Multi-level gene expression profiles affected by thymidylate synthase and 5-fluorouracil in colon cancer

Abstract

Background: Thymidylate synthase (TS) is a critical target for cancer chemotherapy and is one of the most extensively studied biomarkers for fluoropyrimidine-based chemotherapy. In addition to its critical role in enzyme catalysis, TS functions as an RNA binding protein to regulate the expression of its own mRNA translation and other cellular mRNAs, such as p53, at the translational level. In this study, a comprehensive gene expression analysis at the levels of both transcriptional and post-transcriptional regulation was conducted to identify response markers using human genome array with TS-depleted human colon cancer HCT-C18 (TS-) cells and HCT-C18 (TS+) cells stably transfected with the human TS cDNA expression plasmid.

Results: A total of 38 genes were found to be significantly affected by TS based on the expression profiles of steady state mRNA transcripts. However, based on the expression profiles of polysome associated mRNA transcripts, over 149 genes were affected by TS overexpression. This indicates that additional post-transcriptionally controlled genes can be captured with profiling polysome associated mRNA population. This unique approach provides a comprehensive overview of genes affected by TS. Additional novel post-transcriptionally regulated genes affected by 5-fluorouracil (5-FU) treatment were also discovered via similar approach.

Conclusion: To our knowledge, this is the first time that a comprehensive gene expression profile regulated by TS and 5-FU was analyzed at the multiple steps of gene regulation. This study will provide candidate markers that can be potentially used for predicting therapeutic outcomes for fluoropyrimidine-based cancer chemotherapy.

Figure 1

Effect of 5-FU on p53 and TS expression via Western immunoblot analysis. Equal amounts (25 μg) of protein extracts from both control and 5-FU treated HCT-C18 (TS+) cells were loaded and resolved with 10%

Figure 2

Clustering analysis of genes expression affected by TS overexpression with HCT-C18 (TS+) (Lane 1) vs. HCT-C18 (TS-) (Lane 2) based on the profiles of steady state total mRNAs. One-way ANOVA analysis was used for the gene clustering with p < 0.05.

Figure 3

Clustering analysis of genes expression affected by TS overexpression with HCT-C18 (TS+) (Lane 1) vs. HCT-C18 (TS-) (Lane 2) based on the profiles of polysome associated mRNAs. One-way ANOVA analysis was used for the gene clustering with p < 0.05.

Figure 4

Clustering analysis of TS dependent cytotoxicity in HCT-C18 (TS+) cells (Lane 1) treated with 10 μM 5-FU for 4 hrs (Lane 2) and 24 hrs (Lane 3) based on the expression profiles generated from steady state total mRNAs via One-way ANOVA analysis with p < 0.05.

Figure 5

Clustering analysis of TS dependent cytotoxicity in HCT-C18 (TS+) cells (Lane 1) treated with 10 μM 5-FU for 4 hrs (Lane 2) and 24 hrs (Lane 3) based on the expression profiles generated from polysome associated mRNAs via One-way ANOVA analysis with p < 0.05.

Figure 6

Clustering analysis of predictive marker genes for TS independent cytotoxicity in HCT-C18 (TS-) cells (Lane 1) treated with 10 μM 5-FU for 4 hrs (Lane 2) and 24 hrs (Lane 3) via One-way ANOVA analysis with p < 0.05.

Figure 7

Real time pathway analysis of cell-cycle related genes mediated via TS overexpression in HCT-C18 (TS+) cells using actual gene expression data. (Red color indicates overexpression. Blue color indicates decreased expression).