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Dissociation of 5-Fluorouracil-induced DNA Fragmentation from Either Its Incorporation into DNA or Its Cytotoxicity in Murine T-Lymphoma (S-49) Cells¹

Department of Pharmacology, The George Washington University Medical Center, Washington, DC 20037

We have shown previously (W. B. Parker and P. Klubes, Cancer Res., 45: 4249–4256, 1985) that uridine (10 µM) 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 µM). 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 µM [³H]FUra, 1.0 µM [³H]FUra plus 10 µM uridine, or 2.4 µM [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 µM FUra, 1.0 µM FUra plus 10 µM uridine, or 2.4 µM 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 µM thymidine with FUra during the incubation. Similar amounts of DNA fragmentation occurred with 1.0 µM FUra in either the presence or absence of 10 µM uridine. Because 1.0 µM FUra plus 10 µM uridine was more cytotoxic than 1.0 µM FUra alone, these results indicated that the enhancement of FUra cytotoxicity by uridine was not related to increased fragmentation of DNA.

¹ This investigation was supported by Grants CH-160 (P. K.) and CH-274 (K. A. K.) from the American Cancer Society and by Training Grant T-32-CA-09223 from the National Cancer Institute, Department of Health and Human Services.

² In part, from a dissertation presented to the Department of Pharmacology, The Graduate School of Arts and Sciences, The George Washington University, in partial fulfillment of the requirements for the Ph.D. degree. Present address: Department of Pharmacology, 916 FLOB 231-H, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514.

³ To whom requests for reprints should be addressed, at the Department of Pharmacology, The George Washington University Medical Center, 2300 Eye Street, NW, Washington, DC 20037.

Received 11/ 7/85. Revised 4/23/86. Accepted 11/12/86.

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