Related Experiment Video
Updated: Jul 31, 2026
Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer
Published on: July 18, 2017
Isolation and characterization of a thymidylate synthase-deficient human colon tumor cell line
D K Hoganson1, A W Williams, S H Berger
1Department of Basic Pharmaceutical Sciences, University of South Carolina, Columbia 29208, USA.
Abstract:
Following mutagenesis of the human colorectal tumor cell line HCT C with ethyl methanesulfonate, clonal sublines were isolated that survived on medium toxic to cells expressing thymidylate synthase (TS). The subline exhibiting the lowest TS activity, designated as C18, was characterized. Extracts from C18 cells were mixed with extracts from parental C cells to determine whether the TS-deficient phenotype is trans-acting. No effect was observed on the activity of TS in parental extracts. The levels of functional TS in C18 cells were analyzed by the binding of the mechanism-based inhibitor 5-fluoro-2'-deoxyuridylate (FdUMP) under conditions that allowed for the detection of 10 fmol of TS. Only a low level of FdUMP-TS complexes was detected in C18 extracts. The level of TS expression in C18 cells was similar to that in parental C cells, as indicated by immunoblot and RNA analyses. DNA sequence analysis of TS cDNA from C18 cells revealed the existence of a point mutation (C-->T) at nucleotide 647 that predicts the replacement of Ser216 by a leucine residue. That the C18 cell line was homozygous for this mutation was indicated by restriction fragment-length polymorphism analysis and by primer extension analysis. To provide additional evidence that substitution of Ser216 by a leucine residue created a defective protein, a TS-deficient bacterial strain was transformed with an expression vector containing the mutated human TS cDNA. The transformed strain exhibited thymidine auxotrophy, indicating that the mutant TS (Leu216) is nonfunctional.
Insights
A new thymidylate synthase (TS) mutation, Ser216Leu, was identified in colorectal cancer cells (HCT C). This mutation renders the thymidylate synthase protein nonfunctional, impacting cell survival.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- Thymidylate synthase (TS) is a critical enzyme in DNA synthesis.
- Drug resistance in cancer can arise from mutations in key enzymes like TS.
- Understanding TS function is vital for developing targeted cancer therapies.
Purpose of the Study:
- To investigate the molecular basis of drug resistance in a human colorectal tumor cell line (HCT C).
- To characterize a subline (C18) with significantly reduced TS activity.
- To determine the functional impact of a novel TS mutation.
Main Methods:
- Mutagenesis of HCT C cells using ethyl methanesulfonate.
- Biochemical assays to measure TS activity and inhibitor binding (FdUMP).
- Molecular analyses including immunoblotting, RNA analysis, DNA sequencing, RFLP, and primer extension.
- Functional complementation in a TS-deficient bacterial strain.
Main Results:
- A HCT C subline (C18) with low TS activity was isolated.
- C18 cells showed minimal functional TS, despite similar TS expression levels to parental cells.
- A homozygous point mutation (C647T) predicting Ser216Leu substitution in TS was identified.
- The Ser216Leu mutant TS protein was confirmed as nonfunctional in a bacterial complementation assay.
Conclusions:
- The Ser216Leu mutation in thymidylate synthase leads to a nonfunctional enzyme.
- This mutation confers resistance to TS-targeting agents in colorectal cancer cells.
- The findings provide insights into mechanisms of drug resistance and potential therapeutic targets.
