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 microM leucovorin (LV), yielded two stable modulation-resistant sublines, FL2h and FL72h. Although LV potentiated growth inhibition by 5-FU 2-fold in parental HCT-8 cells, it did not potentiate the effect of 5-FU in the FL2h or FL72h sublines. LV modulation of 5-fluorodeoxyuridine (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 incorporation into total cellular RNA were similar to those in parental HCT-8 cells. However, LV significantly (P < 0.01) potentiated the inhibition of TS activity in situ in HCT-8 cells at 24 hr after a 2-hr treatment with either 5-FU or 5-FdUrd, but had no such activity in the FL2h 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 of intracellular TS-FdUMP-methylenetetrahydrofolate ternary complexes, and these complexes dissociated more rapidly (T1/2 > 1.5- to 3-fold faster) in the presence of different concentrations of 5,10-methylenetetrahydropteroylpentaglutamate. Thus, decreased stability of ternary complexes appears to be the mechanism of acquired resistance to the LV modulation of fluoropyrimidine cytotoxicity, possibly due to mutation(s) of TS in these two modulation-resistant HCT-8 sublines.
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K
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Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
McGuire
J J
JJ
Slocum
H K
HK
Rustum
Y M
YM
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D004351:Drug Resistance; D005467:Floxuridine / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D002955:Leucovorin / Q000493:pharmacokinetics / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D005467:Floxuridine; D013940:Thymidylate Synthase; D002955:Leucovorin; D005472:Fluorouracil