Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency

P. J. Houghton, G. S. Germain, B. J. Hazelton, J. W. Pennington, J. A. Houghton

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27 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1377-1381
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume86
Issue number4
DOIs
StatePublished - 1989

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  • General

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