Thymineless death and genetic events in mammalian cells

Thymidylate synthase-negative mutants of cultured mouse FM3A cells were immediately committed to cell death upon thymidine deprivation especially when the cells were synchronized in the S-phase. Thymidine deprivation induced single strand breaks in parental DNA strands, as measured by alkaline sucrose gradient sedimentation, giving rise to two peaks, one with large and the other with short fragments. Increase in the short DNA fragments paralleled that of thymineless death. Thymidine deprivation also accumulated double strand DNA fragments as determined by a method of neutral filter elution, and their extent paralleled that of cell death. Double-strand DNA eluted through the filter sedimented as a single peak both in a neutral and in an alkaline sucrose gradient that coincided with that of the above short DNA fragments. Therefore, the double strand breaks seemed to occur in some defined portions of the genome and in some specific manners in contrast to those induced by X-ray, which occurred rather randomly. Cycloheximide blocked thymineless death and accumulation of the double stranded DNA fragments in parallel. The double strand breaks induced by thymidine starvation were not repaired, but instead advanced on subsequent incubation of the cells in growth medium containing thymidine. Cytogenetically, thymidine deprivation induced chromosome aberrations such as chromatid breaks, chromatid interchanges, and chromosome fragmentation. Also, 5-bromodeoxyuridine deprivation induced sister chromatid exchange. Thymidylate stress also induced loss of a stably integrated human gene in mouse cells, possibly by DNA rearrangements, under the conditions where no point mutations were induced.