Yamada Nazumi A, Farber Rosann A
Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 27599, USA.
Cancer Res. 2002 Nov 1;62(21):6061-4.
Microsatellite instability (MSI) is the condition in which high rates of frameshift mutations are observed in short tandem repeat sequences. Mutations in sequences of this type in coding regions of cancer-related genes can contribute to the development of cancer. Although defects in mismatch repair are usually responsible for high levels of MSI, low levels of MSI have been observed in some cancers with no known mismatch repair defects. We have investigated whether overexpression of an error-prone polymerase, polbeta, is sufficient to induce MSI in the presence of mismatch repair. Because overexpression of polbeta has been observed in several types of cancer, we hypothesized that polbeta overexpression might increase genetic instability and, thus, contribute to carcinogenesis. Microsatellite mutation rate analyses were conducted using a drug-resistance reversion assay, where G(17) or A(17) microsatellites were inserted into a plasmid upstream of a neomycin-resistance gene (neo), such that the neo gene was shifted out of frame. When frameshift mutations occur in the microsatellite, the neo gene can be restored, allowing for selection of revertants in G418. Microsatellite-containing plasmids were transfected into telomerase-immortalized normal human fibroblasts (hTERT-1604), where they integrated into the nuclear genome. polbeta-expressing episomal vectors or empty control vectors were then introduced for analysis of the effect of polbeta overexpression on these microsatellites. Mutation rates were determined by fluctuation analysis. Mutation rates in G(17) repeats were elevated for the polbeta transfectants at all levels of overexpression ( approximately 2-fold to >100-fold compared with vector-only controls), with up to a 3-fold increase in mutation rates compared with the vector-only controls in cells with the highest expression. A similar magnitude of elevation in mutation rates was observed for A(17) microsatellites. No difference was observed between vector-only controls and nontransfected cells in either microsatellite sequence. Cells with high polbeta expression showed an approximately 1.5-fold increase in population doubling time and a 2-fold reduction in mitotic index compared with controls. Cells with both modest and high elevations in microsatellite mutation rates had these altered growth properties. These results suggest that polbeta overexpression may affect cell cycle progression and increase genetic instability.
微卫星不稳定性（MSI）是指在短串联重复序列中观察到高频率移码突变的情况。癌症相关基因编码区中此类序列的突变可促进癌症的发生发展。虽然错配修复缺陷通常是导致高水平MSI的原因，但在一些不存在已知错配修复缺陷的癌症中也观察到了低水平的MSI。我们研究了易错聚合酶polβ的过表达在存在错配修复的情况下是否足以诱导MSI。由于在几种类型的癌症中都观察到了polβ的过表达，我们推测polβ过表达可能会增加遗传不稳定性，从而促进致癌作用。使用耐药性回复分析进行微卫星突变率分析，即将G(17)或A(17)微卫星插入新霉素抗性基因（neo）上游的质粒中，使neo基因移码。当微卫星中发生移码突变时，neo基因可被恢复，从而能够在G418中筛选回复体。将含有微卫星的质粒转染到端粒酶永生化的正常人成纤维细胞（hTERT-1604）中，使其整合到核基因组中。然后引入表达polβ的游离载体或空对照载体，以分析polβ过表达对这些微卫星的影响。通过波动分析确定突变率。在所有过表达水平下，polβ转染细胞中G(17)重复序列的突变率均升高（与仅转染载体的对照相比，约为2倍至>100倍），在表达最高的细胞中，突变率与仅转染载体的对照相比增加了3倍。对于A(17)微卫星，观察到突变率有类似程度的升高。在两种微卫星序列中，仅转染载体的对照与未转染细胞之间均未观察到差异。与对照相比，高表达polβ的细胞群体倍增时间增加了约1.5倍，有丝分裂指数降低了2倍。微卫星突变率适度和高度升高的细胞均具有这些改变的生长特性。这些结果表明，polβ过表达可能会影响细胞周期进程并增加遗传不稳定性。
