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Decreased Stability of Transforming Growth Factor beta Type II Receptor mRNA in RER+ Human Colon Carcinoma Cells
Jiang1, Tillekeratne, Brattain
1Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43699
Abstract:
Transforming growth factor beta (TGF-beta) is a potent inhibitor of cell growth and tumor progression. Previous work has shown that loss of functional TGF-beta type II receptor (RII) due to a frameshift mutation in the 5' half of the RII gene leads to TGF-beta resistance in a highly progressed, RER+ human colon carcinoma cell line designated HCT116. Expression of this mutated RII gene was highly repressed in RER+ cell lines such as HCT116 and RKO, as analyzed by RNase protection assays. Nuclear run-on and RII promoter-reporter (CAT) assays showed that the transcriptional levels of the RII gene in these RER+ cells were not reduced, compared to RII-expressing cells. However, the half-lives of the RII mRNA, as analyzed by RNase protection assays following actinomycin D treatment, were significantly decreased. This suggested that the decreased expression of the RII gene mutant was due to decreased mRNA stability. Furthermore, RII mRNA from HCT116 transfected with wild-type RII had a longer half-life than the endogenous mutated RII mRNA. A dominant negative RII mutant, which encodes a similarly truncated RII protein as HCT116 but lacks the extensive 3' untranslated region of RII mRNA, gave the same half-life as endogenous wild-type RII mRNA. We conclude that the frameshift mutation which results in a premature stop codon in the 5' half of the mRNA transcript accounts for the reduced RII mRNA levels in RER+ cells.
Insights
Loss of functional transforming growth factor beta type II receptor (RII) in colon cancer cells is due to decreased mRNA stability, not reduced transcription. A frameshift mutation causes premature stop codons, leading to reduced RII mRNA levels.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- Transforming growth factor beta (TGF-beta) inhibits cell growth and tumor progression.
- Loss of functional TGF-beta type II receptor (RII) confers TGF-beta resistance.
- A frameshift mutation in the RII gene is implicated in RER+ human colon carcinoma cell lines.
Purpose of the Study:
- To investigate the mechanism of reduced RII gene expression in RER+ colon carcinoma cells.
- To determine if reduced RII expression is due to transcriptional repression or post-transcriptional regulation.
- To identify the role of mRNA stability in RII gene expression.
Main Methods:
- RNase protection assays to analyze RII gene expression and mRNA half-life.
- Nuclear run-on assays to assess transcriptional levels.
- Promoter-reporter (CAT) assays to evaluate RII promoter activity.
- Transfection studies with wild-type and mutant RII constructs.
Main Results:
- RII gene expression was repressed in RER+ cell lines (HCT116, RKO).
- Transcriptional levels of RII were not reduced, indicating post-transcriptional regulation.
- RII mRNA half-life was significantly decreased in RER+ cells.
- A frameshift mutation leading to a premature stop codon in the 5' half of the RII mRNA transcript was identified.
Conclusions:
- Reduced RII mRNA levels in RER+ cells are primarily due to decreased mRNA stability.
- The frameshift mutation and resulting premature stop codon are responsible for the reduced mRNA stability and expression.
- These findings elucidate a key mechanism of TGF-beta resistance in colon cancer progression.