摘要
目的 以复制错误 (replication error,RER+)表型的人结肠癌细胞株 RKO作为靶细胞 ,研究全反式维甲酸 (all- trans- retinoic acid,ATRA)和茶多酚 (polyphenon,PP)对肿瘤细胞微卫星序列(microsatellite sequence,MS)突变的影响 ,并观察肿瘤细胞内碱基错误配对修复 (mismatch repair,MMR)基因 h ML H1 和 h MSH2 的表达情况。方法 将含有外源性 MS(CA) 1 4 的穿梭质粒 p CMV- CAR转染RER+人结肠癌细胞株 RKO。外源性的 (CA) 1 4 的突变可使质粒标记基因 L ac Z恢复正常读码 ,表达产生β-半乳糖苷酶 ,后者使 X- gal变蓝。全反式维甲酸和茶多酚对 MS的影响可直接通过 X- gal染色结果判断。利用逆转录 -聚合酶链反应方法 ,检测经全反式维甲酸和茶多酚处理的 RKO细胞中碱基错误配对修复基因h ML H1 和 h MSH2 的表达情况。结果 全反式维甲酸 1μmol/ L、0 .1μmol/ L ,茶多酚 3μg/ ml,对 RKO细胞的增殖无明显的影响。作用 1周后 ,均显示对外源性 (CA) 1 4 的突变有明显的抑制效应。但不能诱导h ML H1 和 h MSH2 表达。结论 全反式维甲酸和茶多酚能抑制 RER+细胞中外源性 (CA) 1 4 重复序列突变 ,提示两者对人癌细胞 MS遗传不稳定有保护作用。其作用机理可能不是通过影响 h ML H1 和 h MSH2 的表达而起作?
Objective To investigate the effects of all-trans-retinoic acid(ATRA) and polyphenon-100 (PP) on genetic instability of human tumor cells via their role in alteration of microsatellite sequence(MS) and the expression of mismatch repair gene hMLH 1 and hMSH 2 in RER +(replication error) cells. Methods RER +colon cancer cell line was used as a host for lipofection with pCMV-CAR in which a foreign (CA) 14 repeat was inserted in the coding sequence of LacZ reporter gene, resulting in misreading LacZ frame. Any mutation which made the base number of (CA) 14 tract to be 3-fold resumed normal reading frame of LacZ, and thus led to expression of β-galactosidase. Variable expression of LacZ in the transfectant cells resulting from RATA or PP treatment was measured by OD reading at λ 620 after X-gal staining. Expression of mismatch repair genes of hMLH 1 and hMSH 2 was examined at mRNA level by reverse transcription-polymerase chain reaction (RT-PCR). Results ATRA at 1 μmol/L, 0.1 μmol/L and PP at 3 μg/ml had no significant inhibitory effect on cell proliferation. After being treated with ATRA or PP for 1 week, the blue cells of RKO transfectant clones were significantly reduced, and this meant the mutation of exogenous (CA) 14 in RKO cells were inhibited. But no expression of hMLH 1 and hMSH 2 was observed. Conclusion The above data showed both ATRA and PP had inhibitory effects on MS instability of cancer and thus demonstrated directly their beneficial role in stabilization of genomic DNA. However, the present authors have not observed any expression of hMLH 1 and hMSH 2 in RKO cells treated with ATRA or PP.
出处
《中华医学遗传学杂志》
EI
CAS
CSCD
2002年第3期190-193,共4页
Chinese Journal of Medical Genetics
基金
国家自然科学基金 (39670 2 97)~~
关键词
维甲酸
茶多酚
结肠癌
抑制作用
微卫星不稳定性
microsatellite instability
all-trans-retinoic acid
polyphenon
cancer
replication error
