GENETIC INSTABILITY CAUSED BY LOSS OF MSH3 IN COLORECTAL CANCERS

BACKGROUND and AIM. The DNA mismatch repair (MMR) complex MutSβ comprised of a heterodimer of MMR proteins MSH3 and MSH2 recognizes specific DNA strand errors after DNA polymerase function. High levels of insertion/deletion mutations are detected within microsatellite loci containing tetranucleotide repeats in MSH3-deficient cells and termed elevated microsatellite alterations at selected tetranucleotide repeats (EMAST). There are multiple lines of evidence indicating that MutSβ also is involved in double-strand break (DSB) repair through single strand annealing or alternate non-homologous end-joining by promoting excision of unpaired single stranded DNA. We hypothesized that loss of MSH3 not only results in high levels of EMAST but also in chromosomal instability due to impairment of DSB repair. Here we created MSH3-knockout immortalized human diploid colon cells via CRISPR/Cas9 gene editing to address the role of MSH3-deficiency on genetic instability. MATERIALS AND METHODS. We created MSH3-knockout cell lines derived from parental HCEC1CT diploid near normal colon epithelial cells by targeting exon 5 of the MSH3 locus using the CRISPR/Cas9 plasmid PX458. Resultant genetically edited HCEC1CT cells were cultivated in medium supplemented with 2% fetal calf serum and other growth factors. MSH3-negative and control MSH3-positive clones were sub-cultured and chromosomal changes were monitored through passage 25. Rad51 foci formation induced by etoposide (triggering DSBs) was also examined in the MSH3-negative and positive clones. RESULTS. Approximately 100 FACS-sorted EGFP-positive clones were examined for MSH3 expression by immunofluorescence staining for MSH3, and five clones were found to be negative for MSH3 expression. All MSH3-negative clones contained deletion mutations in both alleles of exon 5 of the MSH3 gene and expressed no normal MSH3 protein. There were no off-target events detected in any of the five clones. All five MSH3-negative clones exhibited high levels of EMAST while the MSH3-positive clones did not. However, all but one MSH3-negative clone maintained a normal diploid chromosome number after prolonged passages (through passage 24). Furthermore, all MSH3-negative clones formed Rad 51 foci as efficiently as did MSH3-positive clones after etoposide treatment. DISCUSSION. We demonstrate here that isolated MSH3 deficiency is directly responsible for high levels of EMAST generated in the genome. However, MSH3-deficiency alone does not necessarily result in chromosomal abnormalities or affect Rad51 recruitment for homologous recombination repair of lesions created by etoposide treatment. We surmise that there are additional component aberrations to sole MSH3 deficiency to generate observable chromosomal instability generated within diploid cells.