The effect of dose and interval between 5-fluorouracil and leucovorin on the formation of thymidylate synthase ternary complex in human cancer cells.
We examined the importance of dosing interval between leucovorin (LCV) and 5-fluorouracil (5-FU) on intracellular thymidylate synthase (TS) ternary complex, free TS and total TS protein levels in human MCF-7 breast and NCI H630 colon cancer cell lines. A 2- to 3-fold increase in total TS was noted when either cell line was exposed to 5-FU 10 microM plus LCV (0.01-10 microM) compared with a 1.4- to 1.6-fold increase in total TS due to 5-FU 10 microM alone. The amount of TS ternary complex formed was 2- to 3-fold higher in both cell lines treated with the combination of 5-FU and LCV compared with 5-FU alone. TS complex formation and total TS protein increased with LCV dose (0.1-10 microM). In MCF-7 cells, the maximal increase in total TS protein and TS ternary complex formation was observed when 5-FU was delayed for 4 h after the start of LCV exposure. In NCI H630 cells, maximal total TS protein and ternary complex formation occurred when 5-FU was delayed for 18 h after the start of LCV exposure. The amount of free TS did not change in either cell line whether 5-FU was given concurrently with LCV or delayed for up to 24 h. The accumulation rate of intracellular folates in the form of higher glutamates Glu3-Glu5 was rapid in MCF-7 cells (maximal formation after 4 h), whereas in H630 cells accumulation of higher polyglutamates continued to increase up to 18 h. The time of peak folate polyglutamate (Glu3-Glu5) formation coincided with the time of peak TS complex formation and total TS protein in each cell line. In these human carcinoma cell lines, the LCV dose and interval between 5-FU and LCV play a role in increased TS total protein and TS ternary complex; however, the amount of free TS is independent of the interval between 5-FU and LCV. The time-and dose-dependent increases in TS ternary complex and TS total protein are associated with differences in the accumulation of folate polyglutamates in these cell lines.
Drake
J C
JC
NCI-Navy Medical Oncology Branch, Bethesda, Maryland 20889-5105, USA.
Voeller
D M
DM
Allegra
C J
CJ
Johnston
P G
PG
D015153:Blotting, Western; D001943:Breast Neoplasms; D002460:Cell Line; D003110:Colonic Neoplasms; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D002955:Leucovorin / Q000494:pharmacology*; D046911:Macromolecular Substances; D013940:Thymidylate Synthase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D046911:Macromolecular Substances; D013940:Thymidylate Synthase; D002955:Leucovorin; D005472:Fluorouracil