Frequency of aberrant V(D)J recombinase mediated rearrangements following in vitro genotoxic exposure

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Aberrant V(D)J recombinase mediated genomic rearrangements at cryptic recombination signal sequences (RSSs) has been implicated as an important genetic mechanism responsible for the recombination events observed in lymphoid malignancies. Normally, the V(D)J recombinase system mediates RSS directed rearrangements of (V), diversity (D) and joining (J) germline gene segments that leads to the generation of diversified T cell receptors and immunoglobulin proteins in lymphoid cells. We have previously reported that aberrant V(D)J recombinase mediated deletions at cryptic RSSs of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus are significantly increased in newborn infants following transplacental exposure to environmental tobacco smoke when compared to unexposed newborns. The goal of this work is to investigate the association between the frequency of aberrant V(D)J recombinase mediated genomic rearrangements, environmental exposures and the development of pediatric lymphoid malignancies. We observed a two to five fold increase in the frequency of V(D)J recombinase mediated rearrangements of the V(D)J shuttle vector pGG51 transfected in the Reh pre-B lymphocyte cell line following exposure to ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS). In contrast we saw no increase in the frequency of V(D)J rearrangements following exposure to methyl sulfone, an MMS/EMS analogue unable to alkylate DNA, nor was there any significant response to exposure to gamma radiation. The observed increase in V(D)J rearrangements were not associated with an increase in the expression of RAG 1/2 compared to unexposed controls. These observations demonstrate that exposure to alkylating agents is associated with an increase in the frequency of V(D)J recombinase mediated recombination events in vitro. This increase in recombination frequency does not appear to be associated with increased expression of RAG 1/2 and therefore may be linked to other regulatory factors involved with V(D)J recombinase activity or non-homologous end joining (NHEJ) repair of genomic double strand breaks. Future studies will include an assessment of the frequency of aberrant V(D)J mediated rearrangements in response to additional alkylating agents such as cyclophosphamide, as well as topoisomerase inhibitors, which together comprise the two main treatments related exposures associated with secondary leukemia. These studies should provide insight into the relationship between genotoxic exposures and aberrant V(D)J rearrangements, which have been implicated in the development of primary and secondary lymphoid malignancies.