Effect of adding the topoisomerase I poison 7-ethyl-10-hydroxycamptothecin (SN-38) to 5-fluorouracil and folinic acid in HCT-8 cells: elevated dTTP pools and enhanced cytotoxicity.
OBJECTIVE
To determine the effect of combined treatment with 7-ethyl-10-hydroxycamptothecin (SN-38, the active metabolite of irinotecan) and 5-fluorouracil/ folinic acid (5FU/FA) in vitro using HCT-8 human intestinal adenocarcinoma cells.


METHODS
Cell survival was examined using colony forming assays. Cell cycle distribution before and after treatment was assessed by flow microfluorimetry. Levels of thymidylate synthase (TS) and topoisomerase I (topo I) in untreated and treated cells were determined by immunoblotting. Changes in deoxynucleotide pools were examined by high-performance liquid chromatography.


RESULTS
Clonogenic assays revealed that colony formation was decreased by 50% after a 24-h exposure to 8+/-2 nM SN-38 or 12+/-3 microM 5FU, the latter being assayed in the presence of 2 microM FA. When treatment with 5FU/FA was followed by SN-38, the cytotoxicity was similar to that observed with 5FU/FA alone. In contrast, when HCT-8 cells were exposed to both agents simultaneously or to SN-38 followed by 5FU/FA, the cytotoxicity was greater than that of SN-38 or 5FU/FA treatment alone. Investigation of the mechanistic basis for this sequence dependence revealed that SN-38 treatment was associated with a dose- and time-dependent decrease in conversion of [5-3H]-2'-deoxyuridine to [3H]-H2O and thymidylate in intact cells. Immunoblotting failed to reveal any decrease in TS protein that could account for the decreased activity. High-performance liquid chromatography revealed that SN-38 treatment was associated with increased levels of the deoxynucleotide dTTP and decreased levels of dUTP. Flow microfluorimetry revealed that a 24-h treatment wit 10 nM SN-38 resulted in accumulation of HCT-8 cells in late S and G2 phases of the cell cycle, with a further increase in the G2 fraction during the 24 h after SN-38 removal.


CONCLUSIONS
These observations are consistent with a model in which SN-38 sequentially induces diminished DNA synthesis, elevated dTTP pools, inhibition of dUMP synthesis and enhanced toxicity of 5FU/FA. Accordingly, sequencing of irinotecan and 5FU/FA might be important in combining these agents into an effective treatment for colorectal cancer.
Mullany
S
S
Division of Oncology Research, Mayo Clinic, Rochester, MN 55905, USA.
Svingen
P A
PA
Kaufmann
S H
SH
Erlichman
C
C
D000230:Adenocarcinoma; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D002166:Camptothecin / Q000031:analogs & derivatives / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D007414:Intestinal Neoplasms; D000077146:Irinotecan; D002955:Leucovorin / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors; D013942:Thymine Nucleotides / Q000378:metabolism; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured / Q000187:drug effects
D003856:Deoxyuracil Nucleotides; D004791:Enzyme Inhibitors; D013942:Thymine Nucleotides; D059004:Topoisomerase I Inhibitors; C027078:deoxyuridine triphosphate; D000077146:Irinotecan; D013940:Thymidylate Synthase; D004264:DNA Topoisomerases, Type I; D002955:Leucovorin; C024157:thymidine 5'-triphosphate; D005472:Fluorouracil; D002166:Camptothecin