Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency

GC₃/cl human colon adenocarcinoma cells were treated with the mutagen ethyl methanesulfonate, and three clones deficient in thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) activity were selected and characterized. Growth in medium deficient in thymidine caused cell death in two clones (TS^-c₁ and TS^-c₃), whereas one clone (TS^-c₂) showed limited growth. Growth correlated with thymidine synthase activity and 5-fluoro-2'-deoxyuridine 5'-monophosphate-binding capacity and with incorporation of 2'-deoxy[6-³H]uridine into DNA. In the presence of optimal thymidine, growth rates were only 5-18% that of parental clone (GC₃/c1), which grew equally well in thymidine-deficient or -replete medium. Analysis of poly(A)⁺ RNA showed normal levels of a 1.6-kilobase transcript in TS^-c₁ and TS^-c₂ but decreased levels (~6% control) in TS^-c₃. Clone TS^-c₃ was 32-, 750-, and >100,000-fold more resistant than the parental clone to 5-fluorouracil, 5-fluoro-2'-deoxyuridine, and methotrexate, respectively. When inoculated into athymic nude mice, each TS^- clone produced tumors, demonstrating continued ability to proliferate in vivo.