Because of its susceptibility to apoptosis on Ag receptor cross-linking, cells of the mouse cell line WEHI-231 have been classified as immature B cells. Surprisingly, however, the cell line expresses activation-induced cytidine deaminase, the enzyme that mediates hypermutation and Ig class switch recombination in activated B cells. Although both cDNA sequence and protein expression of activation-induced cytidine deaminase appear normal, the cell line does not hypermutate an indicator plasmid. For the readout, the indicator plasmid depends on the removal of deoxyuridine after transition from C to U and, therefore, on functional expression of uracil N-glycosylase 2, which is normal in WEHI-231. At the endogenous Ig locus, however, WEHI-231 does undergo the canonical hypermutation of G · C to A · T base pairs to some extent. The cell line also expresses the germline transcripts of the Ig γ2b, ε, and α loci, but it does not switch its IgM surface Ig.

Mismatch repair contributes to hypermutation in B lymphocytes, both by increasing the frequency of mutations and by changing the mutational patterns. In this paper, we investigated whether or not mismatch repair influences activation-induced cytidine deaminase (AID)-mediated hypermutation in a non-B lymphocyte line. We did so by regulating expression of MutL homologue MLH 1, which is essential in mismatch repair, in a kidney cell line that had been transduced by an AID-containing vector. Whether or not MLH1 was expressed, we found no difference in the mutation rates of an indicator gene. We conclude that in order to contribute to hypermutation, mismatch repair needs additional factors that are present in activated B lymphocytes, but absent in the cell line investigated. IntroductionSomatic hypermutation contributes to antibody diversity through introduction of point mutations into the variable (V) region of immunoglobulin sequences [1]. The mutation rate in mice and humans is about 10 6 -fold higher than the spontaneous mutation rate in most other genes [2]. Targeting of somatic hypermutation to the Ig loci remains a subject of debate, since genomic sites other than the Ig loci have been reported to support hypermutation to various degrees [3][4][5][6]. However, somatic hypermutation is triggered by and restricted to the expression of the activation-induced cytidine deaminase (AID) enzyme in activated germinal center B cells [7][8][9][10]. Ectopic expression of AID in hybridomas [11], non-B cells [12,13] and even E. coli [14] results in a mutator phenotype. In conjunction with replication protein A (RPA) [15], AID acts on single-stranded DNA during transcription and deaminates dC into dU [16][17][18]. If the mismatch is not repaired, it results in a C?T transition, which is the predominant mutation during ectopic AID expression in cell lines and E. coli. After introduction of the G-U mismatch, various pathways fix the mutation in the genome (reviewed in [19]). For example, the uracil N-glycosylase (UNG) removes the uracil generated by AID [20]. If the resulting abasic site is converted by an AP-endonuclease into a singlestrand nick and subsequently repaired via the base excision pathway, no mutation is generated. But if the abasic site is not removed, any base can be introduced during replication [21]. Alternatively, although debated [22], mismatch repair (MMR) may sense G-U mispairing and set off a sequence of events that introduces further mutations rather than restore the original base pair sequence [23] (reviewed in [24] [39]. Cancer predisposition is linked to germ-line mutations in MMR genes; most mutations associated with HNPCC were found in hMLH1, hMSH2, and hMSH6 genes [40], while mutations in hPMS2 were reported sporadically. The majority of all HNPCC-causing mutations were found in hMLH1, focusing the interest on the analysis of pathogenic hMLH1 variants [41]. Although loss of EXO1, FEN1 and PCNA function also results in a mutator phenotype, it does not seem to significantly contribute to HNPCC forma...

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Because of its susceptibility to apoptosis upon Ag receptor cross-linking and lack of IgD expression, cells of the mouse cell line WEHI-231 have been classified as immature B cells. In this study we show that early freezings of the WEHI-231 line express IgD but not CD93, which classifies the cells as more similar to mature B cells. Another, later line obviously has differentiated in culture and has all the hallmarks of activated B cells. But despite activation-induced cytidine deaminase expression, there is no switch in isotype; instead we found switching from one μ allele to the other. As a consequence of these findings, we now view the apoptosis studies in the WEHI-231 line to reflect properties of mature and activated B lymphocytes, respectively.