Apoptosis, reproductive failure, and oxidative stress in Chinese hamster ovary cells with compromised genomic...

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 clones for relationships between chromosome stability, reproductive integrity, and apoptosis. All clones were generated from the GM10115 cell line and derived from single progenitor cells surviving 10 Gy of X-rays, and all measurements were made approximately 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 clones (P < 0.05; ANOVA and Student's t test). Furthermore, statistical analyses of the biological end points of persistent reproductive cell death and apoptosis were consistent, showing R2 values of 0.78 and 0.76, respectively. These results suggest that persistent reproductive cell death can, in part, be explained by the predisposition of a fraction of the clonal population to undergo apoptosis or necrosis. Immunological blot analyses of protein levels and DNA bandshift assays confirmed the mutant status of p53 in the host cell line, implying an apoptotic 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 stress. Similar findings were observed in several subclones showing persistent apoptosis. These results suggest that the elevated levels of free radical damage that we detected were derived from the fraction of cells dying by apoptotic or necrotic processes. Possible mechanisms whereby oxidative stress may contribute indirectly to the perpetuation of chromosomal instability are discussed.