Dissociation of 5-fluorouracil-induced DNA fragmentation from either its incorporation into DNA or its cytotoxicity in murine T-lymphoma (S-49) cells.
We have shown previously (W. B. Parker and P. Klubes, Cancer Res., 45:4249-4256, 1985) that uridine (10 microM) enhanced the cytotoxicity of 5-fluorouracil (FUra) in cultured mouse T-lymphoma (S-49) cells. Here we show, by the use of colony formation assays, that approximately 50% of the cytotoxicity of FUra plus uridine could be prevented by the simultaneous administration of thymidine (2.5 to 10 microM). In order to explain our observation of a thymidine-preventable component of the cytotoxicity of the FUra plus uridine combination, we examined the incorporation of FUra into DNA. The DNA from FUra-treated S-49 cells was purified by cesium chloride gradient centrifugation and degraded to nucleosides by DNase I and Crotalus atrox snake venom. 5-[3H]-Fluoro-2'-deoxyuridine was not detected by high-pressure liquid chromatography in the hydrolysate of DNA from S-49 cells treated with 1.0 microM [3H]FUra, 1.0 microM [3H]FUra plus 10 microM uridine, or 2.4 microM [3H]FUra. In contrast, 5-[3H]fluoro-2'-deoxyuridine was detected in the DNA of L1210 cells treated with cytotoxic concentrations of either [3H]FUra or 5-[3H]fluoro-2'-deoxyuridine. Thus incorporation of FUra into the DNA of S-49 cells treated with cytotoxic concentrations of FUra was shown to be minimal or insignificant. Using alkaline elution techniques, however, fragmentation of the DNA was detected in S-49 cells treated with 1.0 microM FUra, 1.0 microM FUra plus 10 microM uridine, or 2.4 microM FUra (115-, 107-, and 159-rad equivalent single strand breaks, respectively). Most of the DNA fragmentation caused by FUra could be prevented by the inclusion of 2.5 microM thymidine with FUra during the incubation. Similar amounts of DNA fragmentation occurred with 1.0 microM FUra in either the presence or absence of 10 microM uridine. Because 1.0 microM FUra plus 10 microM uridine was more cytotoxic than 1.0 microM FUra alone, these results indicated that the enhancement of FUra cytotoxicity by uridine was not related to increased fragmentation of DNA.
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003114:Colony-Forming Units Assay; D003435:Crotalid Venoms / Q000494:pharmacology; D004247:DNA / Q000187:drug effects*; D003850:Deoxyribonuclease I / Q000378:metabolism; D005472:Fluorouracil / Q000494:pharmacology*; D008223:Lymphoma / Q000235:genetics / Q000473:pathology*; D051379:Mice; D013936:Thymidine / Q000494:pharmacology; D014529:Uridine / Q000494:pharmacology
D003435:Crotalid Venoms; D004247:DNA; D003850:Deoxyribonuclease I; D005472:Fluorouracil; D013936:Thymidine; D014529:Uridine