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

Abstract

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/C1 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 µm 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⁻ C1/Cl clone in the absence of dThd. Activation of dTMP biosynthesis de novo was demonstrated within 6 h of exposing cells to 20 µm [6R,S]5-CHO-H₄PteGlu, 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 [6R,S]5-CHO-H₄PteGlu, whereas 33.5% labeled in the presence of 20 µm exogenous folate. Relative to the parental (TS⁺) clone, there was a >87,500-, an 8,182-, and a 425-fold higher requirement for 5-methyltetrahydrofolate ([6R,S]5-CH₃-H₄PteGlu), PteGlu, and [6R,S]5-CH₃-H₄PteGlu to support 50% maximal colony formation in the absence of dThd.

Quantitative analysis of the combined pools of 5,10-methylenetrahydrofolate (CH₂-H₄PteGlu_a) and H₄PteGlu_a showed that parental GC₃/C1 cells had higher endogenous folate pools compared to TS⁻C1/C1 cells [168 ± 40 (SD) and 10.9 ± 0.3 fmol/10⁶ cells, respectively]. Qualitatively the distribution of polyglutamate species and their redistribution in cells exposed to 20 µm [6R,S]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 CH₂-H₄PteGlu_n and H₄PteGlu_n. 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/C1 (TS⁺) cells, [6R]CH₂-H₄PteGlu₁ acted as a cofactor in the release of ³H₂O 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⁻ C1/C1 cells in the presence of [6R]CH₂-H₄PteGlu₅. The apparent K_m for [6R]CH₂-H₄PteGlu₅ was 14.2 ± 3.3 µm for the parental cells and 71 ± 15 µm for enzyme in TS⁻C1/C1.

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-H₄PteGlu_a. 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 CH₂-H₄PteGlu_a 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.