Limits to thymidylate synthase and TP53 genes as predictive determinants for fluoropyrimidine sensitivity and further evidence for RNA-based toxicity as a major influence

Abstract

The major determinants of 5-flurouracil (5-FU) response would seem, based on accumulated literature, to be thymidylate synthase (TYMS, TS) expression levels, TS gene modifications, and TP53 status. We tested 5-FU sensitivity in yeast and human cancer cell models in which TS or TP53 alleles and expression were varied. Polymorphic TS tandem repeat status, TS expression levels reported, TS intragenic mutations, and TP53 status in outbred and experimental cancer cell lines did not predict 5-FU sensitivity or resistance. Novel observations included a dose-resistant persistence of unbound TS protein in many cancers and, upon 5-FU treatment of the colon cancer cell line, HCT116, evidence of allelic switching favoring transcripts of the mutant TS allele. The reported alleles having an intragenic mutation could not be causally associated with major degrees of 5-FU sensitivity. In yeast, TS protein was altered upon treatment with FdUMP, but 5-FU toxicity seemed to be largely RNA-based, being rescued by uridine rather than by thymidine. Cancer cell lines were also rescued from 5-FU toxicity with uridine rather than thymidine. Additionally, a TS (CDC21) knockout yeast strain, obviating any potential role for TS protein as a target, was hypersensitive to 5-FU. When denatured proteins from cancer cells treated with radiolabeled 5-FU were labeled, species with alternative molecular weights other than TS were visualized, providing further evidence for alternative 5-FU protein targets. These data emphasize that TS and TP53 status do not consistently explain the variance in responses of fluoropyrimidine-treated cancer cells, in part due to RNA-based toxicity.

Figure 1

TS status and detection of classic complexes in cell culture. (A) and (B) We determined the TS protein levels of the cell lines generated for survival assays. Non-specific bands (NS) reflect equal loading of protein per lane. Cell lines were treated as indicated with 5-FU (+) or untreated (-) before cell lysates were made for immunoblot analysis. (C) The TS-TR assay distingishes among the upper band (3R) and lower band (2R) as separated on an agarose gel. (D) Minimal immunodetectable TS protein exists in the the mutated HCT C 18 cell line. Equal protein was loaded.

Figure 2

Survival assays of cancer cell lines treated with 5-FU. Each panel is a representative single experiment, one of at least three independent experiments. Each data point represents the mean of three replicate wells from the experiment (error bars, S.E.M.). (A) From left to right: unrelated pancreatic cancer cell lines; MSI colorectal cancer lines; HCT116 lines evolved to be drug-resistant as compared to parental HCT116 and DLD-1 cells. (B) From left to right: manipulated TS status in isogenic RKO cell lines; manipulated TS status in the background of the TS+/- cell line; cells having stable overexpression of the HCT116 mutation as compared to other HCT116 cell lines. (C) From left to right: isogenic p53-null and wild type p53 cell lines; manipulated TS status in isogenic HCT116 cell lines, HCT1162 represents a second flask of HCT116 parental cells; HCT C isogenic cell lines. (D) From left to right: uridine (open bars), but not thymidine (filled bars), rescued cancer cells from 5-FU toxicity. Cell line clones having different genetically manipulated TS levels were derived from the same parental line (RKO) and tested for uridine and thymidine rescue from 5-FU toxicity (25μM to 100 μM). A, an RKO parental clone; B, the TS+-.add back clone; C, TS+/- cells. The parental RKO line was tested at an extended range of 5-FU (up to 200μM). Bars of a given graph represent individual determinations obtained in a single experiment.

Figure 3

Upregulation of TS expression and allele-specific expression in HCT116 cells. (A) TS gene sequences of bulk cDNA generated from three 5-FU-treated and one untreated HCT116 cultures. (B) TS expression levels measured by real-time PCR from cDNA of the indicated 5-FU-treated and untreated HCT116 and RKO cells. The expression of TS was first normalized to that of GAPDH, and the fold change calculated between treated and untreated cultures. 116.R2 (- 5-FU) represents a resistant line withdrawn from exposure to 5-FU. Columns heights reflect the mean, and the error bars depict the SEM, among three separate experiments.

Figure 4

Evidence for alternative 5-FU targets from yeast and cancer cells. (A) The yeast TS knockout strain is sensitive to 5-FU as compared to the wild type parental strain. (B) Uridine can rescue yeast TS-knockout strains from 5-FU toxicity. (C) Model validation: No detectable TS protein exists in the yeast knockout strain. Equal protein was loaded in each lane. (D) Two cancer cell lines (HCT116 and RKO) were treated with C¹⁴-labeled 5-FU, lysed, and proteins separated on an SDS-polyacrylamide gel. Three species were detected by autoradiography at approximately 50kDa, 36 kDa (TS), and 20 kDa.