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Published on: July 28, 2010
MSH3-deficiency initiates EMAST without oncogenic transformation of human colon epithelial cells
Christoph Campregher1, Gerald Schmid, Franziska Ferk
1Christian Doppler Laboratory for Molecular Cancer Chemoprevention, Division of Gastroenterology and Hepatology, Department of Medicine 3, Medical University of Vienna, Vienna, Austria.
Background/Aim:
Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency.
Methods:
HCT116 and HCT116+chr3 (both MSH3-deficient) and primary human colon epithelial cells (HCEC, MSH3-wildtype) were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs) were assessed by Comet assay.
Results:
Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10(-4)) at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50), apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold.
Conclusions:
MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon carcinogenesis.
Insights
MSH3-deficiency in human colon cells causes microsatellite instability and DNA damage but does not lead to cancer. This suggests MSH3-deficiency alone is insufficient for driving human colon carcinogenesis.
Area of Science:
- Molecular biology
- Genetics
- Cancer research
Background:
- Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is linked to MSH3-deficiency in sporadic colorectal cancer.
- The role of EMAST in human oncogenesis is debated, as Msh3-deficient mice do not show enhanced cancer development.
Purpose of the Study:
- To investigate the molecular consequences of MSH3-deficiency in human colon epithelial cells.
- To determine if MSH3-deficiency alone can drive oncogenic transformation in humans.
Main Methods:
- Utilized reporter plasmids to detect frameshift mutations in MSH3-deficient and wildtype human colon cells.
- Employed shRNA to silence MSH3, followed by proteomics to analyze protein expression changes.
- Assessed oncogenic transformation using colony formation assays and DNA damage via Comet assays.
Main Results:
- MSH3-deficiency led to increased frameshift mutations at [AAAG]17 repeats, consistent with EMAST.
- Significant proteome alterations were observed, including changes in DNA repair and apoptosis pathways.
- Despite increased double-strand breaks, MSH3-silencing did not induce oncogenic transformation.
Conclusions:
- MSH3-deficiency in human colon cells results in EMAST, DNA damage, and proteomic changes.
- MSH3-deficiency alone is unlikely to be the sole driver of human colon cancer development.
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