title: Biological activity of the multitargeted antifolate, MTA (LY231514), in human cell lines with different resistance mechanisms to antifolate drugs.
abstract: Prior studies have indicated that MTA requires intracellular polyglutamation for optimal cytotoxic effect and that these polyglutamates potently inhibit several key enzymes of folate metabolism, including thymidylate synthase (TS), dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase (GARFT). In the present studies, we have investigated the mechanistic basis for resistance to MTA in several human tumor cell lines. The cell lines were developed for resistance by the gradual exposure to stepwise (fivefold) increases in the concentration of MTA over a 5-month period. The degree of resistance was 140-fold for GC3 colon carcinoma, 117-fold for HCT-8 ileocecal carcinoma, and 729-fold for CCRF-CEM leukemia cells adapted to 2 micromol/L MTA. The lines had strong cross-resistance (>3,200-fold) to raltitrexed. Only modest resistance was noted for methotrexate and the GARFT inhibitor, LY309887. The cytotoxicity of MTA in wild-type cells was only partially alleviated by thymidine addition (5 micromol/L) and complete protection required the addition of both hypoxanthine (100 micromol/L) and thymidine. In contrast, thymidine alone totally lacked protective activity in the MTA-resistant lines. The cells either demonstrated a GARFT-like reversal pattern (complete protection by hypoxanthine) for GC3MTA or a dihydrofolate reductase-like reversal pattern (complete protection by the combination of hypoxanthine and thymidine) for HCT-8MTA and CCRF-CEM(MTA) cells. Cellular resistance was multifactorial and stable on removal of selective pressure. Only GC3MTA cells showed increased TS activity (approximately 40-fold). Accumulations of 3H-MTA at 24 hours in CCRF-CEM(MTA), HCT-8MTA, and GC3MTA cells were 2%, 6%, and 46% of wild-type values, respectively. We also evaluated the cytotoxic activity of MTA in MCF-7 breast carcinoma and H630 colon carcinoma cells selected for resistance to raltitrexed and 5-fluorouracil, respectively, via TS amplification (provided by Dr P.G. Johnston, Belfast, Ireland). These cells demonstrated more than 200-fold less resistance to MTA compared with raltitrexed and MTA-induced cytotoxicity was prevented by hypoxanthine. These studies suggest that in addition to TS modulation, secondary targets emerge during the development of MTA resistance.
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Schultz
authors.forename: V J
authors.initials: VJ
authors.lastname: Chen
authors.forename: J R
authors.initials: JR
authors.lastname: Bewley
authors.forename: E F
authors.initials: EF
authors.lastname: Roberts
authors.forename: C
authors.initials: C
authors.lastname: Shih
authors.forename: J A
authors.initials: JA
authors.lastname: Dempsey
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000378:metabolism / Q000494:pharmacology*; D001692:Biological Transport; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D005493:Folic Acid Antagonists / Q000378:metabolism / Q000494:pharmacology*; D005971:Glutamates / Q000378:metabolism / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D000068437:Pemetrexed; D011799:Quinazolines / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D005971:Glutamates; D011799:Quinazolines; D013876:Thiophenes; D000068437:Pemetrexed; D006147:Guanine; D013940:Thymidylate Synthase; C068874:raltitrexed