Steady-state regulation of the human DNA mismatch repair system

Steady-state levels of human DNA mismatch repair (MMR) transcripts and prot eins were measured in MMR-proficient and -deficient cell lines by the newly developed competitive quantitative reverse transcription- polymerase chain reaction and Western analysis normalized with purified proteins. In MMR-pr oficient cells, hMSH2 is the most abundant MMR protein and is expressed 3 t o 5 times more than hMLH1. The hMLH1 protein was expressed 1.5 to 2.5 times more than hPMS2. Steady-state levels of mRNA expression correlated well wi th protein expression, hMSH2-mutated LoVo cells did not express detectable hMSH3 or hMSH6 proteins, Similarly, hMLH1-mutated HCT116 cells did not expr ess detectable hMLH1 or hPMS2 protein, whereas in hMLH1-restored HCT116+ch3 cells, hPMS2 protein was reexpressed. In hMSH6-mutated HCT15 cells, both h MSH3 protein and mRNA were Increased. In SV40-transformed lung fibroblasts, all MMR mRNAs and proteins examined were expressed at levels 1.5-5-fold hi gher than in their nontransformed counterpart. The steady-state levels of M MR proteins indicate that substantially more hMutS proteins, which are invo lved in DNA mismatch recognition, are present in comparison with the hMutL proteins. Stability of hMSH3 and hMSH6 proteins appears to depend upon the presence of the hMSH2 protein, and, similarly, the stability of the hPMS2 p rotein depends upon hMLH1. When the hMSH6 is mutationally inactivated, hMSH 3 increases by both transcriptional up-regulation and enhanced protein stab ility. A balanced up-regulation of all of the components was seen after vir al transformation in a fibroblast model. Quantitative changes of the MMR co mponents are a potential mechanism to modify the DNA MMR capabilities of a cell.