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PMC:1310901 / 13049-21662 JSONTXT 20 Projects

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Id Subject Object Predicate Lexical cue
T100 0-7 Sentence denotes RESULTS
T6214 9-89 Sentence denotes Absence of IRF-4 expression in leukemia cells is not due to promoter alterations
T101 9-89 Sentence denotes Absence of IRF-4 expression in leukemia cells is not due to promoter alterations
T6215 90-206 Sentence denotes We have previously demonstrated a lack of IRF-4 expression in leukemia patients and specifically in CML T-cells (3).
T102 90-206 Sentence denotes We have previously demonstrated a lack of IRF-4 expression in leukemia patients and specifically in CML T-cells (3).
T6216 207-666 Sentence denotes Here, we demonstrate the absence of IRF-4 expression in various hematopoietic cell lines, such as Jurkat, a T-cell leukemia, CML-T1, a bcr-abl-positive T-cell line, K-562, a bcr-abl-positve erythroleukemia, U-937, a monocytic leukemia, EM-2 and LAMA-84, bcr-abl-positve myeloid leukemia, but not in SD-1, a bcr-abl-positive acute lymphoblastic leukemia (pre B-ALL), RPMI-8226, a multiple myeloma and BV-173, a bcr-abl-positive B-cell line (Figures 1A and 5D).
T103 207-666 Sentence denotes Here, we demonstrate the absence of IRF-4 expression in various hematopoietic cell lines, such as Jurkat, a T-cell leukemia, CML-T1, a bcr-abl-positive T-cell line, K-562, a bcr-abl-positve erythroleukemia, U-937, a monocytic leukemia, EM-2 and LAMA-84, bcr-abl-positve myeloid leukemia, but not in SD-1, a bcr-abl-positive acute lymphoblastic leukemia (pre B-ALL), RPMI-8226, a multiple myeloma and BV-173, a bcr-abl-positive B-cell line (Figures 1A and 5D).
T6217 667-823 Sentence denotes After sequencing of the IRF-4 promoter, it could be excluded that absence of IRF-4 expression in any of the above cell lines was due to genetic aberrations.
T104 667-823 Sentence denotes After sequencing of the IRF-4 promoter, it could be excluded that absence of IRF-4 expression in any of the above cell lines was due to genetic aberrations.
T6218 824-996 Sentence denotes However, 2 bp changes (nucleotide −1081, T→C and −1068, A→C) could be detected in both the IRF-4-positive BV-173 and the IRF-4-negative LAMA-84, EM-2 and K-562 (Figure 1B).
T105 824-996 Sentence denotes However, 2 bp changes (nucleotide −1081, T→C and −1068, A→C) could be detected in both the IRF-4-positive BV-173 and the IRF-4-negative LAMA-84, EM-2 and K-562 (Figure 1B).
T6219 997-1202 Sentence denotes At position −116 an A→C substitution was found in EM-2, K-562 and CML-T1, whereas Jurkat, BV-173 and SD-1 exhibited a mixed A/C sequence and U-937, LAMA-84 and RPMI-8226 no substitution at all (Figure 1B).
T106 997-1202 Sentence denotes At position −116 an A→C substitution was found in EM-2, K-562 and CML-T1, whereas Jurkat, BV-173 and SD-1 exhibited a mixed A/C sequence and U-937, LAMA-84 and RPMI-8226 no substitution at all (Figure 1B).
T6220 1203-1275 Sentence denotes Consequently, these alterations are unlikely to affect IRF-4 expression.
T107 1203-1275 Sentence denotes Consequently, these alterations are unlikely to affect IRF-4 expression.
T7004 1277-1358 Sentence denotes Increase of IRF-4 expression in hematopoietic cells after demethylating treatment
T108 1277-1358 Sentence denotes Increase of IRF-4 expression in hematopoietic cells after demethylating treatment
T7005 1359-1466 Sentence denotes We next analyzed whether promoter methylation could be responsible for down-regulation of IRF-4 expression.
T109 1359-1466 Sentence denotes We next analyzed whether promoter methylation could be responsible for down-regulation of IRF-4 expression.
T7006 1467-1573 Sentence denotes A region including exon1 in the IRF-4 promoter exhibited a large number of CpG-rich sequences (Figure 3A).
T110 1467-1573 Sentence denotes A region including exon1 in the IRF-4 promoter exhibited a large number of CpG-rich sequences (Figure 3A).
T7007 1574-1770 Sentence denotes Several chemical substances such as 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) inhibit de novo and maintenance methylation, and thus can be used to discern promoter methylation (32,33).
T111 1574-1770 Sentence denotes Several chemical substances such as 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) inhibit de novo and maintenance methylation, and thus can be used to discern promoter methylation (32,33).
T7008 1771-1814 Sentence denotes We used AzadC to generate unmethylated DNA.
T112 1771-1814 Sentence denotes We used AzadC to generate unmethylated DNA.
T7009 1815-1998 Sentence denotes A 72 h AzadC-treatment resulted in a concentration-dependent activation of IRF-4 transcription in Jurkat and CML-T1 T-cells as well as in U-937, K-562 and EM-2 cell lines (Figure 2A).
T113 1815-1998 Sentence denotes A 72 h AzadC-treatment resulted in a concentration-dependent activation of IRF-4 transcription in Jurkat and CML-T1 T-cells as well as in U-937, K-562 and EM-2 cell lines (Figure 2A).
T7010 1999-2166 Sentence denotes IRF-4 transcription was induced in a time-dependent manner and was observed as early as 24 h after treatment with AzadC and increased over time until 72 h (Figure 2B).
T114 1999-2166 Sentence denotes IRF-4 transcription was induced in a time-dependent manner and was observed as early as 24 h after treatment with AzadC and increased over time until 72 h (Figure 2B).
T7011 2167-2317 Sentence denotes Time and strength of the appearance of IRF-4 transcripts varied among cell lines, i.e. CML-T1 responded strongest to AzadC-treatment (data not shown).
T115 2167-2317 Sentence denotes Time and strength of the appearance of IRF-4 transcripts varied among cell lines, i.e. CML-T1 responded strongest to AzadC-treatment (data not shown).
T7012 2318-2453 Sentence denotes In line with this, AzadC-treatment of CML-T1 and LAMA-84 cells also translated in an induction of IRF-4 protein expression (Figure 2C).
T116 2318-2453 Sentence denotes In line with this, AzadC-treatment of CML-T1 and LAMA-84 cells also translated in an induction of IRF-4 protein expression (Figure 2C).
T7013 2454-2593 Sentence denotes Accordingly, treatment of the IRF-4-positive cell line BV-173, SD-1 and RPMI-8226 with AzadC had no effect on IRF-4 expression (Figure 2D).
T117 2454-2593 Sentence denotes Accordingly, treatment of the IRF-4-positive cell line BV-173, SD-1 and RPMI-8226 with AzadC had no effect on IRF-4 expression (Figure 2D).
T7014 2594-2735 Sentence denotes There was no difference in the effects of AzaC versus AzadC, as both increased the IRF-4 mRNA level in CML-T1 cells as well (data not shown).
T118 2594-2735 Sentence denotes There was no difference in the effects of AzaC versus AzadC, as both increased the IRF-4 mRNA level in CML-T1 cells as well (data not shown).
T7015 2736-2921 Sentence denotes This implied that promoter methylation may control IRF-4 expression, but an alternative explanation may be activation of positive transcriptional regulators of IRF-4 by AzadC (or AzaC).
T119 2736-2921 Sentence denotes This implied that promoter methylation may control IRF-4 expression, but an alternative explanation may be activation of positive transcriptional regulators of IRF-4 by AzadC (or AzaC).
T7831 2923-3023 Sentence denotes Methylation-sensitive enzymes do not cut specific sites in the IRF-4 promoter in hematopoietic cells
T120 2923-3023 Sentence denotes Methylation-sensitive enzymes do not cut specific sites in the IRF-4 promoter in hematopoietic cells
T7832 3024-3263 Sentence denotes To further investigate promoter methylation as a regulatory mechanism of IRF-4 gene expression, restriction-PCR-assays were performed (20,24), where only methylated DNA would not be cut enabling subsequent PCR amplification and vice versa.
T121 3024-3263 Sentence denotes To further investigate promoter methylation as a regulatory mechanism of IRF-4 gene expression, restriction-PCR-assays were performed (20,24), where only methylated DNA would not be cut enabling subsequent PCR amplification and vice versa.
T7833 3264-3441 Sentence denotes Genomic DNA from leukemic cells Jurkat, CML-T1, U-937, K-562, EM-2 and BV-173 was digested with the methylation-sensitive enzymes HpaII, Bsh1236I and HaeII-isochizomer Bsp143II.
T122 3264-3441 Sentence denotes Genomic DNA from leukemic cells Jurkat, CML-T1, U-937, K-562, EM-2 and BV-173 was digested with the methylation-sensitive enzymes HpaII, Bsh1236I and HaeII-isochizomer Bsp143II.
T7834 3442-3567 Sentence denotes EcoRI, which has no recognition site within the IRF-4 promoter, and the methylation-resistant enzyme MspI served as controls.
T123 3442-3567 Sentence denotes EcoRI, which has no recognition site within the IRF-4 promoter, and the methylation-resistant enzyme MspI served as controls.
T7835 3568-3669 Sentence denotes Two separate amplification reactions were performed, generating two fragments, F1 and F2 (Figure 3A).
T124 3568-3669 Sentence denotes Two separate amplification reactions were performed, generating two fragments, F1 and F2 (Figure 3A).
T7836 3670-3956 Sentence denotes After digestion with HpaII and Bsp143II a sufficient PCR amplification of F1 and F2 was detected in DNA from IRF-4-negative Jurkat, CML-T1, U-937, K-562 and EM-2 cells, suggesting a promoter methylation (and restriction protection) at the respective recognition sites (Figure 3B and C).
T125 3670-3956 Sentence denotes After digestion with HpaII and Bsp143II a sufficient PCR amplification of F1 and F2 was detected in DNA from IRF-4-negative Jurkat, CML-T1, U-937, K-562 and EM-2 cells, suggesting a promoter methylation (and restriction protection) at the respective recognition sites (Figure 3B and C).
T7837 3957-4092 Sentence denotes Notably, in IRF-4-positive SD-1 cells digestion with the methylation-sensitive enzymes completely inhibited amplification of F1 and F2.
T126 3957-4092 Sentence denotes Notably, in IRF-4-positive SD-1 cells digestion with the methylation-sensitive enzymes completely inhibited amplification of F1 and F2.
T7838 4093-4289 Sentence denotes In IRF-4-positive BV-173 cells a HpaII, but not a Bsh1236I digestion, significantly reduced the amplifiable DNA message of F2 (Figure 3C), whereas amplification of F1 was not affected (Figure 3B).
T127 4093-4289 Sentence denotes In IRF-4-positive BV-173 cells a HpaII, but not a Bsh1236I digestion, significantly reduced the amplifiable DNA message of F2 (Figure 3C), whereas amplification of F1 was not affected (Figure 3B).
T7839 4290-4485 Sentence denotes This implied that IRF-4 transcription in SD-1 and BV-173 cells is associated with less promoter methylation (in BV-173 especially at HpaII sites) as compared with the tested IRF-4-negative cells.
T128 4290-4485 Sentence denotes This implied that IRF-4 transcription in SD-1 and BV-173 cells is associated with less promoter methylation (in BV-173 especially at HpaII sites) as compared with the tested IRF-4-negative cells.
T8559 4487-4565 Sentence denotes Specific CpG sites in the IRF-4 promoter are methylated in hematopoietic cells
T129 4487-4565 Sentence denotes Specific CpG sites in the IRF-4 promoter are methylated in hematopoietic cells
T8560 4566-4897 Sentence denotes In order to exactly map the methylation sites within the IRF-4 promoter, we treated DNA of Jurkat, CML-T1, U-937, K-562 and EM-2 cells as well as of SD-1, RPMI-8226 and BV-173 control cells with bisulfite, which chemically converts unmethylated cytosine to uracil, whereas it has no effect on methylated cytosine, i.e. in CpG (34).
T130 4566-4897 Sentence denotes In order to exactly map the methylation sites within the IRF-4 promoter, we treated DNA of Jurkat, CML-T1, U-937, K-562 and EM-2 cells as well as of SD-1, RPMI-8226 and BV-173 control cells with bisulfite, which chemically converts unmethylated cytosine to uracil, whereas it has no effect on methylated cytosine, i.e. in CpG (34).
T8561 4898-4980 Sentence denotes This technique is especially useful for detection of unknown methylation patterns.
T131 4898-4980 Sentence denotes This technique is especially useful for detection of unknown methylation patterns.
T8562 4981-5160 Sentence denotes PCR amplification, cloning and sequencing of the bisulfite-treated DNA showed a specific methylation pattern of the analyzed 62 CpG sites in all cell lines (Figure 4 and Table 1).
T132 4981-5160 Sentence denotes PCR amplification, cloning and sequencing of the bisulfite-treated DNA showed a specific methylation pattern of the analyzed 62 CpG sites in all cell lines (Figure 4 and Table 1).
T8563 5161-5360 Sentence denotes In general, the methylational status ranged from one cell line with a nearly non-methylated IRF-4 promoter (SD-1, IRF-4-positive) to a completely methylated IRF-4 promoter in CML-T1 (IRF-4-negative).
T133 5161-5360 Sentence denotes In general, the methylational status ranged from one cell line with a nearly non-methylated IRF-4 promoter (SD-1, IRF-4-positive) to a completely methylated IRF-4 promoter in CML-T1 (IRF-4-negative).
T8564 5361-5555 Sentence denotes Interestingly, the percentage of CpG methylation in the IRF-4 promoter from IRF-4-positive cells was very low (mean 24%) as compared with IRF-4-negative cells (mean 94%) (Figure 4A and Table 1).
T134 5361-5555 Sentence denotes Interestingly, the percentage of CpG methylation in the IRF-4 promoter from IRF-4-positive cells was very low (mean 24%) as compared with IRF-4-negative cells (mean 94%) (Figure 4A and Table 1).
T8565 5556-5922 Sentence denotes A 5′-region (R1) with 13 hypermethylated CpG sites (mean number of methylated clones 5.5 of 8 with 77% methylated CpGs) was found in most cells (except SD-1 and RPMI-8226) and a 3′-region (R3) of 6 hypomethylated CpG sites (mean number of methylated clones 1.7 of 8 with 33% methylated CpGs) was found in most cells (except CML-T1 and U-937) (Figure 4A and Table 1).
T135 5556-5922 Sentence denotes A 5′-region (R1) with 13 hypermethylated CpG sites (mean number of methylated clones 5.5 of 8 with 77% methylated CpGs) was found in most cells (except SD-1 and RPMI-8226) and a 3′-region (R3) of 6 hypomethylated CpG sites (mean number of methylated clones 1.7 of 8 with 33% methylated CpGs) was found in most cells (except CML-T1 and U-937) (Figure 4A and Table 1).
T8566 5923-6200 Sentence denotes Intriguingly, a stretch of 13 CpG sites (#10–22; R2) was detected in between these regions, which were highly methylated in IRF-4-negative (mean number of methylated clones 7.1 of 8 with 89% methylated CpGs) but totally non-methylated in IRF-4-positive cells (Figure 4A and B).
T136 5923-6200 Sentence denotes Intriguingly, a stretch of 13 CpG sites (#10–22; R2) was detected in between these regions, which were highly methylated in IRF-4-negative (mean number of methylated clones 7.1 of 8 with 89% methylated CpGs) but totally non-methylated in IRF-4-positive cells (Figure 4A and B).
T8567 6201-6394 Sentence denotes Furthermore, three CpG sites at the 5′ end (#54, 56, 58) and two CpG motifs at the 3′ end (#1, 2) showed this direct correlation between high methylation status and absence of IRF-4 expression.
T137 6201-6394 Sentence denotes Furthermore, three CpG sites at the 5′ end (#54, 56, 58) and two CpG motifs at the 3′ end (#1, 2) showed this direct correlation between high methylation status and absence of IRF-4 expression.
T8568 6395-6571 Sentence denotes In addition, two CpG sites located in a NFκB (#48) and a SP1 element (#45) are less methylated in IRF-4-positive than in IRF-4-negative cells (mean number of methylated clones:
T138 6395-6571 Sentence denotes In addition, two CpG sites located in a NFκB (#48) and a SP1 element (#45) are less methylated in IRF-4-positive than in IRF-4-negative cells (mean number of methylated clones:
T8569 6572-6588 Sentence denotes 1/8 versus 8/8).
T139 6572-6588 Sentence denotes 1/8 versus 8/8).
T8570 6589-6703 Sentence denotes These results indicate the involvement of CpG methylation in the regulation of IRF-4 expression in leukemic cells.
T140 6589-6703 Sentence denotes These results indicate the involvement of CpG methylation in the regulation of IRF-4 expression in leukemic cells.
T9690 6705-6788 Sentence denotes In vitro methylation of an IRF-4 promoter-reporter construct decreases its activity
T141 6705-6788 Sentence denotes In vitro methylation of an IRF-4 promoter-reporter construct decreases its activity
T9691 6789-6989 Sentence denotes To provide evidence for a direct effect of methylational status on IRF-4 promoter activity we performed reporter gene assays with IRF-4 promoter constructs before and after their in vitro methylation.
T142 6789-6989 Sentence denotes To provide evidence for a direct effect of methylational status on IRF-4 promoter activity we performed reporter gene assays with IRF-4 promoter constructs before and after their in vitro methylation.
T9692 6990-7121 Sentence denotes A complete methylation of these constructs was checked via restriction assays with methylation-sensitive endonucleases (Figure 5A).
T143 6990-7121 Sentence denotes A complete methylation of these constructs was checked via restriction assays with methylation-sensitive endonucleases (Figure 5A).
T9693 7122-7262 Sentence denotes Intriguingly, methylation of the IRF-4 promoter significantly decreased promoter activity in IRF-4-positive SD-1 cells by 85.0% (Figure 5B).
T144 7122-7262 Sentence denotes Intriguingly, methylation of the IRF-4 promoter significantly decreased promoter activity in IRF-4-positive SD-1 cells by 85.0% (Figure 5B).
T9694 7263-7464 Sentence denotes The silencing effect of CpG methylation was not restricted to IRF-4-positive cells, since in vitro methylation led to a 92.9% abrogation of promoter activity in IRF-4-negative Jurkat cells (Figure 5C).
T145 7263-7464 Sentence denotes The silencing effect of CpG methylation was not restricted to IRF-4-positive cells, since in vitro methylation led to a 92.9% abrogation of promoter activity in IRF-4-negative Jurkat cells (Figure 5C).
T9695 7465-7638 Sentence denotes In contrast, control methylation of a reporter construct with a different promoter (FasL) as well as an empty vector had no effect on the reporter activity (data not shown).
T146 7465-7638 Sentence denotes In contrast, control methylation of a reporter construct with a different promoter (FasL) as well as an empty vector had no effect on the reporter activity (data not shown).
T9696 7639-7733 Sentence denotes These data proved a direct association between methylation and activity of the IRF-4 promoter.
T147 7639-7733 Sentence denotes These data proved a direct association between methylation and activity of the IRF-4 promoter.
T10244 7735-7862 Sentence denotes mRNA expression of DNA methyltransferases and methyl-CpG-binding proteins may not be associated with IRF-4 promoter methylation
T148 7735-7862 Sentence denotes mRNA expression of DNA methyltransferases and methyl-CpG-binding proteins may not be associated with IRF-4 promoter methylation
T10245 7863-8089 Sentence denotes Since abundance of DNMT and MBP contribute to promoter regulation via methylation (25,26,28), we studied their mRNA expression to investigate a possible mechanism for the observed methylation differences in the IRF-4 promoter.
T149 7863-8089 Sentence denotes Since abundance of DNMT and MBP contribute to promoter regulation via methylation (25,26,28), we studied their mRNA expression to investigate a possible mechanism for the observed methylation differences in the IRF-4 promoter.
T10246 8090-8287 Sentence denotes To this end, we did not detect a significant difference in DNMT (DNMT1, DNMT3A and DNMT3B) or MBP (MBD1, MBD2, MBD4 and MeCP) mRNA expression between IRF-4-positive and -negative cells (Figure 5D).
T150 8090-8287 Sentence denotes To this end, we did not detect a significant difference in DNMT (DNMT1, DNMT3A and DNMT3B) or MBP (MBD1, MBD2, MBD4 and MeCP) mRNA expression between IRF-4-positive and -negative cells (Figure 5D).
T10247 8288-8443 Sentence denotes In fact, all analyzed cells had moderate to high mRNA levels of these tested DNMT/MBPs and differences in expression were not correlated with IRF-4 status.
T151 8288-8443 Sentence denotes In fact, all analyzed cells had moderate to high mRNA levels of these tested DNMT/MBPs and differences in expression were not correlated with IRF-4 status.
T10248 8444-8612 Sentence denotes These results indicate a distinct cause of the methylation differences in IRF-4-positive and -negative cells rather than changes in the DNMT and MBP mRNA transcription.
T152 8444-8612 Sentence denotes These results indicate a distinct cause of the methylation differences in IRF-4-positive and -negative cells rather than changes in the DNMT and MBP mRNA transcription.