Replicative bypass of many DNA adducts is dependent on the interaction of hREV1 with DNA polymerase and potentially with members of the Y family of DNA polymerases. To examine the role of hREV1 in the development of cisplatin (DDP) resistance, a subline (2008-shREV1-3.3) of the ovarian carcinoma cell line 2008 was isolated in which stable expression of a short hairpin RNA suppressed hREV1 expression to 20% and reduced hREV1 protein level to 43% of that found in the parental cells. The 2008-shREV1-3.3 cells were 1.5-fold more sensitive to the cytotoxic effect of DDP but less sensitive to the mutagenic effect of DDP as evidenced by a 2.6-or 2.7-fold reduction in the ability to induce clones highly resistant to 6-thioguanine or DDP itself, respectively, in the surviving population. Reduction of hREV1 did not alter the initial rate of DDP adduct removal from DNA but did impair both spontaneous and DDPinduced extra-chromosomal homologous recombination, as measured by the recombination-sensitive reporter vector pBHRF. DDP induced an increase in hREV1 protein level. DDP resistance at the population level evolved 2.8-fold more slowly in the 2008-shREV1-3.3 cells than in the parental cells during repeated cycles of drug exposure. The results indicate that hREV1 functions to enhance both cell survival and the generation of drug-resistant variants in the surviving population. DDP up-regulates hREV1, suggesting that it may enhance its own mutagenicity. Most importantly, hREV1 controls the rate of emergence of resistance to DDP at the population level. Thus, hREV1 is an important contributor to DDP-induced genomic instability and the subsequent emergence of resistance.

REV1 interacts with Y-type DNA polymerases (Pol) and Pol to bypass many types of adducts that block the replicative DNA polymerases. This pathway accounts for many of the mutations induced by cisplatin (cis-diamminedichloroplatinium II, DDP). This study sought to determine how increasing human REV1 (hREV1) affects the cytotoxicity and mutagenicity of DDP. Human ovarian carcinoma 2008 cells were transfected with an hREV1 expression vector and 4 sublines developed in which the hREV1 mRNA level was increased by 6.3-to 23.4-fold and hREV1 protein by 2.7-to 6.2-fold. The sublines were 1.3-to 1.7-fold resistant to the cytotoxic effect of DDP and 2.3-to 5.1-fold hypersensitive to the mutagenic effect of DDP. The hREV1-transfected sublines were 1.5-to 1.8-fold better than the parental 2008 cells at managing DDP adducts as assessed by their ability to express Renilla reniformis luciferase from a vector that had been extensively loaded with DDP adducts before transfection. Increased hREV1 expression was associated with a 1.5-fold increase in the rate at which the whole population acquired resistance to DDP during sequential cycles of drug exposure. Increasing the abundance of hREV1 thus resulted in both resistance to DDP and a significant elevation in DDP-induced mutagenicity. This was accompanied by an enhanced capacity to synthesize a functional protein from a DDPdamaged gene and, most importantly, by more rapid development of resistance during sequential cycles of DDP exposure that mimic clinical schedules of DDP administration. We conclude that hREV1-dependent processes are important determinants of DDP-induced genomic instability and the development of resistance.

The mutagenicity of cis-diamminedichloroplatinum(II) (DDP; cisplatin) and the rate at which resistance develops with repeated exposure to DDP are dependent on mutagenic translesional replication across DDP DNA adducts, mediated in part by DNA polymerase~, and on the integrity of the DNA mismatch repair (MMR) system. The aim of this study was to determine whether disabling Pol~by suppressing expression of its hREV3 subunit in human cancer cells can reduce the mutagenicity of DDP and whether loss of MMR facilitates mutagenic Pol~-dependent translesional bypass. The HCT116+ch3 (MMR + /REV3 + ) and HCT116 (MMR À /REV3 + ) human colon carcinoma cell lines were engineered to suppress hREV3 mRNA by stable expression of a short hairpin interfering RNA targeted to hREV3. The effect of knocking down REV3 expression was to completely offset the DDP resistance mediated by loss of MMR. Knockdown of REV3 also reduced the mutagenicity of DDP and eliminated the enhanced mutagenicity of DDP observed in the MMR À /REV3 + cells. Similar results were obtained when the ability of the cells to express luciferase from a platinated plasmid was measured. We conclude that Pol~plays a central role in the mutagenic bypass of DDP adducts and that the DDP resistance, enhanced mutagenicity, and the increased capacity of MMR À /REV3 + cells to express a gene burdened by DDP adducts are all dependent on the Pol~pathway.