Radiation-Induced Mutagenesis and Apoptotic Regulation

University Of Calif-Lawrenc Berkeley Lab, Berkeley CA

Investigators

Abstract

DESCRIPTION (provided by applicant): Programmed cell death (PCD) is a fundamental process that eliminates extraneous or potentially dangerous cells. Many human tumors have found ways to evade PCD. We discovered a novel role for PCD in limiting mutagenesis in human cells. Over-expression of BCL-2 or BCL-X L increased x-ray-induced mutation at the autosomal TK1 locus in TK6 cells. We also saw an increased frequency of loss of heterozygosity (LOH) mutations with duplications of the inactive allele - suggesting that more cells were mutated via homologous recombinational repair (HRR). Our goal is to understand the basis for increased autosomal mutagenes:s mediated by BCL-2 family members. Four aims are proposed: 1) We will test the hypothesis that the elevated level of LOH mutations at the TK1 locus in TK6 cells that over-express BCL-2 or BCL-X L is associated with an increase in homology directed repair (HDR) of DNA double-strand breaks (DSBs). We will use an integrated DR-GFP reporter to measure gene conversion following a single, site-specific DSB. We recently showed that high BCL-X L expression promotes HDR in TK6 cells. We plan to generalize this finding to other BCL-2 family members and to FL5.12 cells in which PCD regulation by BCL-2 family members has been well studied. 2) We will test the hypothesis that the elevated frequencies of TK1 mutations in TK6-bclXL cells result from a novel activity of BCL-X L distinct from its anti-apoptotic function. We will develop isogenic TK6 cells that express mutated BCL-X L that can't block PCD to assess if x-ray-induced TK1 mutagenesis is modulated. 3) We will test the hypothesis that BCL-X L promotes x-ray-induced TK1 mutations by maintaining higher levels of HsRAD51 in TK6 cells post-IR. We will use isogenic TK6 cells that express a mutant HsRAD51 (HsRAD51D-A)'insensitive to caspase cleavage that promotes HRR, wildtype HsRAD51, or a cleavage fragment of HsRAD51 that can't promote HRR. If HsRAD51D-A elevates x-ray- induced TK1 mutagenesis, we will determine if it acts in the same pathway as BCL-X L by co-expressing these proteins in TK6 cells. New strategies are included in the event that an elevated level of HsRAD51 is toxic. 4) We will test the hypothesis that PCD suppression per se can enhance x-ray-induced TK1 mutagenesis. We will use TK6 cells expressing mutant procaspase-9 (cys287ala) that acts as a dominant negative to suppress PCD. Elucidating the mechanisms through which BCL-2 and BCL-X L mediate mutagenesis in vitro should illuminate mechanisms of mutation in B-cell follicular lymphomas that express high levels of BCL-2. It is the secondary mutations that lead to advance, aggressive disease in people.