title: The roles of thymidylate synthase and p53 in regulating Fas-mediated apoptosis in response to antimetabolites.
abstract: Fas (CD95/Apo-1) is a member of the tumor necrosis factor receptor family. Receptor binding results in activation of caspase 8, leading to activation of proapoptotic downstream molecules. We found that expression of Fas was up-regulated >10-fold in MCF-7 breast and HCT116 and RKO colon cancer cell lines after treatment with IC(60) doses of 5-fluorouracil (5-FU) and raltitrexed (RTX). Combined treatment with the agonistic Fas antibody CH-11 and either 5-FU or RTX resulted in a highly synergistic induction of apoptosis in these cell lines. Similar results were obtained for another antifolate, Alimta. Induction of thymidylate synthase expression inhibited Fas induction in response to RTX and Alimta, but not in response to 5-FU. Furthermore, thymidylate synthase induction abrogated the synergy between CH-11 and both antifolates but had no effect on the synergistic interaction between 5-FU and CH-11. Inactivation of p53 in MCF-7 and HCT116 cell lines blocked 5-FU- and antifolate-mediated up-regulation of Fas. Furthermore, Fas was not up-regulated in response to 5-FU or antifolates in the p53-mutant H630 colon cancer cell line. Lack of Fas up-regulation in the p53-null and -mutant lines abolished the synergistic interaction between 5-FU and CH-11. Interestingly, synergy was still observed between the antifolates and CH-11 in the p53-null HCT116 and p53-mutant H630 cell lines, although this was significantly reduced compared with the p53 wild-type cell lines. Our results indicate that Fas is an important mediator of apoptosis in response to both 5-FU and antifolates.
authors.affiliation: Department of Oncology, Cancer Research Centre, Queen's University Belfast, Belfast, Northern Ireland.
authors.forename: Daniel Broderick
authors.initials: DB
authors.lastname: Longley
authors.forename: Wendy Louise
authors.initials: WL
authors.lastname: Allen
authors.forename: Ultan
authors.initials: U
authors.lastname: McDermott
authors.forename: Timothy Richard
authors.initials: TR
authors.lastname: Wilson
authors.forename: Tariq
authors.initials: T
authors.lastname: Latif
authors.forename: John
authors.initials: J
authors.lastname: Boyer
authors.forename: Maria
authors.initials: M
authors.lastname: Lynch
authors.forename: Patrick Gerard
authors.initials: PG
authors.lastname: Johnston
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000963:Antimetabolites / Q000494:pharmacology*; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005472:Fluorouracil / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000378:metabolism / Q000494:pharmacology; D016158:Genes, p53; D006801:Humans; D020128:Inhibitory Concentration 50; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D015398:Signal Transduction; D013752:Tetracycline / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000502:physiology*; D019076:Transgenes; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism / Q000502:physiology*; D015854:Up-Regulation; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D000963:Antimetabolites; C481004:CH-11 anti-fas antibody, human; D005493:Folic Acid Antagonists; D016159:Tumor Suppressor Protein p53; D019014:fas Receptor; D013940:Thymidylate Synthase; D013752:Tetracycline; D005472:Fluorouracil