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|Results and Discussion|
A panel of eight HL-derived cell lines was screened for IκBα defects by Western blot, reverse transcription PCR amplification and sequencing of IκBα transcripts, and, if relevant, characterization of the exons of the IκBα gene (Table ). The reported defects in IκBα protein expression in two lines (L428, KMH-2 ) turned out to be due to deleterious mutations in one copy of the IκBα gene and apparent loss of the other copy, in agreement with a recent study 14. However, all other cell lines (including two not previously analyzed B lineage–derived lines) harbor wild-type IκBα transcripts and express full-length IκBα protein. Notably, the only cell line for which the derivation from H/RS cells is unequivocally proven (L1236 17), expresses wild-type IκBα from both alleles of the gene, indicating that IκBα mutations are not present in the H/RS cells in all cases of HL, if at all. As the limited number and questionable origin of HL-derived cell lines allow no solid conclusions on this issue, we turned to the analysis of H/RS cells from tumor biopsies.
Five cases of HL were chosen for analysis. In two of them (cases 4 and 5), EBV-encoded small RNA (EBER) in situ hybridization detected EBV in the H/RS cells. To determine the germline sequence of the coding parts of the IκBα gene in the patients, DNA was extracted from whole sections of the infiltrated lymph nodes (i.e., primarily from the nonmalignant inflammatory cells in the tissue) and used for amplification and direct sequencing of the six IκBα exons. Three deviations from the published IκBα sequence 18 were consistently found in all five cases (738G, 2111T, 2734T), while at another six positions we detected frequent germline polymorphisms (Table ). For determination of the sequence of the IκBα gene in the malignant cells, individual H/RS cells micromanipulated from immunostained histological sections were used for seminested PCR amplification. To avoid the detection of mutations introduced by Taq DNA polymerase, direct sequencing of the gel-purified PCR products was performed. In most cases ≥4 PCR products of each exon were analyzed, since the single cell PCR approach may stochastically miss either of the two copies of the gene in individual reactions.
In case 1, mutations were detected in exons 1 and 4: the deletion of two and one nucleotides, respectively, leads to frameshifts (Table ). Both mutations were repeatedly detected in PCR products from H/RS cells (see also Table ), yet not in the germline sequences of the respective patient. We conclude that they represent clonal somatic mutations in the malignant cells. Notably, the PCR products from H/RS cells contain wild-type as well as mutant copies of the respective exons, indicating the presence of two distinct alleles of the IκBα gene. To assign the two mutations to the two alleles, we separated the IκBα loci of H/RS cells before coamplification of exons 1 and 4. Single H/RS cells were incubated with protease in buffer, which was then distributed to six tubes for PCR analysis. Most cells analyzed contain several copies of both wild-type and mutated exons 1 and 4 (Table ), presumably due to polyploidy, a characteristic feature of H/RS cells 19. Aliquots with evidence for the presence of more than one copy of either exon were disregarded. In most other cases, the mutant exon 1 was coamplified with wild-type exon 4, and vice versa, mutant exon 4 with wild-type exon 1. We conclude that the two mutations are located on the two different alleles of the gene, implying that no full-length IκBα protein can be synthesized in the H/RS cells. The coamplification of wild-type exons 1 and 4 in two aliquots (ID, IIIB) is apparently due to amplification of only the wild-type exons from tubes containing both wild-type and mutated copies.
In case 2, a deletion of two nucleotides, leading to a frameshift, was detected in all exon 5 products amplified from the H/RS cells (Table ). Again, both wild-type and mutant copies of the exon are evident in the H/RS cells, but no mutation was seen in the germline sequence. Thus, one of the two alleles of the IκBα gene in the malignant cells is inactivated by a somatic mutation. For all other exons, the germline sequence was obtained, but an assignment of these wild-type products to either of the two alleles is not possible. Analysis of PCR products spanning exons 3–5 and 5–6, respectively (allowing discrimination of the two alleles due to the mutation in exon 5), revealed no other point mutation or deletion in that region in either of the two alleles. Since exons 1 and 2 could not be included in this analysis (see Materials and Methods), it is unclear whether the entire coding region of the second IκBα allele is unmutated. In the third EBV-negative case (case 3), as well as in the two EBV-positive cases (cases 4 and 5), no clonal mutations in any of the IκBα exons were detected (Table ). For cases 3 and 5, we conclude that all coding exons of both alleles are unmutated, since germline polymorphisms allow their discrimination.
In several PCR products amplified from the H/RS cells of cases 2–5, in particular those spanning exons 1 and 2, unique nucleotide exchanges were detected. They were not seen in any of the other products of the respective exon of the respective case, and are thus apparently not due to mutations present in all malignant cells. These mutations might indicate enhanced mutability of the IκBα gene, reflect the genomic instability of H/RS cells 20, or be derived from Taq DNA polymerase errors (although the latter seems unlikely). Be that as it may, the frequent occurrence of this type of mutation emphasizes an important issue: one can only invoke clonal genetic defects (i.e., those that can be assigned to all malignant cells in primary tumors) as an early event in tumorigenesis. This is particularly critical in HL, as also nonneoplastic cells in the affected patients frequently harbor genetic aberrations 21. Taking this into account, IκBα mutations found in an undefined minute cell population from a relapsed tumor 14 or in only a fraction of the H/RS cells are inconclusive, in particular with respect to the role of IκBα defects in the pathogenesis of the original tumor.
To assess whether (deleterious) IκBα mutations are frequent in other B cell lymphomas, the six exons of the IκBα gene were analyzed in a total of 20 non-HL specimens (11 B cell chronic lymphocytic leukemias and 9 Burkitt's lymphoma cell lines; for details, see Materials and Methods). No mutations were found in any of the samples, suggesting that IκBα mutations are not common features in these non-HLs. Since H/RS cells are derived from germinal center B cells, we have also assessed whether mutations in the IκBα gene are frequently introduced in B cells during the germinal center reaction, as reported for the 5′ region of the bcl-6 gene 22 23. However, no evidence for an enhanced frequency of mutations in germinal center B cells was found in the 5′ region of the IκBα gene (exons 1 and 2), compared with naive B cells and the expected polymerase error (Table ).
In this study, clonal deleterious somatic mutations in the IκBα gene were detected in the H/RS cells in two of three EBV-negative and none of two EBV-positive cases of HL. Reminiscent of the mutations in HL-derived cell lines, they lead to the synthesis of truncated IκBα proteins lacking a part of the ankyrin repeat and/or the COOH-terminal PEST domain (Fig. 1), which are required for interaction of IκB proteins with NF-κB and inhibition of its DNA binding, respectively 24 25 26. This suggests that loss or severe impairment of these functions was selected for during the pathogenesis of the tumor cell clone: it leads to constitutive nuclear activity of NF-κB. The severe phenotype of IκBα knockout mice indicates that none of the other IκB family members may fully take over IκBα function 27.
Loss of IκBα function presumably requires the inactivation of both copies of the gene. In the H/RS cells in most of the primary cases we investigated, two distinct alleles of the IκBα gene are clearly detectable, making proof of loss of function dependent on evidence for inactivation of both of them. Our single cell PCR approach, which is dictated by the peculiar histology of HL, does allow the detection of biallelic gene inactivation in the H/RS cells (in contrast to a previous one ), but it misses several ways of tumor suppressor gene inactivation, such as large deletions or chromosomal translocations. In light of this, we consider the detection of three inactivating mutations in the cases studied, and evidence for loss of IκBα function in one of these cases, to be a highly significant finding.
The data presented here establish deleterious IκBα mutations as the first recurrent genetic defect found in HL. It is intriguing that such mutations were only identified in EBV-negative cases (and lines)—a major subset of this disease for which no mechanism of transformation could be pinpointed to date. However, the limited capacity of the single cell PCR approach does not allow definite conclusions about whether IκBα inactivation is restricted to this subset, nor about its overall frequency in HL. Our attempts to analyze a larger panel of cases by specific detection of full-length IκBα protein in tissue sections failed, due to apparent cross-reactivity of the available antibody against the IκBα COOH terminus (C-21; Santa Cruz Biotechnology). Certainly, not IκBα inactivation but rather constitutive NF-κB (p50/p65) activation may be the unifying feature of H/RS cells 8 9 28, and it may apparently be brought about by different means. The finding that H/RS cells are derived from germinal center B cells that may survive the loss of B cell receptor expression warrants testing of the role of constitutive NF-κB activation in the rescue of such “crippled” B cells from apoptosis. Evidently, constitutive activation of NF-κB (p50/p65) in the context of a B cell provides an intriguing explanation for several of the distinctive clinical and pathological features of HL. It likely precipitates the secretion of a battery of cytokines and chemokines leading to massive attraction of inflammatory cells and profound disturbances in immunoregulation, contributes to the peculiar activated phenotype of H/RS cells, and, most importantly, confers apoptosis resistance and continuous proliferation as prerequisites of malignant transformation 10 11 12. The tumorigenic potential of members of the NF-κB family and of factors inducing NF-κB activity is well established. In contrast, ex vivo proof of a tumor suppressor function of an IκB family member has not been provided to date (but see reference 29). This study gives evidence for recurrent inactivation of the prototypic IκB family member, IκBα, in the most common lymphoma of the Western world. It will be interesting to see whether functional impairment of IκBs plays a role in the pathogenesis of other human malignancies.
|Unselected / annnotation||Selected / annnotation|