The antigen-binding regions of immunoglobulins and T-cell receptors are somatically assembled from separate V (variable)-, D (diversity)- and J (joining)-gene segments through a process called """"""""V(D)J recombination"""""""". V(D)J recombination involves site-specific cleavages to generate double-strand breaks and imprecise end joinings of these breaks. Defects in this recombination can lead to a disease syndrome known as severe combined immune deficiency (SCID). Affected individuals lack functional T- and B-cells. The scid mouse mutation results in a defect in double-strand break repair. Cells with such a defect manifest both a hypersensitivity to DNA damaging agents (such as, gamma-irradiation) and an inability to join V, D and J elements. Similar to DNA damaging agents, initiation of V(D)J recombination could be deleterious to scid lymphocytes due to accumulation of unresolved DNA breaks. These breaks could also be targets for secondary recombination which lead to chromosomal deletions and translocations. Thus, aberrant resolution of recombination-associated breaks is likely to contribute to the pathogenesis of lymphoid malignancies. To directly investigate the effect of recombination-associated breaks on survival, growth and differentiation of both normal and scid lymphocytes, we will employ two culture systems in which activation of VDJ recombination or B-cell differentiation can be induced by in vitro manipulation. One system involves the use of temperature sensitive transformed pre-B-cell lines. The other makes use of progenitors/stromal cell culture in the presence of interleukin-7 (IL-7). Specifically, we propose: 1) to examine how scid lymphocytes resolve double strand breaks made in situ as a result of V(D)J recombination; 2) to determine how unresolved breaks may affect scid cells in vitro; and 3) to investigate how a bcl-2 transgene influences survival and differentiation of break-bearing scid B lymphocytes. The long term goal of this research is to discern the role of V(D)J recombination in the pathogenesis of immunodeficiency and lymphoid malignancies. The results should provide new insights for earlier detection and intervention of these immunological diseases.
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