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Induction of a Low Level of Microsatellite Instability by Overexpression of DNA Polymerase ß¹

Department of Pathology and Laboratory Medicine [N. A. Y., R. A. F.], Department of Genetics [R. A. F.], and Lineberger Comprehensive Cancer Center [R. A. F.], University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

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, polß, is sufficient to induce MSI in the presence of mismatch repair. Because overexpression of polß has been observed in several types of cancer, we hypothesized that polß overexpression might increase genetic instability and, thus, contribute to carcinogenesis. Microsatellite mutation rate analyses were conducted using a drug-resistance reversion assay, where G₁₇ or A₁₇ 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. polß-expressing episomal vectors or empty control vectors were then introduced for analysis of the effect of polß overexpression on these microsatellites. Mutation rates were determined by fluctuation analysis. Mutation rates in G₁₇ repeats were elevated for the polß transfectants at all levels of overexpression (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 A₁₇ microsatellites. No difference was observed between vector-only controls and nontransfected cells in either microsatellite sequence. Cells with high polß expression showed an 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 polß overexpression may affect cell cycle progression and increase genetic instability.

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