Differential DNA fragmentation patterns induced by fluoropyrimidines: Implications for intrinsic sensitivity and resistance to cytotoxicity.

Abstract

Current evidence suggests that DNA damage plays an integral role in mediating cytotoxicity that results from thymidylate depletion. Using the technique of pulsed field gel electrophoresis, both qualitative and quantitative differences in DNA fragmentation patterns induced by fluorodeoxyuridine (FdUrd) were detected among three human colorectal tumor (HCT) cell lines. Thymidylate synthase (TS) inhibition induced two patterns of DNA fragmentation in HT29, SW620 and HuTu80 cells, and these patterns reflected the sensitivity of each cell line to FdUrd-induced cytotoxicity. The HT29/SW620 pattern, a broad but apparently nonrandom distribution of fragments ranging from 50 kb to 5 Mb, appeared to be specifically caused by TS inhibition. Even though both cell lines were equally sensitive to TS inhibition, SW620 cells displayed a delayed response to both double-strand break formation and cytotoxicity. SW620 resistance correlated with delayed induction of DNA single-strand breaks, enhanced deoxyuridine triphosphatase activity and a failure to accumulate dUTP, as compared to HT29 cells. Therefore, SW620 resistance is attributable to its response to TS inhibition, rather than the extent of TS inhibition. The HuTu80 pattern of DNA fragmentation consisted of a discrete population of fragments ranging from 50 to 200 kb, the estimated size of a DNA replicon. Not only were HuTu80 DNA fragments induced by TS inhibitors, they were also induced by a variety of agents that perturb the cell cycle. This characteristic DNA fragmentation response is coupled with enhanced sensitivity to cytotoxicity induced by these agents as compared to the other two cell lines. This relationship between enhanced sensitivity to cytotoxicity and a particular DNA fragmentation response is analogous to that observed in cells that are susceptible to drug-induced apoptosis, although oligonucleosomal DNA fragments were not detected. Therefore, these results suggest that there are at least two different mechanisms for the conversion of TS inhibition to DNA damage and variations in these processes may determine the sensitivity of a particular tumor cell to fluoropyrimidine-induced cytotoxicity.