MED1 MUTATIONS IN COLORECTAL CANCER

转移性结直肠癌的治疗在很大程度上是不令人满意的，迫切需要新的治疗方法或改进现有治疗方法的患者选择。由于DNA修复基因与结直肠癌的生物学和发病机制密切相关，也可能影响对DNA损伤化疗的反应，因此它们作为新的预后和预测因素具有可行性。在以错配修复蛋白MLH1为诱饵的酵母双杂交筛选中，我们克隆了一个新的人类DNA修复基因MED1。MED1蛋白在功能上与人类细胞中的MLH1相互作用，与细菌DNA修复糖基酶/裂解酶同源，并结合cpg甲基化的DNA，这表明胞嘧啶甲基化在哺乳动物DNA修复中起着新的作用。MED1显示胸腺嘧啶和尿嘧啶糖基酶活性，从G:T和G:U错配中去除这些碱基。这表明MED1可能通过5-甲基胞嘧啶和胞嘧啶分别自发脱胺为胸腺嘧啶和尿嘧啶来抵消突变。我们还发现MED1有效地从DNA中去除5-氟尿嘧啶，而5-氟尿嘧啶是结肠直肠癌全身治疗的主要成分。最近，我们获得的证据表明，MED1基因在人类结直肠癌和结外癌中经常发生突变（25%的病例），表现出微卫星不稳定性。在结直肠癌标本中，我们也检测到MED1位点的杂合性缺失。本项目的假设是：MED1是确保人类细胞基因组保真度的机制的重要组成部分；MED1突变与结直肠癌的发病机制有关；并且它可能在对化疗药物的耐药性/敏感性中起作用。我们提出的实验旨在解决：1)MED1突变在结直肠癌中的性质和频率；2) MED1突变是否为预后因素并影响治疗反应。

The therapy of metastatic colorectal cancer is largely unsatisfactory and new treatments or improved patient selection for the currently available treatments are greatly needed. DNA repair genes represent viable candidates as novel prognostic and predictive factors, because they are intimately connected with colorectal cancer biology and pathogenesis, and may also affect response to DNA-damaging chemotherapy. In a yeast two-hybrid screening with the mismatch repair protein MLH1 as "bait", we cloned MED1, a novel human DNA repair gene. The MED1 protein functionally interacts with MLH1 in human cells, is homologous to bacterial DNA repair glycosylases/lyases, and binds to CpG-methylated DNA, suggesting that cytosine methylation plays a novel role in mammalian DNA repair. MED1 displays thymine and uracil glycosylase activity, removing these bases from G:T and G:U mismatches. This suggests that MED1 may counteract mutagenesis by spontaneous deamination of 5-methylcytosine and cytosine to thymine and uracil, respectively. We also found that MED1 efficiently removes from DNA 5-fluorouracil, the mainstay of systemic therapy of colorectal cancer. Recently, we obtained evidence that the MED1 gene is frequently mutated (25% of the cases) in human colorectal and extracolonic carcinomas exhibiting microsatellite instability. In colorectal cancer specimens, we also detected loss of heterozygosity at the MED1 locus. The hypotheses underlying the present project are: that MED1 is an important component of the machinery ensuring genomic fidelity in human cells; that MED1 mutations are relevant to the pathogenesis of colorectal cancer; and that it may play a role in resistance/sensitivity to chemotherapeutic agents. Experiments are proposed to address: 1) the nature and frequency of MED1 mutations in colorectal cancer; 2) whether MED1 mutations are a prognostic factor and influence response to treatment.