Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency.
GC3/c1 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 the 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 (approximately 6% control) in TS- c3. Clone TS- c3 was 32-, 750-, and greater than 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.
Houghton
P J
PJ
Department of Biochemical and Clinical Pharmacology, Saint Jude Children's Research Hospital, Memphis, TN 38101.
Germain
G S
GS
Hazelton
B J
BJ
Pennington
J W
JW
Houghton
J A
JA
D000230:Adenocarcinoma / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D000818:Animals; D002455:Cell Division; D002460:Cell Line; D002470:Cell Survival; D002999:Clone Cells; D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D004261:DNA Replication; D003857:Deoxyuridine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D009154:Mutation; D009368:Neoplasm Transplantation; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D013940:Thymidylate Synthase / Q000172:deficiency*; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000201:enzymology*
D013940:Thymidylate Synthase; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase; D013936:Thymidine; D003857:Deoxyuridine