sentence

denotes

Clonal expansion of B-1 cells can be detected in aging mice above 18 mo of age, and B-1 cells are thought to be the murine progenitor of B cell chronic lymphocytic leukemia [30].

T250

36841-37019

Sentence

denotes

Clonal expansion of B-1 cells can be detected in aging mice above 18 mo of age, and B-1 cells are thought to be the murine progenitor of B cell chronic lymphocytic leukemia [30].

T14519

37020-37194

Sentence

denotes

The data presented in this study indicate that although LMP1 is expressed in all B lymphocytes in the transgenic mice, malignancy develops in this specific subset of B cells.

T251

37020-37194

Sentence

denotes

The data presented in this study indicate that although LMP1 is expressed in all B lymphocytes in the transgenic mice, malignancy develops in this specific subset of B cells.

T14520

37195-37316

Sentence

denotes

The elevated expression of LMP1 in B-1a cells and the activation of specific pathways apparently induce malignant growth.

T252

37195-37316

Sentence

denotes

The elevated expression of LMP1 in B-1a cells and the activation of specific pathways apparently induce malignant growth.

T14521

37317-37429

Sentence

denotes

These same pathways can also become sporadically activated in aged mice and also result in lymphoma development.

T253

37317-37429

Sentence

denotes

These same pathways can also become sporadically activated in aged mice and also result in lymphoma development.

T14522

37430-37609

Sentence

denotes

This is similar to EBV-associated cancers in vivo, where pathways that are activated by LMP1 are also activated in the less prevalent EBV-negative forms of the cancers [40,55–58].

T254

37430-37609

Sentence

denotes

This is similar to EBV-associated cancers in vivo, where pathways that are activated by LMP1 are also activated in the less prevalent EBV-negative forms of the cancers [40,55–58].

T14523

37610-37798

Sentence

denotes

Thus, the contribution of EBV and LMP1 to tumor development is apparently the continuous activation of pathways that can also be sporadically activated and contribute to tumor development.

T255

37610-37798

Sentence

denotes

Thus, the contribution of EBV and LMP1 to tumor development is apparently the continuous activation of pathways that can also be sporadically activated and contribute to tumor development.

T14524

37799-37982

Sentence

denotes

The lymphomas were marked by the upregulation of IL10, constitutive activation of Stat3 signaling, and a requirement for activation of Akt, NFκB, and Stat3 pathways (Figures 5 and 7).

T256

37799-37982

Sentence

denotes

The lymphomas were marked by the upregulation of IL10, constitutive activation of Stat3 signaling, and a requirement for activation of Akt, NFκB, and Stat3 pathways (Figures 5 and 7).

T14525

37983-38180

Sentence

denotes

Induction of IL10 is associated with the transformation of B-1 lymphomas in mice [59,60] and is frequently associated with EBV-positive B cell malignancies acting as a B cell growth factor [35–38].

T257

37983-38180

Sentence

denotes

Induction of IL10 is associated with the transformation of B-1 lymphomas in mice [59,60] and is frequently associated with EBV-positive B cell malignancies acting as a B cell growth factor [35–38].

T14526

38181-38282

Sentence

denotes

In addition, LMP1 has been shown to stimulate IL10 expression in Burkitt lymphoma cell lines [61,62].

T258

38181-38282

Sentence

denotes

In addition, LMP1 has been shown to stimulate IL10 expression in Burkitt lymphoma cell lines [61,62].

T14527

38283-38489

Sentence

denotes

This suggests that although Stat3 is constitutively activated in the lymphoma cells, the induction of IL10 may further enhance Stat3 activation or may contribute to other IL10-responsive signaling pathways.

T259

38283-38489

Sentence

denotes

This suggests that although Stat3 is constitutively activated in the lymphoma cells, the induction of IL10 may further enhance Stat3 activation or may contribute to other IL10-responsive signaling pathways.

T14528

38490-38703

Sentence

denotes

LMP1 activates both the canonical and non-canonical pathways of NFκB signaling [14,63–65], and inhibition of NFκB blocked the survival of LMP1 transgenic lymphocytes and LMP1-positive and -negative lymphoma cells.

T260

38490-38703

Sentence

denotes

LMP1 activates both the canonical and non-canonical pathways of NFκB signaling [14,63–65], and inhibition of NFκB blocked the survival of LMP1 transgenic lymphocytes and LMP1-positive and -negative lymphoma cells.

T14529

38704-38909

Sentence

denotes

NFκB and PI3K signaling are crucial for CD40-induced proliferation, and mice deficient for cRel or the p85 regulatory subunit of PI3K are unresponsive to mitogenic stimuli, including CD40 ligation [66–68].

T261

38704-38909

Sentence

denotes

NFκB and PI3K signaling are crucial for CD40-induced proliferation, and mice deficient for cRel or the p85 regulatory subunit of PI3K are unresponsive to mitogenic stimuli, including CD40 ligation [66–68].

T14530

38910-39101

Sentence

denotes

We have previously shown that cRel is specifically activated in both wild-type and LMP1 transgenic lymphomas, suggesting that activation of cRel is associated with B cell transformation [27].

T262

38910-39101

Sentence

denotes

We have previously shown that cRel is specifically activated in both wild-type and LMP1 transgenic lymphomas, suggesting that activation of cRel is associated with B cell transformation [27].

T14531

39102-39285

Sentence

denotes

Our observations suggest that similar to CD40-induced proliferation, LMP1 induces proliferation through PI3K-mediated activation of Akt and activation of NFκB components such as cRel.

T263

39102-39285

Sentence

denotes

Our observations suggest that similar to CD40-induced proliferation, LMP1 induces proliferation through PI3K-mediated activation of Akt and activation of NFκB components such as cRel.

T14532

39286-39508

Sentence

denotes

CD40 also induces downregulation of the cell cycle inhibitor p27 through a PI3K-dependent manner, and the LMP1 lymphoma cells also had decreased levels of p27 with phosphorylation of Rb and increased Cdk2 (Figure 6C) [66].

T264

39286-39508

Sentence

denotes

CD40 also induces downregulation of the cell cycle inhibitor p27 through a PI3K-dependent manner, and the LMP1 lymphoma cells also had decreased levels of p27 with phosphorylation of Rb and increased Cdk2 (Figure 6C) [66].

T14533

39509-39676

Sentence

denotes

Although LMP1 has been shown to deregulate the Rb pathway in epithelial cells [69], to our knowledge this is the first demonstration of this property in B lymphocytes.

T265

39509-39676

Sentence

denotes

Although LMP1 has been shown to deregulate the Rb pathway in epithelial cells [69], to our knowledge this is the first demonstration of this property in B lymphocytes.

T14534

39677-39808

Sentence

denotes

The requirement for Akt activation was confirmed by the striking inhibition of lymphoma viability by triciribine, an Akt inhibitor.

T266

39677-39808

Sentence

denotes

The requirement for Akt activation was confirmed by the striking inhibition of lymphoma viability by triciribine, an Akt inhibitor.

T14535

39809-39928

Sentence

denotes

However, the activated pAkt did not lead to phosphorylation and inactivation of the downstream target GSK3 (Figure 6A).

T267

39809-39928

Sentence

denotes

However, the activated pAkt did not lead to phosphorylation and inactivation of the downstream target GSK3 (Figure 6A).

T14536

39929-39998

Sentence

denotes

This effect has also been described in EBV-positive HD biopsies [40].

T268

39929-39998

Sentence

denotes

This effect has also been described in EBV-positive HD biopsies [40].

T14537

39999-40178

Sentence

denotes

In contrast, rapamycin, U0126, and SB202190 did not affect the survival of LMP1 transgenic lymphocytes or the wild-type and LMP1 transgenic lymphoma cells (Figure 7A, 7C, and 7E).

T269

39999-40178

Sentence

denotes

In contrast, rapamycin, U0126, and SB202190 did not affect the survival of LMP1 transgenic lymphocytes or the wild-type and LMP1 transgenic lymphoma cells (Figure 7A, 7C, and 7E).

T14538

40179-40273

Sentence

denotes

This lack of effect by rapamycin confirmed the absence of activated p-mTOR levels (Figure 6B).

T270

40179-40273

Sentence

denotes

This lack of effect by rapamycin confirmed the absence of activated p-mTOR levels (Figure 6B).

T14539

40274-40356

Sentence

denotes

These findings suggest that other Akt targets contribute to malignant progression.

T271

40274-40356

Sentence

denotes

These findings suggest that other Akt targets contribute to malignant progression.

T14540

40357-40432

Sentence

denotes

One key target is likely the inhibition of the Fox01 transcription factors.

T272

40357-40432

Sentence

denotes

One key target is likely the inhibition of the Fox01 transcription factors.

T14541

40433-40609

Sentence

denotes

Repression of the pro-apoptotic transcription factor FoxO1 in a PI3K-dependent manner can inhibit expression of bcl6, a transcription factor necessary for GC formation [49,70].

T273

40433-40609

Sentence

denotes

Repression of the pro-apoptotic transcription factor FoxO1 in a PI3K-dependent manner can inhibit expression of bcl6, a transcription factor necessary for GC formation [49,70].

T14542

40610-40720

Sentence

denotes

It has been shown that overstimulation of CD40 signaling with agonistic antibodies inhibits GC formation [71].

T274

40610-40720

Sentence

denotes

It has been shown that overstimulation of CD40 signaling with agonistic antibodies inhibits GC formation [71].

T14543

40721-40830

Sentence

denotes

Similarly, due to mimicry of CD40 signaling, transgenic LMP1 mice are also defective in GC formation [23,49].

T275

40721-40830

Sentence

denotes

Similarly, due to mimicry of CD40 signaling, transgenic LMP1 mice are also defective in GC formation [23,49].

T14544

40831-40924

Sentence

denotes

The constitutive signaling by LMP1 likely blocks GC formation through downregulation of bcl6.

T276

40831-40924

Sentence

denotes

The constitutive signaling by LMP1 likely blocks GC formation through downregulation of bcl6.

T14545

40925-41057

Sentence

denotes

Interestingly, clinical studies indicate that expression of LMP1 and bcl6 are mutually exclusive in non-HD and classical HD [72,73].

T277

40925-41057

Sentence

denotes

Interestingly, clinical studies indicate that expression of LMP1 and bcl6 are mutually exclusive in non-HD and classical HD [72,73].

T14546

41058-41127

Sentence

denotes

Thus, the LMP1 transgenic lymphomas mirror aspects of EBV-induced HD.

T278

41058-41127

Sentence

denotes

Thus, the LMP1 transgenic lymphomas mirror aspects of EBV-induced HD.

T14547

41128-41418

Sentence

denotes

Although the activation of Akt and the lack of Fox01 in the lymphoma cells suggest that LMP1 affects bcl6 and GC formation through this pathway, regulation of other Forkhead targets involved in cell cycle progression, such as p27 and CyclinD2, likely contribute to malignant transformation.

T279

41128-41418

Sentence

denotes

Although the activation of Akt and the lack of Fox01 in the lymphoma cells suggest that LMP1 affects bcl6 and GC formation through this pathway, regulation of other Forkhead targets involved in cell cycle progression, such as p27 and CyclinD2, likely contribute to malignant transformation.

T14548

41419-41519

Sentence

denotes

Indeed, loss of FoxO1 expression in lymphoma cells correlated with a loss of p27 (Figure 6B and 6C).

T280

41419-41519

Sentence

denotes

Indeed, loss of FoxO1 expression in lymphoma cells correlated with a loss of p27 (Figure 6B and 6C).

T14549

41520-41625

Sentence

denotes

CyclinD2 has also been shown to be upregulated by LMP1 through release of FoxO1-mediated repression [70].

T281

41520-41625

Sentence

denotes

CyclinD2 has also been shown to be upregulated by LMP1 through release of FoxO1-mediated repression [70].

T14550

41626-41785

Sentence

denotes

In summary, in this transgenic model of lymphomagenesis, LMP1 promotes malignancy in B-1a cells, a population that is predisposed to clonal expansion with age.

T282

41626-41785

Sentence

denotes

In summary, in this transgenic model of lymphomagenesis, LMP1 promotes malignancy in B-1a cells, a population that is predisposed to clonal expansion with age.

T14551

41786-42012

Sentence

denotes

The malignant lymphocytes were distinguished by constitutively active Stat3 signaling, decreased p27, and activated Akt and NFκB pathways, properties that are associated with promoting the growth and survival of B lymphocytes.

T283

41786-42012

Sentence

denotes

The malignant lymphocytes were distinguished by constitutively active Stat3 signaling, decreased p27, and activated Akt and NFκB pathways, properties that are associated with promoting the growth and survival of B lymphocytes.

T14552

42013-42181

Sentence

denotes

Importantly, Akt, NFκB, and Stat3 pathways were critically required for the growth and survival of malignant lymphocytes as well as healthy LMP1 transgenic lymphocytes.

T284

42013-42181

Sentence

denotes

Importantly, Akt, NFκB, and Stat3 pathways were critically required for the growth and survival of malignant lymphocytes as well as healthy LMP1 transgenic lymphocytes.

T14553

42182-42351

Sentence

denotes

The growth of EBV-transformed lymphocytes also requires activation of NFκB, and these studies provide insight into how LMP1 contributes to EBV-associated transformation.

T285

42182-42351

Sentence

denotes

The growth of EBV-transformed lymphocytes also requires activation of NFκB, and these studies provide insight into how LMP1 contributes to EBV-associated transformation.

T14554

42352-42520

Sentence

denotes

The transgenic lymphomas mirror multiple aspects of EBV-induced tumors and suggest that in vivo these properties of LMP1 are major factors in the development of cancer.

T286

42352-42520

Sentence

denotes

The transgenic lymphomas mirror multiple aspects of EBV-induced tumors and suggest that in vivo these properties of LMP1 are major factors in the development of cancer.

T287

42522-42543

Sentence

denotes

Materials and Methods

T18288

42545-42561

Sentence

denotes

Transgenic mice.

T288

42545-42561

Sentence

denotes

Transgenic mice.

T18289

42562-42728

Sentence

denotes

Generation of LMP1 mice under the Ig heavy chain promoter and enhancer have been described previously and were maintained as heterozygotes on a Balbc background [26].

T289

42562-42728

Sentence

denotes

Generation of LMP1 mice under the Ig heavy chain promoter and enhancer have been described previously and were maintained as heterozygotes on a Balbc background [26].

T18290

42729-42829

Sentence

denotes

LMP1 mice were genotyped by Southern blot and PCR analysis of tail DNA as described previously [26].

T290

42729-42829

Sentence

denotes

LMP1 mice were genotyped by Southern blot and PCR analysis of tail DNA as described previously [26].

T18291

42830-43005

Sentence

denotes

Spleen and liver sections were fixed in 4% paraformaldehyde and embedded in paraffin, and 5-μm sections were stained with hematoxylin and eosin for histopathological analysis.

T291

42830-43005

Sentence

denotes

Spleen and liver sections were fixed in 4% paraformaldehyde and embedded in paraffin, and 5-μm sections were stained with hematoxylin and eosin for histopathological analysis.

T18292

43006-43152

Sentence

denotes

Lymphomas were passaged by intraperitoneal injection of 1 × 108 splenocytes into SCID mice and sacrificed upon development of an extended abdomen.

T292

43006-43152

Sentence

denotes

Lymphomas were passaged by intraperitoneal injection of 1 × 108 splenocytes into SCID mice and sacrificed upon development of an extended abdomen.

T293

43153-43317

Sentence

denotes

Animals were housed in the Association for Assessment and Accreditation for Animal Care–approved animal facility at the University of North Carolina at Chapel Hill.

T18293

43153-43397

Sentence

denotes

Animals were housed in the Association for Assessment and Accreditation for Animal Care–approved animal facility at the University of North Carolina at Chapel Hill. All protocols were approved by the Institutional Animal Care and Use Committee.

T294

43318-43397

Sentence

denotes

All protocols were approved by the Institutional Animal Care and Use Committee.

T18609

43399-43431

Sentence

denotes

Isolation and growth of B cells.

T295

43399-43431

Sentence

denotes

Isolation and growth of B cells.

T18610

43432-43567

Sentence

denotes

Splenocytes were prepared by homogenizing spleen tissue with two frosted slides and debris was filtered through a 100-μm cell strainer.

T296

43432-43567

Sentence

denotes

Splenocytes were prepared by homogenizing spleen tissue with two frosted slides and debris was filtered through a 100-μm cell strainer.

T18611

43568-43698

Sentence

denotes

Erythrocytes were lysed using 0.8% ammonium chloride solution (StemCell Technologies) for 10 min on ice and washed twice with PBS.

T297

43568-43698

Sentence

denotes

Erythrocytes were lysed using 0.8% ammonium chloride solution (StemCell Technologies) for 10 min on ice and washed twice with PBS.

T18612

43699-43928

Sentence

denotes

B cells were isolated using CD19-MACS beads according to the manufacturer's instructions (Miltenyi Biotec) and grown in Iscove's medium supplemented with heat-inactivated 10% fetal bovine serum and antibiotic/antimycotic (GIBCO).

T298

43699-43928

Sentence

denotes

B cells were isolated using CD19-MACS beads according to the manufacturer's instructions (Miltenyi Biotec) and grown in Iscove's medium supplemented with heat-inactivated 10% fetal bovine serum and antibiotic/antimycotic (GIBCO).

T18613

43929-44098

Sentence

denotes

Splenocytes isolated from SCID-passaged lymphomas consisted of 80%–90% B cells as determined by flow cytometry, and were hence not further purified with CD19-MACS beads.

T299

43929-44098

Sentence

denotes

Splenocytes isolated from SCID-passaged lymphomas consisted of 80%–90% B cells as determined by flow cytometry, and were hence not further purified with CD19-MACS beads.

T18614

44099-44334

Sentence

denotes

Splenocytes were seeded at 1.25 × 106 cells/ml and where applicable, recombinant mouse IL4 was added at 100 ng/ml, recombinant mouse IL10 at 10 ng/ml, and rat IgG1 anti-mouse IL10 and rat IgG1 isotype control at 10 μg/ml (R&D Systems).

T300

44099-44334

Sentence

denotes

Splenocytes were seeded at 1.25 × 106 cells/ml and where applicable, recombinant mouse IL4 was added at 100 ng/ml, recombinant mouse IL10 at 10 ng/ml, and rat IgG1 anti-mouse IL10 and rat IgG1 isotype control at 10 μg/ml (R&D Systems).

T18615

44335-44432

Sentence

denotes

For BrdU incorporation assays, splenocytes were pulsed for 24 h with 10 μM BrdU 1 d post-harvest.

T301

44335-44432

Sentence

denotes

For BrdU incorporation assays, splenocytes were pulsed for 24 h with 10 μM BrdU 1 d post-harvest.

T19038

44434-44444

Sentence

denotes

MTS assay.

T302

44434-44444

Sentence

denotes

MTS assay.

T19039

44445-44731

Sentence

denotes

The 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) cell cytotoxicity/proliferation assays were performed using the CellTiter 96 aqueous one-solution cell proliferation assay (Promega), according to manufacturer's instructions.

T303

44445-44731

Sentence

denotes

The 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) cell cytotoxicity/proliferation assays were performed using the CellTiter 96 aqueous one-solution cell proliferation assay (Promega), according to manufacturer's instructions.

T19040

44732-44827

Sentence

denotes

For IL4 studies, splenocytes were cultured for 3 d in the presence or absence of 100 ng/ml IL4.

T304

44732-44827

Sentence

denotes

For IL4 studies, splenocytes were cultured for 3 d in the presence or absence of 100 ng/ml IL4.

T19041

44828-44929

Sentence

denotes

Cells were seeded on day 3 in triplicate in a 96-well plate at 2.5 × 106 cells/ml at 100 μl per well.

T305

44828-44929

Sentence

denotes

Cells were seeded on day 3 in triplicate in a 96-well plate at 2.5 × 106 cells/ml at 100 μl per well.

T19042

44930-45038

Sentence

denotes

MTS reagent was added for 4 h and absorbance was read at 540 nm; values plotted were subtracted from blanks.

T306

44930-45038

Sentence

denotes

MTS reagent was added for 4 h and absorbance was read at 540 nm; values plotted were subtracted from blanks.

T19043

45039-45281

Sentence

denotes

For neutralization assays, splenocytes were seeded at 5 × 106 cells/ml at 100 μl per well on day of harvest and IL4, rat IgG1 anti-mouse IL4, or rat IgG1 isotype control (R&D Systems) were added at the concentrations indicated in the figures.

T307

45039-45281

Sentence

denotes

For neutralization assays, splenocytes were seeded at 5 × 106 cells/ml at 100 μl per well on day of harvest and IL4, rat IgG1 anti-mouse IL4, or rat IgG1 isotype control (R&D Systems) were added at the concentrations indicated in the figures.

T19044

45282-45345

Sentence

denotes

Cultures were pulsed for 4 h with MTS reagent 1 d post-seeding.

T308

45282-45345

Sentence

denotes

Cultures were pulsed for 4 h with MTS reagent 1 d post-seeding.

T19045

45346-45524

Sentence

denotes

For inhibitor studies, splenocytes were seeded at 1 × 107 cells/ml at 100 μl per well on day of harvest, and inhibitors were added at the concentrations indicated in the figures.

T309

45346-45524

Sentence

denotes

For inhibitor studies, splenocytes were seeded at 1 × 107 cells/ml at 100 μl per well on day of harvest, and inhibitors were added at the concentrations indicated in the figures.

T310

45525-45648

Sentence

denotes

The inhibitors BAY11–7085, rapamycin, triciribine, U0126, SB202190, and cucurbitacin I were purchased from EMD Biosciences.

T19046

45525-45787

Sentence

denotes

The inhibitors BAY11–7085, rapamycin, triciribine, U0126, SB202190, and cucurbitacin I were purchased from EMD Biosciences. For non-malignant splenocyte cultures, B cell activation was induced with 10 μg/ml of goat F(ab') anti-mouse IgM (Jackson ImmunoResearch).

T311

45649-45787

Sentence

denotes

For non-malignant splenocyte cultures, B cell activation was induced with 10 μg/ml of goat F(ab') anti-mouse IgM (Jackson ImmunoResearch).

T19047

45788-45851

Sentence

denotes

Cultures were pulsed for 4 h with MTS reagent 1 d post-seeding.

T312

45788-45851

Sentence

denotes

Cultures were pulsed for 4 h with MTS reagent 1 d post-seeding.

T19642

45853-45874

Sentence

denotes

Immunohistochemistry.

T313

45853-45874

Sentence

denotes

Immunohistochemistry.

T19643

45875-45999

Sentence

denotes

Paraffin-embedded spleen sections were deparaffinized in Histoclear (National Diagnostics) and rehydrated in graded ethanol.

T314

45875-45999

Sentence

denotes

Paraffin-embedded spleen sections were deparaffinized in Histoclear (National Diagnostics) and rehydrated in graded ethanol.

T19644

46000-46130

Sentence

denotes

Sections were antigen retrieved by microwaving in citrate buffer (pH 6.0) for 15 min (LMP1 staining) and 10 min (pStat3 staining).

T315

46000-46130

Sentence

denotes

Sections were antigen retrieved by microwaving in citrate buffer (pH 6.0) for 15 min (LMP1 staining) and 10 min (pStat3 staining).

T19645

46131-46270

Sentence

denotes

For LMP1 staining, sections were blocked with 1% BSA and 0.1% cold fish skin gelatin followed with streptavidin/biotin block (Vector Labs).

T316

46131-46270

Sentence

denotes

For LMP1 staining, sections were blocked with 1% BSA and 0.1% cold fish skin gelatin followed with streptavidin/biotin block (Vector Labs).

T19646

46271-46582

Sentence

denotes

Rat IgG anti-LMP1 (clones 8G3 and 1G6, Ascenion) were used at 1:10 dilution from tissue culture supernatants, followed with 8 μg/ml biotinylated mouse F(ab') anti-rat IgG (H+L) pre-adsorbed to mouse serum (Jackson ImmunoResearch) and 2 μg/ml streptavidin-alkaline phosphatase conjugate (Jackson ImmunoResearch).

T317

46271-46582

Sentence

denotes

Rat IgG anti-LMP1 (clones 8G3 and 1G6, Ascenion) were used at 1:10 dilution from tissue culture supernatants, followed with 8 μg/ml biotinylated mouse F(ab') anti-rat IgG (H+L) pre-adsorbed to mouse serum (Jackson ImmunoResearch) and 2 μg/ml streptavidin-alkaline phosphatase conjugate (Jackson ImmunoResearch).

T19647

46583-46665

Sentence

denotes

Stains were developed with BCIP/NBT and counterstained in Nuclear Fast Red (Dako).

T318

46583-46665

Sentence

denotes

Stains were developed with BCIP/NBT and counterstained in Nuclear Fast Red (Dako).

T19648

46666-46818

Sentence

denotes

Phospho-Stat3 was detected with 4 μg/ml of pStat3 antibody (Tyr705, Cell Signaling) and detected with anti-rabbit Poly-HRP IHC detection kit (Chemicon).

T319

46666-46818

Sentence

denotes

Phospho-Stat3 was detected with 4 μg/ml of pStat3 antibody (Tyr705, Cell Signaling) and detected with anti-rabbit Poly-HRP IHC detection kit (Chemicon).

T20038

46820-46835

Sentence

denotes

Flow cytometry.

T320

46820-46835

Sentence

denotes

Flow cytometry.

T20039

46836-47000

Sentence

denotes

One million splenocytes were stained with the appropriate primary antibody unconjugated or conjugated to FITC, PE, or APC diluted in stain buffer (PBS with 3% FBS).

T321

46836-47000

Sentence

denotes

One million splenocytes were stained with the appropriate primary antibody unconjugated or conjugated to FITC, PE, or APC diluted in stain buffer (PBS with 3% FBS).

T20040

47001-47103

Sentence

denotes

For BrdU detection, the FITC-BrdU flow kit was used as instructed by the manufacturer (BD Bioscience).

T322

47001-47103

Sentence

denotes

For BrdU detection, the FITC-BrdU flow kit was used as instructed by the manufacturer (BD Bioscience).

T20041

47104-47275

Sentence

denotes

Briefly, cells were exposed to EMA (Molecular Probes) for exclusion of dead cells, stained for surface antigens, fixed in paraformaldehyde, and permeabilized with saponin.

T323

47104-47275

Sentence

denotes

Briefly, cells were exposed to EMA (Molecular Probes) for exclusion of dead cells, stained for surface antigens, fixed in paraformaldehyde, and permeabilized with saponin.

T20042

47276-47383

Sentence

denotes

To expose BrdU epitopes, cells were treated with Dnase and stained with FITC-conjugated anti-BrdU antibody.

T324

47276-47383

Sentence

denotes

To expose BrdU epitopes, cells were treated with Dnase and stained with FITC-conjugated anti-BrdU antibody.

T20043

47384-47475

Sentence

denotes

Flow cytometry was performed on FACScalibur using the CellQuest program (Becton Dickinson).

T325

47384-47475

Sentence

denotes

Flow cytometry was performed on FACScalibur using the CellQuest program (Becton Dickinson).

T20044

47476-47541

Sentence

denotes

Further analysis was conducted on the Summit v4.2 program (Dako).

T326

47476-47541

Sentence

denotes

Further analysis was conducted on the Summit v4.2 program (Dako).

T20334

47543-47566

Sentence

denotes

Rnase protection assay.

T327

47543-47566

Sentence

denotes

Rnase protection assay.

T20335

47567-47738

Sentence

denotes

Total RNA was isolated from CD19+ MACS-purified B cells using the Rneasy midi purification kit with Dnase treatment, according to the manufacturer's instructions (Qiagen).

T328

47567-47738

Sentence

denotes

Total RNA was isolated from CD19+ MACS-purified B cells using the Rneasy midi purification kit with Dnase treatment, according to the manufacturer's instructions (Qiagen).

T20336

47739-47896

Sentence

denotes

The mCK-1 probe template set (BD Biosciences) was labeled using the In Vitro Transcription Kit according to the manufacturer's instructions (BD Biosciences).

T329

47739-47896

Sentence

denotes

The mCK-1 probe template set (BD Biosciences) was labeled using the In Vitro Transcription Kit according to the manufacturer's instructions (BD Biosciences).

T20337

47897-48054

Sentence

denotes

Briefly, 50 ng of mCK-1 probe set was labeled with [α-32P]UTP using T7 RNA polymerase and purified using Sephadex G-50 columns (NucAway Spin column, Ambion).

T330

47897-48054

Sentence

denotes

Briefly, 50 ng of mCK-1 probe set was labeled with [α-32P]UTP using T7 RNA polymerase and purified using Sephadex G-50 columns (NucAway Spin column, Ambion).

T20338

48055-48224

Sentence

denotes

The labeled probe was quantitated using a scintillation counter, and 6 × 105 cherenkov cpm was used to hybridize to 4 μg of total RNA using the RPA kit (BD Biosciences).

T331

48055-48224

Sentence

denotes

The labeled probe was quantitated using a scintillation counter, and 6 × 105 cherenkov cpm was used to hybridize to 4 μg of total RNA using the RPA kit (BD Biosciences).

T20339

48225-48291

Sentence

denotes

Samples were denatured at 90 °C and hybridized at 56 °C overnight.

T332

48225-48291

Sentence

denotes

Samples were denatured at 90 °C and hybridized at 56 °C overnight.

T20340

48292-48394

Sentence

denotes

Single-stranded RNA was digested with a mixture of Rnase A and T1, and precipitated using isopropanol.

T333

48292-48394

Sentence

denotes

Single-stranded RNA was digested with a mixture of Rnase A and T1, and precipitated using isopropanol.

T20341

48395-48526

Sentence

denotes

Protected probes were resolved on a denaturing 4.75% acrylamide gel, dried, and imaged using a phosphorImager (Molecular Dynamics).

T334

48395-48526

Sentence

denotes

Protected probes were resolved on a denaturing 4.75% acrylamide gel, dried, and imaged using a phosphorImager (Molecular Dynamics).

T20342

48527-48609

Sentence

denotes

Densitometry was performed using the ImageQuant TL v2005 software (GE Healthcare).

T335

48527-48609

Sentence

denotes

Densitometry was performed using the ImageQuant TL v2005 software (GE Healthcare).

T20768

48611-48649

Sentence

denotes

PCR analysis of κ chain rearrangement.

T336

48611-48649

Sentence

denotes

PCR analysis of κ chain rearrangement.

T20769

48650-48743

Sentence

denotes

DNA was isolated from splenocytes using the Dneasy Tissue Kit (Qiagen), with Rnase treatment.

T337

48650-48743

Sentence

denotes

DNA was isolated from splenocytes using the Dneasy Tissue Kit (Qiagen), with Rnase treatment.

T20770

48744-48899

Sentence

denotes

PCR reactions contained 100 ng of genomic DNA, 0.2 μM each primer, 0.2 mM dNTPs, and 2.5 U Taq DNA polymerase (NEB) performed in 1X ThermoPol buffer (NEB).

T338

48744-48899

Sentence

denotes

PCR reactions contained 100 ng of genomic DNA, 0.2 μM each primer, 0.2 mM dNTPs, and 2.5 U Taq DNA polymerase (NEB) performed in 1X ThermoPol buffer (NEB).

T20771

48900-48975

Sentence

denotes

Primers used were Vκcon and Jκ5–1° and have been described previously [74].

T339

48900-48975

Sentence

denotes

Primers used were Vκcon and Jκ5–1° and have been described previously [74].

T20772

48976-49118

Sentence

denotes

PCR conditions were 94 °C for 2 min, 40 cycles of 94 °C for 30 s, 63 °C for 90 s, and 72 °C for 1 min, followed by 1 cycle of 72 °C for 5 min.

T340

48976-49118

Sentence

denotes

PCR conditions were 94 °C for 2 min, 40 cycles of 94 °C for 30 s, 63 °C for 90 s, and 72 °C for 1 min, followed by 1 cycle of 72 °C for 5 min.

T21027

49120-49140

Sentence

denotes

Immunoblot analysis.

T341

49120-49140

Sentence

denotes

Immunoblot analysis.

T21028

49141-49444

Sentence

denotes

Whole cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate) supplemented with 2 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1:100 protease/phosphatase inhibitor cocktails (Sigma).

T342

49141-49444

Sentence

denotes

Whole cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate) supplemented with 2 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1:100 protease/phosphatase inhibitor cocktails (Sigma).

T21029

49445-49566

Sentence

denotes

Crude lysates were centrifuged at 13,000 rpm for 10 min at 4 °C and the supernatants were collected for further analysis.

T343

49445-49566

Sentence

denotes

Crude lysates were centrifuged at 13,000 rpm for 10 min at 4 °C and the supernatants were collected for further analysis.

T21030

49567-49647

Sentence

denotes

Protein concentrations were determined with the Bio-Rad DC protein assay system.

T344

49567-49647

Sentence

denotes

Protein concentrations were determined with the Bio-Rad DC protein assay system.

T21031

49648-49840

Sentence

denotes

Lysates were boiled in the presence of 2.5% β-mecaptoethanol, separated by denaturing SDS-PAGE, and transferred to 0.45-μm Optitran membranes (Schleicher & Schuell) in a Bio-Rad transfer unit.

T345

49648-49840

Sentence

denotes

Lysates were boiled in the presence of 2.5% β-mecaptoethanol, separated by denaturing SDS-PAGE, and transferred to 0.45-μm Optitran membranes (Schleicher & Schuell) in a Bio-Rad transfer unit.

T21032

49841-50063

Sentence

denotes

Membranes were immunoblotted with the appropriate primary antibody followed by horseradish peroxidase–tagged secondary antibodies (Amersham Biosciences and Dako) and detected with the SuperSignal West Pico System (Pierce).

T346

49841-50063

Sentence

denotes

Membranes were immunoblotted with the appropriate primary antibody followed by horseradish peroxidase–tagged secondary antibodies (Amersham Biosciences and Dako) and detected with the SuperSignal West Pico System (Pierce).

T21448

50065-50076

Sentence

denotes

Antibodies.

T347

50065-50076

Sentence

denotes

Antibodies.

T348

50077-50371

Sentence

denotes

FITC-conjugated goat anti-mouse IgM, rat IgG2aκ anti-mouse IgD (clone 11–26), rat IgG1κ anti-mouse kappa (clone 187.1), rat IgG2bκ anti-mouse lambda (clone JC5–1); PE-conjugated goat anti-mouse IgG; un-conjugated goat anti-mouse kappa and anti-mouse lambda were purchased from Southern Biotech.

T21449

50077-50668

Sentence

denotes

FITC-conjugated goat anti-mouse IgM, rat IgG2aκ anti-mouse IgD (clone 11–26), rat IgG1κ anti-mouse kappa (clone 187.1), rat IgG2bκ anti-mouse lambda (clone JC5–1); PE-conjugated goat anti-mouse IgG; un-conjugated goat anti-mouse kappa and anti-mouse lambda were purchased from Southern Biotech. APC-conjugated rat IgG2aκ anti-mouse CD19 (clone 6D5), PE-conjugated mouse IgG2aκ anti-LMP1 (clone S12), un-conjugated mouse IgG2a anti-Rb (clone 2), and anti-Cdk2 (clone 55) were purchased from BD Bioscience. PE-conjugated rat IgG2aκ anti-mouse CD5 (clone 53–7.3) was purchased from eBioscience.

T349

50372-50581

Sentence

denotes

APC-conjugated rat IgG2aκ anti-mouse CD19 (clone 6D5), PE-conjugated mouse IgG2aκ anti-LMP1 (clone S12), un-conjugated mouse IgG2a anti-Rb (clone 2), and anti-Cdk2 (clone 55) were purchased from BD Bioscience.

T350

50582-50668

Sentence

denotes

PE-conjugated rat IgG2aκ anti-mouse CD5 (clone 53–7.3) was purchased from eBioscience.

T351

50669-50744

Sentence

denotes

Rat anti-LMP1 (clones 8G3, 1G6, 7E10, and 7G8) was purchased from Ascenion.

T21450

50669-51190

Sentence

denotes

Rat anti-LMP1 (clones 8G3, 1G6, 7E10, and 7G8) was purchased from Ascenion. Rabbit anti-pAkt (Ser473), anti-pGSK3α/β (Ser21/9), anti-pStat3 (Tyr705), anti-pStat6 (Tyr641), anti-pmTOR (Ser2448), anti-Stat6, anti-Akt, anti-FoxO1, and anti-p27 were purchased from Cell Signaling. Rabbit anti-Stat3 (H-190), anti-IκBα (C-21), and goat anti-β actin (I-19) were purchased from Santa Cruz Biotechnology. Mouse IgG1κ anti-GSK3 was purchased from Upstate Biotechnology. Rabbit anti-pRb (Thr373) was purchased from EMD Biosciences.

T352

50745-50945

Sentence

denotes

Rabbit anti-pAkt (Ser473), anti-pGSK3α/β (Ser21/9), anti-pStat3 (Tyr705), anti-pStat6 (Tyr641), anti-pmTOR (Ser2448), anti-Stat6, anti-Akt, anti-FoxO1, and anti-p27 were purchased from Cell Signaling.

T353

50946-51065

Sentence

denotes

Rabbit anti-Stat3 (H-190), anti-IκBα (C-21), and goat anti-β actin (I-19) were purchased from Santa Cruz Biotechnology.

T354

51066-51129

Sentence

denotes

Mouse IgG1κ anti-GSK3 was purchased from Upstate Biotechnology.

T355

51130-51190

Sentence

denotes

Rabbit anti-pRb (Thr373) was purchased from EMD Biosciences.

T356

51192-51214

Sentence

denotes

Supporting Information

T357

51215-51277

Sentence

denotes

Figure S1 Lymphomas Are Clonal by κ Light Chain Rearrangement

T358

51278-51341

Sentence

denotes

PCR analysis of total spleen genomic DNA for the mouse κ locus.

T359

51342-51432

Sentence

denotes

Primers were designed to amplify all V-J rearrangements, including VJ1, VJ2, VJ4, and VJ5.

T360

51433-51547

Sentence

denotes

Specific PCR products for the four rearrangements corresponding to the calculated sizes are indicated with arrows.

T361

51548-51602

Sentence

denotes

Bands of other sizes are due to non-specific products.

T362

51603-51733

Sentence

denotes

If a tumor was present in the background of normal spleen cells, a single band appears brighter than all the other specific bands.

T363

51734-51852

Sentence

denotes

Wild-type and LMP1 transgenic lymphocytes are positive for all four V-J rearrangements and serve as positive controls.

T364

51853-51968

Sentence

denotes

Clonality is determined by the appearance of a predominant band at the correct size for a single V-J rearrangement.

T365

51969-52018

Sentence

denotes

(739 KB PDF) Click here for additional data file.

T366

52020-52037

Sentence

denotes

Accession Numbers

T367

52038-52146

Sentence

denotes

The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession number for the EBV genome sequence is AJ507799.

T368

52147-52185

Sentence

denotes

The gene identifier for LMP1 is BNLF1.

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