Basic study for establishment of chemotherapy of DNA mismatch repair-deficient colorectal cancers : Specificity of DNA damage and drug sensitivity

癌基因和抑癌基因突变的积累是结直肠癌发生的必要条件。已知DNA错配修复（MMR）基因的缺陷是对这些癌症相关基因中积累的突变之一作出反应的。已鉴定出包括hMLH1和hMSH2在内的7个人类MMR基因。mmr缺陷肿瘤细胞表现出对一些抗癌药物如顺铂（CDDP）的耐药性。然而，mmr缺陷细胞对许多用于结直肠癌化疗的抗癌药物和诱导mmr肿瘤细胞的药物的敏感性尚不清楚。我们研究了MMR缺陷的结直肠癌细胞系对抗癌药物的敏感性，以及MMR系统的生理作用如下：1)MMR缺陷细胞HCT116和LoVo细胞系中6种抗癌药物的细胞毒性与配对的MMR正常细胞HCT116+ch3和LoVo+ch2的细胞毒性进行了比较。HCT116对5-FU、替加富和伊立替康表现出更强的耐药性，而吉米他滨对HCT116和LoVo的细胞毒性大于对mmr精通的细胞。2) mmr缺陷细胞的高敏感性机制为了探究为什么mmr缺陷细胞具有高敏感性，我们对阿希霉素和氢脲进行了多次实验。结果表明MMR系统与DNA复制完成检查点密切相关，但需要进一步研究。3)建立一种快速简便的细胞毒测定方法。为了评估抗癌药物在mmr缺陷细胞和mmr精通细胞之间的细胞毒性差异，我们开发了一种使用带有表达质粒的大肠杆菌的快速检测系统。4)患者肿瘤组织中存在mmr蛋白。为了了解化疗药物对mmr缺陷细胞外观的影响，我们通过免疫组织化学染色筛选了100多个标本。虽然所有病例都是原发肿瘤，但有几个样本的染色呈阴性。少

Accumulation of mutations in oncogenes and tumor suppressor genes is esential for generation of colorectal cancer. Defect of a DNA mismatch repair (MMR) gene is known to be responding to one of the accumulated mutations in these cancer-related genes. Seven human MMR genes including hMLH1 and hMSH2 have been identified. MMR-deficient tumor cells exhibit a drug-resistance to some anticancer agents such as cisplatin (CDDP). However, sensitivities of MMR-deficient cells to many of anticancer drugs, which are utilizing in chemotherapy for colorectal cancers, and agents that induce MMR-tumor cells are not clear. We investigated here sensitivity of MMR-deficient colorectal cancer cell lines to the anticancer agents and a physiological role of the MMR system as follows :1) Drug-sensitivity of MMR-deficient cellsCytotoxicities of 6 anticancer agents in the MMR-deficient HCT116 and LoVo cell lines were compared to those of paired MMR-proficient cells, HCT116+ch3 and LoVo+ch2. HCT116 exhibited re … More sistance to 5-FU, tegafur, and irinotecan, while cytotoxicity of gemicitabine in HCT116 and LoVo was greater than that of MMR-proficient cells.2) Mechanism of high sensitivity of MMR-deficient cellsTo explore why MMR-deficient cells show high sensitivity, we had several experiments with aphidicolin and hydroxurea. The results strongly indicate that MMR system is closely involved in the DNA replication completion checkpoint, though further investigation is required.3) Establishment of a rapid and easy method for cytotoxic assay.To estimate how different cytotoxicity of anticancer agents between MMR-deficient and -proficient cells, we developed a rapid assay system using E. coli with expression plasmids.4) Presence of MMR-protein in tumor tissues of patients.To know the contribution of chemotherapeutic agents to appearance of MMR-deficient cells, we screened more than 100 specimens by immunohistochemical staining. Several samples was negative for the staining, though all cases were primary tumor. Less