Flohr T, Dai J C, Büttner J, Popanda O, Hagmüller E, Thielmann H W
Division of Interaction of Carcinogens with Biologial Macromolecules, German Research Center, Heidelberg.
Int J Cancer. 1999 Mar 15;80(6):919-29. doi: 10.1002/(sici)1097-0215(19990315)80:6<919::aid-ijc19>3.0.co;2-u.
To test the hypothesis whether DNA polymerases acquire mutator properties during tumor development (mutator hypothesis), we examined DNA polymerase delta mRNA in 6 colon cancer cell lines (DLD-1, HCT116, SW48, HT29, SW480 and SW620) and 7 sporadic human colorectal cancers. For analysis we used amplification of cDNA by polymerase chain reaction, single-strand conformation polymorphism and sequencing techniques. In 5 of the cell lines, 9 mutations leading to changes of the amino acid sequence of DNA polymerase delta were detected. Most mutations were found in the cell lines DLD-1, HCT116 and SW48 for which defects in mismatch repair genes had been identified previously. In the majority of cases, wild type and mutated sequences were present. In 2 cell lines (HCT116 and SW48), a single-nucleotide deletion occurred at the same position. This resulted in a premature termination codon by which the DNA interaction domain of the enzyme was eliminated. Furthermore, sequence deviations were found in the tumor tissues of 4 colon cancer patients. Wild-type and altered sequences were present simultaneously. The deviations included missense mutations (2 cases) and silent mutations (2 cases). The missense mutations and one of the silent mutations were found in normal mucosa as well. In addition, the mutation clustered region of a tumor suppressor gene, often found to be defective in colon cancer, the adenomatous polyposis coli (APC) gene, was investigated in surgical specimens and cell lines. One carcinoma and 2 cell lines exhibited amino acid changes in both the DNA polymerase delta gene and in the mutation clustered region of the APC gene. Since most of the mutations detected in the DNA polymerase delta mRNA are likely to alter the structure of the protein, the enzyme is expected to be functionally impaired. In particular, copying fidelity might be decreased, thus contributing to the high mutation rate observed in colorectal cancer.
为了验证DNA聚合酶在肿瘤发生过程中是否获得致突变特性这一假说（致突变假说），我们检测了6种结肠癌细胞系（DLD-1、HCT116、SW48、HT29、SW480和SW620）以及7例散发性人类结肠直肠癌中的DNA聚合酶δ信使核糖核酸。分析时我们采用聚合酶链反应扩增互补脱氧核糖核酸、单链构象多态性和测序技术。在5种细胞系中，检测到9个导致DNA聚合酶δ氨基酸序列改变的突变。大多数突变见于先前已确定错配修复基因存在缺陷的DLD-1、HCT116和SW48细胞系。在大多数情况下，野生型和突变序列同时存在。在2种细胞系（HCT116和SW48）中，同一位置发生了单核苷酸缺失。这导致产生一个提前终止密码子，该酶的DNA相互作用结构域因此被消除。此外，在4例结肠癌患者的肿瘤组织中发现了序列偏差。野生型和改变后的序列同时存在。这些偏差包括错义突变（2例）和沉默突变（2例）。错义突变和其中一个沉默突变在正常黏膜中也有发现。此外，在手术标本和细胞系中研究了一个肿瘤抑制基因的突变聚集区域，该区域在结肠癌中常存在缺陷，即腺瘤性息肉病 coli（APC）基因。1例癌组织和2种细胞系在DNA聚合酶δ基因以及APC基因的突变聚集区域均出现了氨基酸变化。由于在DNA聚合酶δ信使核糖核酸中检测到的大多数突变可能会改变蛋白质结构，预计该酶功能受损。特别是，复制保真度可能会降低，从而导致在结肠直肠癌中观察到的高突变率。
