Chang D K, Ricciardiello L, Goel A, Chang C L, Boland C R
Department of Medicine and Cancer Center, University of California, San Diego, California 92093, USA.
J Biol Chem. 2000 Jun 16;275(24):18424-31. doi: 10.1074/jbc.M001140200.
Steady-state levels of human DNA mismatch repair (MMR) transcripts and proteins 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-proficient cells, hMSH2 is the most abundant MMR protein and is expressed 3 to 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 with protein expression. hMSH2-mutated LoVo cells did not express detectable hMSH3 or hMSH6 proteins. Similarly, hMLH1-mutated HCT116 cells did not express detectable hMLH1 or hPMS2 protein, whereas in hMLH1-restored HCT116+ch3 cells, hPMS2 protein was re-expressed. In hMSH6-mutated HCT15 cells, both hMSH3 protein and mRNA were increased. In SV40-transformed lung fibroblasts, all MMR mRNAs and proteins examined were expressed at levels 1.5-5-fold higher than in their nontransformed counterpart. The steady-state levels of MMR proteins indicate that substantially more hMutS proteins, which are involved 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 protein depends upon hMLH1. When the hMSH6 is mutationally inactivated, hMSH3 increases by both transcriptional up-regulation and enhanced protein stability. A balanced up-regulation of all of the components was seen after viral transformation in a fibroblast model. Quantitative changes of the MMR components are a potential mechanism to modify the DNA MMR capabilities of a cell.
通过新开发的竞争性定量逆转录-聚合酶链反应和用纯化蛋白标准化的蛋白质印迹分析，在错配修复功能正常和缺陷的细胞系中检测人DNA错配修复（MMR）转录本和蛋白的稳态水平。在错配修复功能正常的细胞中，hMSH2是最丰富的MMR蛋白，其表达量比hMLH1高3至5倍。hMLH1蛋白的表达量比hPMS2高1.5至2.5倍。mRNA表达的稳态水平与蛋白表达密切相关。hMSH2突变的LoVo细胞不表达可检测到的hMSH3或hMSH6蛋白。同样，hMLH1突变的HCT116细胞不表达可检测到的hMLH1或hPMS2蛋白，而在hMLH1恢复的HCT116+ch3细胞中，hPMS2蛋白重新表达。在hMSH6突变的HCT15细胞中，hMSH3蛋白和mRNA均增加。在SV40转化的肺成纤维细胞中，所有检测的MMR mRNA和蛋白的表达水平比未转化的对应细胞高1.5至5倍。MMR蛋白的稳态水平表明，与hMutL蛋白相比，参与DNA错配识别的hMutS蛋白的含量要多得多。hMSH3和hMSH6蛋白的稳定性似乎取决于hMSH2蛋白的存在，同样，hPMS2蛋白的稳定性取决于hMLH1。当hMSH6发生突变失活时，hMSH3通过转录上调和增强的蛋白稳定性而增加。在成纤维细胞模型中，病毒转化后所有组分均出现平衡上调。MMR组分的定量变化是改变细胞DNA错配修复能力的潜在机制。
