Response of DNA thymine synthesis in human tumor and normal tissue to 5-fluorouracil.

Thymidine-methyl-3 H and either formate-' @ C or @ C were added to incubations containing various concentrations of 5-fluorouracil. The thymine in DNA was isolated, and precursor incorporation was determined by dual-label liquid scintillation counting. MATERIALS AND METHODS The methods used in this study have been previously described (12). In summary, tissues obtained from patients who had not received prior chemotherapy were minced promptly after surgical removal. In the case of normal bowel tissue, only the epithelium was used for the studies. Duplicate incubations containing either no drug or 5-fluorouracil, 1.8 X l0@ or 1.8 X l06 M, were prepared. After I hr, thymidine-methyl-3 H (19.5 Ci/mmole) and either sodium formate-'4C (25 mCi/mmole) or serine-3-'4C (48 mCi/mmole) were added. Incubation flasks were removed after 23 hr. The medium was removed by centrifugation, and 4% perchionc acid was added. Acid-soluble compounds were removed, and the DNA was isolated. Hydrolysis in formic acid liberated the free bases from DNA. Paper chromatography with carrier added was used to isolate the thymine. The amount of each labeled precursor present in the thymine spot was determined by dual-label liquid scintillation counting. Conversions to nmoles incorporated per ml of tissue were calculated on the basis of precursor specific activity and the volume of tissue used in each incubation. RESULTS It was necessary to establish that the biological activity of 5-fluorouracil was accurately reflected in the observed effects on precursor incorporation into DNA thymine. The effect of 5-fluorouracil on incorporation of 1-carbon units from 2 different sources was compared in a number of tumors (Chart 1). At the same 5-fluorouracil concentrations, formate-1 4C and @ C incorporations were similarly inhibited. The compensatory increase in thymidine incorporation observed in the presence of 5-fluorouracil was further evidence that the data obtained accurately reflected drug effect (Charts 2 and 3). In view of these findings, @ C and thymidine-methyl-3 H were used in subsequent experiments as DNA thymine precursors. The uptake patterns and drug effect were strikingly similar in colon tumors and autochthonous normal tissue from 7 SUMMARY Human tumor tissue and the normal tissue from which it arose similarly incorporated DNA thymine precursors. The effect of 5-fluorouracil on alternative pathways of DNA thymine synthesis was similar in both tumor and normal tissue. Differential drug sensitivity was not demonstrated.