title: Oxidative stress induces nuclear-to-cytosol shift of hMSH3, a potential mechanism for EMAST in colorectal cancer cells.
abstract: BACKGROUND
  Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable CRCs where hypermethylation of the DNA mismatch repair (MMR) gene hMLH1's promoter is causal, the precise cause of EMAST is not clearly defined but points towards hMSH3 deficiency.
  
  
  OBJECTIVE
  To examine if hMSH3 deficiency causes EMAST, and to explore mechanisms for its deficiency.
  
  
  METHODS
  We measured -4 bp framshifts at D8S321 and D20S82 loci within EGFP-containing constructs to determine EMAST formation in MMR-proficient, hMLH1⁻/⁻, hMSH6⁻/⁻, and hMSH3⁻/⁻ CRC cells. We observed the subcellular location of hMSH3 with oxidative stress.
  
  
  RESULTS
  D8S321 mutations occurred 31-and 40-fold higher and D20S82 mutations occurred 82-and 49-fold higher in hMLH1⁻/⁻ and hMSH3⁻/⁻ cells, respectively, than in hMSH6⁻/⁻ or MMR-proficient cells. hMSH3 knockdown in MMR-proficient cells caused higher D8S321 mutation rates (18.14 and 11.14×10⁻⁴ mutations/cell/generation in two independent clones) than scrambled controls (0 and 0.26×10⁻⁴ mutations/cell/generation; p<0.01). DNA sequencing confirmed the expected frameshift mutations with evidence for ongoing mutations of the constructs. Because EMAST-positive tumors are associated with inflammation, we subjected MMR-proficient cells to oxidative stress via H₂O₂ to examine its effect on hMSH3. A reversible nuclear-to-cytosol shift of hMSH3 was observed upon H₂O₂ treatment.
  
  
  CONCLUSIONS
  EMAST is dependent upon the MMR background, with hMSH3⁻/⁻ more prone to frameshift mutations than hMSH6⁻/⁻, opposite to frameshift mutations observed for mononucleotide repeats. hMSH3⁻/⁻ mimics complete MMR failure (hMLH1⁻/⁻) in inducing EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, hMSH3-deficiency appears to be the event that commences EMAST. Oxidative stress, which causes a shift of hMSH3's subcellular location, may contribute to an hMSH3 loss-of-function phenotype by sequestering it to the cytosol.
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Stephanie S
authors.initials: SS
authors.lastname: Tseng-Rogenski
authors.forename: Heekyung
authors.initials: H
authors.lastname: Chung
authors.forename: Maike B
authors.initials: MB
authors.lastname: Wilk
authors.forename: Shuai
authors.initials: S
authors.lastname: Zhang
authors.forename: Moriya
authors.initials: M
authors.lastname: Iwaizumi
authors.forename: John M
authors.initials: JM
authors.lastname: Carethers
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D003600:Cytosol / Q000378:metabolism*; D053843:DNA Mismatch Repair / Q000235:genetics; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D016368:Frameshift Mutation / Q000235:genetics; D055785:Gene Knockdown Techniques; D056426:Genetic Loci / Q000235:genetics; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D000074081:MutS Homolog 3 Protein; D018384:Oxidative Stress / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; C078444:MSH3 protein, human; D000074081:MutS Homolog 3 Protein