MECHANISMS OF ACQUIRED-RESISTANCE TO MODULATION OF 5-FLUOROURACIL BY LEUCOVORIN IN HCT-8 HUMAN ILEOCECAL CARCINOMA-CELLS

Repeated (10x) exposure of HCT-8 human ileocecal carcinoma cells to 5- fluorouracil (5-FU) for 2 or 72 hr, both incubations in the continuous presence of 20 mu M leucovorin (LV), yielded two stable modulation-re sistant sublines, FL2h and FL72h. Although LV potentiated growth inhib ition by 5-FU 2-fold in parental HCT-8 cells, it did not potentiate th e effect of 5-FU in the FL2h or FL72h sublines. LV modulation of 5-flu orodeoxyuridine (5-FdUrd) was also reduced (FL72h) or eliminated (FL2h ). In the FL2h and FL72h sublines, the level of thymidylate synthase ( TS) protein and TS activity in cell extracts, TS activity in situ, the rate of cellular uptake and metabolism of LV, and the level of 5-FU i ncorporation into total cellular RNA were similar to those in parental HCT-8 cells. However, LV significantly (P < 0.01) potentiated the inh ibition of TS activity in situ in HCT-8 cells at 24 hr after a 2-hr tr eatment with either 5-FU or 5-FdUrd, but had no such activity in the F L2h and FL72h sublines (P > 0.1). Resistance to modulation of 5-FU by LV was associated with the inability of LV to increase the formation o f intracellular TS-FdUMP-methylenetetrahydrofolate ternary complexes, and these complexes dissociated more rapidly (T-1/2 > 1.5- to 3-fold f aster) in the presence of different concentrations of 5,10-methylenete trahydropteroylpentaglutamate Thus, decreased stability of ternary com plexes appears to be the mechanism of acquired resistance to the LV mo dulation of fluoropyrimidine cytotoxicity, possibly due to mutation(s) of TS in these two modulation-resistant HCT-8 sublines. (C) 1997 Else vier Science Inc.