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MSH2 and MLH1 have a central role in correcting mismatches in DNA occurring during DNA replication and have been implicated in the engagement of apoptosis induced by a number of cytotoxic anticancer agents. The function of MLH1 is not clearly defined, although it is required for mismatch repair (MMR) and engagement of apoptosis after certain types of DNA damage. In order to identify other partners of MLH1 that may be involved in signalling MMR or apoptosis, we used human MLH1 in yeast two-hybrid screens of normal human breast and ovarian cDNA libraries. As well as known partners of MLH1 such as PMS1, MLH3 and MBD4, we identified the carboxy terminus of the human c-MYC proto-oncogene as an interacting sequence. We demonstrate, both in vitro by yeast two-hybrid and GST-fusion pull-down experiments, as well as in vivo by coimmunoprecipitation from human tumour cell extracts, that MLH1 interacts with the c-MYC protein. We further demonstrate that the heterodimeric partner of c-MYC, MAX, interacts with a different MMR protein, MSH2, both in vitro and in vivo. Using an inducible c-MYC-ER™ fusion gene, we show that elevated c-MYC expression leads to an increased HGPRT mutation rate of Rat1 cells and an increase in the number of frameshift mutants at the HGPRT locus. The effect on HGPRT mutation rate is small (2–3-fold), but is consistent with deregulated c-MYC expression partially inhibiting MMR activity.

Activation of nuclear β-catenin and expression of its transcriptional targets promotes chronic myeloid leukemia (CML) progression, tyrosine kinase inhibitor (TKI) resistance, and leukemic stem cell self-renewal. We report that nuclear β-catenin has a role in leukemia cell-intrinsic but not -extrinsic BCR-ABL1 kinase-independent TKI resistance. Upon imatinib inhibition of BCR-ABL1 kinase activity, β-catenin expression was maintained in intrinsically resistant cells grown in suspension culture and sensitive cells cultured in direct contact (DC) with bone marrow (BM) stromal cells. Thus, TKI resistance uncouples β-catenin expression from BCR-ABL1 kinase activity. In β-catenin reporter assays, intrinsically resistant cells showed increased transcriptional activity versus parental TKI-sensitive controls, and this was associated with restored expression of β-catenin target genes. In contrast, DC with BM stromal cells promoted TKI resistance, but had little effects on Lef/Tcf reporter activity and no consistent effects on cytoplasmic β-catenin levels, arguing against a role for β-catenin in extrinsic TKI resistance. N-cadherin or H-cadherin blocking antibodies abrogated DC-based resistance despite increasing Lef/Tcf reporter activity, suggesting that factors other than β-catenin contribute to extrinsic, BM-derived TKI resistance. Our data indicate that, while nuclear β-catenin enhances survival of intrinsically TKI-resistant CML progenitors, it is not required for extrinsic resistance mediated by the BM microenvironment.

Loss of expression of the human DNA mismatch repair (MMR) gene, hMLH1, is seen in a number of tumour cell lines resistant to a variety of cytotoxic drugs. The aim of this study was to identify other proteins that interact with hMLH1 to attempt to further elucidate its role in MMR and the engagement of downstream damage response pathways. A yeast two-hybrid system, an in vivo system for detecting protein-protein interactions was utilised for this purpose. Fifteen known and five unknown genes were identified as encoding proteins interacting with hMLH1. These included three known hMLH1 binding proteins, hMLH3, hPMS1 and MED1. Amongst the other genes identified was the proto-oncogene c-MYC, a gene previously implicated in genetic instability and apoptosis. Using in vitro derived mutants of c-MYC, it has been shown that hMLH1 interacts with the leucine-zipper domain of c-MYC. The effect of elevated c-MYC expression on functional MMR was examined. An inducible c-MYC expression system, Rat-1 fibroblasts expressing c-MYCERTM, a fusion of c-MYC to the hormone binding domain of the oestrogen receptor was utilised. Elevated expression of c-MYC did not effect the mismatch specific binding complex activity in these cells as measured in EMSA experiments. However c-MYC overexpression utilising the Rat-1 cMYCERTM system was shown to result in a mutator phenotype in these cells. The results suggest there may be a link between the mutator phenotype, induced through overexpression of c-MYC, and loss of MMR. Overexpression of c-MYC, which is associated with many cancers, may result in the sequestration of hMLH1 preventing functional MMR. The interaction between hMLH1 and c-MYC is proposed to act in a DNA damage response pathway which is disrupted upon aberrant c-MYC expression.