APOPTOSIS, REPRODUCTIVE FAILURE, AND OXIDATIVE STRESS IN CHINESE-HAMSTER OVARY CELLS WITH COMPROMISED GENOMIC INTEGRITY

Chromosomal instability and persistent reproductive cell death show a significant correlation after cells are exposed to ionizing radiation. To examine the possible role of apoptosis in persistent reproductive cell death, we analyzed subsets of chromosomally stable and unstable c lones for relationships between chromosome stability, reproductive int egrity, and apoptosis. All clones were generated from the GM10115 cell line and derived from single progenitor cells surviving 10 Gy of X-ra ys, and all measurements were made similar to 60-80 generations after irradiation. The incidence of apoptosis, as measured by both annexin V binding of phosphatidylserine residues and terminal deoxynucleotidyl transferase labeling of DNA strand breaks, was significantly higher in chromosomally unstable clones than it was in chromosomally stable clo nes (P < 0.05; ANOVA and Student's t test). Furthermore, statistical a nalyses of the biological end points of persistent reproductive cell d eath and apoptosis were consistent, showing R-2 values of 0.78 and 0.7 6, respectively. These results suggest that persistent reproductive ce ll death can, in Dart, be explained by the predisposition of a fractio n of the clonal population to undergo apoptosis or necrosis. Immunolog ical blot analyses of protein Levels and DNA bandshift assays confirme d the mutant status of p53 in the host cell line, implying an apoptoti c pathway that is independent of p53. Induction of apoptosis by agents such as actinomycin D, etoposide, and staurosporine and induction of necrosis by sodium azide were accompanied by an increase in the level of intracellular peroxy radicals and lipid peroxidation products, two independent end points that are typically associated with oxidative st ress, Similar findings were observed in several subclones showing pers istent apoptosis. These results suggest that the elevated levels of fr ee radical damage that we detected mere derived from the fraction of c ells dying by apoptotic or necrotic processes. possible mechanisms whe reby oxidative stress mag contribute indirectly to the perpetuation of chromosomal instability are discussed.