2636

Abstract

After exposure to ionizing irradiation individual cells may die by accessing a range of specific cell death pathways, such as apoptosis and mitotic cell death. It has been a commonly held belief that once a cell has initiated the apoptotic cell death program and begun to degrade its own DNA, the process proceeds irreversibly to completion. If this were found to be incorrect, and a fraction of cells survive apoptotic nuclease attack, then this would offer a potentially novel source of genomic instability within affected cells of either normal or tumor origin. The non-transformed human lymphoblastoid TK6 cell line was used for most of these studies. Apoptosis was initiated with either 2 Gy of X-irradiation or by exposure to 0.5μg/ml anti-CD95 antibody, the latter signals the Fas/Fas ligand signaling pathway that activates apoptosis via a non-genotoxic process. We have shown before in a number of cell types, including cells of human carcinoma origin that activation of apoptosis initiates cleavage within the MLL gene, located at 11q23. Small DNA fragments ranging in size from 0.3 - 8.3 kbp that include the DNA cleavage site were placed within the pCEP4 episome, transfected into TK6 cells and subsequently exposed to apoptotic stimuli. The episomal constructs were subsequently modified by the inclusion of an initially out of frame neomycin selection gene, immediately adjacent to the cleavable fragment. Cleavage of the included DNA fragment and subsequent mis-repair has the potential to drive neomycin transcription and translation, allowing the affected cell to survive exposure to G418 selection. Exposure of TK6 cells to both ionizing irradiation and anti-CD95 antibody resulted in DNA cleavage of 11q23 and the detection of a range of mis-repair events up to three weeks after treatment - mostly translocations. Small fragments that contained the 11q23 cleavage site within the episomal constructs were also successfully cleaved when exposed to apoptotic triggers. Episomes that contained a cleavable fragment of 11q23 adjacent to an out of frame neomycin gene were also exposed to either 2 Gy or anti CD-95 antibody and subsequently grown in G418 selection media. It was found that the presence of the potent apoptotic triggers and selection construct was essential for the subsequent survival of the treated pools of cells. Cells containing either no construct with or without irradiation, or selection construct without pro-apoptotic treatment all died during G418 selection. Interrogation of surviving pools of cells showed indications that some of the episomes had become incorporated into genomic DNA, losing some or all of the episomal backbone. However clones were found that had undergone the specific type of rearrangement predicted, suggesting that apoptotic nuclease-induced rearrangements were the direct cause of the cells survival. Transient activation of apoptotic nucleases may occur within populations of cells exposed to pro-apoptotic stimuli. Though the number of cells that survive may be small, it may be sufficient to contribute to genetic diversity within a growing tumor mass and thus facilitate tumor development to more a more aggressive or drug and radiation resistant phenotype.