Mutation(s) of the Thymidylate Synthase Gene of Human Adenocarcinoma Cells Causes a Thymidylate Synthase-negative Phenotype That Can Be Attenuated by Exogenous Folates

Peter J. Houghton, Atiqur Rahman, Janet A. Houghton

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18 Scopus citations


Biological characterization of a human colon adenocarcinoma cell line deficient in thymidylate synthase (TS~) is described. The clone, designated TS~C1/C1, was derived from the parental line GC3/CI by selection in medium containing aminopterin, thymidine (dThd), and low concentrations of 5-formyltetrahydrofolate (5-CHO-H4PteGlu), and was subsequently reselected by single-step cloning in 500 MM methotrexate in the presence of dThd. This clone retained its TS” phenotype, was highly resistant to methotrexate (>100,000-fold), and remained tumorigenic in mice (P. J. Houghton, et al., Proc. Natl. Acad. Sci. USA, 86:1377-1381, 1989). In studies reported, it is shown that high levels of exogenous folate can support the growth of the TS” Cl/Cl clone in the absence of dThd. Activation of dTMP biosynthesis de novo was demonstrated within 6 h of exposing cells to 20 MM [6,S]5-CHO-FL,PteGlu, and >80% of activity was lost within 24 h of removing this folate from the medium. The labeling index was determined by autoradiographic techniques using [6-3H]2′-deoxyuridine. None of the >6,000 cells radiolabeled in the absence of [6RvS15-CHO-H4PteGlu, whereas 33.5% labeled in the presence of 20 MM exogenous folate. Relative to the parental (TS*) clone, there was a >87,500-, an 8,182-, and a 425-fold higher requirement for 5-methylte-trahydrofolate ([6RvS15-CH3-H4PteGlu), PteGlu, and [6RyS]5-CH3-HUPteGlu to support 50% maximal colony formation in the absence of dThd. Quantitative analysis of the combined pools of 5,10-methylenetetra-hydrofolate (CHi-r ^ PteGluJ and H4PteGlu. showed that parental GC3/ CI cells had higher endogenous folate pools compared to TSX1/C1 cells [168 ± 40 (SD) and 10.9 ± 03 fmol/106 cells, respectively]. Qualitatively the distribution of polyglutamate species and their redistribution in cells exposed to 20 MM [6RyS]5-CHO-H4PteGlu were similar in the two lines. Analysis of pools in a second, independently derived, TS” clone (TS“C3/ C3, a transcription-negative mutant) demonstrated undetectable levels of CHrl ^ PteGlu,, and HiPteGlu*. This line cannot be rescued by exogenous folate. The data thus suggest that deletion of dTMP synthase activity may cause redistribution of reduced folate pools. In cytosolic extracts from parental GC3/CI (TS+) ceUs, [6/qCHr-FUPteGlu, acted as a cofactor in the release of 3H20 from [5-3H]dUMP, whereas no activity was detected in cytosols from TS~C1/C1. In contrast dTMP synthase activity was detected in cytosols from TS” Cl/Cl cells in the presence of [6RlCH2-rL.PteGlu5. The apparent Ka for [6R]CH2-HUPteGlu5 was 14.2 ± 33 MM for the parental ceUs and 71 ± 15 MM for enzyme inTS“Cl/Cl. The sequence of dTMP synthase complementary DNA from base positions 297 to 974 (relative to the first base of the translation initiation codon) was determined after polymerase chain reaction amplification and by primer extension analysis. Two mutations were determined. At base position 652 there was a G to A transition and at base position 766 a C to T mutation. These would result in an Asp to Asn substitution at codon 218 and a His to Tyr alteration at amino acid residue 256, both highly conserved residues. Data support the hypothesis that the TS“C1/C1 clone is deficient in functional dTMP synthase activity due to a mutation that reduces the affinity for CH2-H4PteGluJl. Under physiological folate concentrations the phenotype is TS”, whereas in high concentrations of exogenous folate the TS+ phenotype is induced. It is likely that reduced affinity for CH ^ r ^ PteGlu, contributes to the “folate-sensitive” phenotype in this clone. It is of significance that, due to the ability to maintain this clone either as the TS” or TS* phenotype, it will be possible to determine the significance of the de novo synthesis of dTMP for repair processes subsequent to cellular damage by different classes of cytotoxic agents without the use of inhibitors that may have sites of action in addition to dTMP synthase.

Original languageEnglish (US)
Pages (from-to)558-565
Number of pages8
JournalCancer Research
Issue number3
StatePublished - Feb 1992
Externally publishedYes

ASJC Scopus subject areas

  • Oncology
  • Cancer Research


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