Hrev1 Modulates The Cytotoxicity And Mutagenicity Of Cisplatin In Human Ovarian Carcinoma Cells
CANCER RESEARCH(2006)
Abstract
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 6.3to 23.4-fold and hREV1 protein by 2.7to 6.2-fold. The sublines were 1.3to 1.7-fold resistant to the cytotoxic effect of DDP and 2.3to 5.1-fold hypersensitive to the mutagenic effect of DDP. The hREV1-transfected sublines were 1.5to 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 damage-induced mutagenesis pathway is an important mechanism which generates mutations when a genome that is burdened with lesions is replicated. Most mutations induced by UV irradiation are generated when DNA containing residual unrepaired damage is replicated during S phase of the cell cycle. Such lesions perturb the structure of DNA (Rice et al., 1988; Park et al., 2002) and are likely to block replicative DNA polymerases that have stringent base-pairing requirements. In Saccharomyces cerevisiae, mutagenic bypass of DNA damage is equivalent to error-prone translesion replication (Broomfield et al., 2001). Virtually all mutations induced by UV irradiation are dependent on the activity of DNA polymerase (Pol ), acting in concert with a protein encoded by the Rev1 gene (Lawrence and Maher, 2001; Lawrence, 2002). REV1 is required for UV mutagenesis in vivo and interacts with Pol in vitro to stimulate translesion replication activity (Lawrence, 2002). It has been proposed that in human cells, hREV1 protein functions in this pathway through its interaction with Pol and possibly with other members of the Y family of DNA polymerases (Murakumo et al., 2001; Gibbs et al., 2005; Ross et al., 2005). The damage-induced mutagenesis pathway is conserved evolutionarily. The genes of this pathway identified in yeast have mammalian homologs that include HHR6A, HHR6B, hRAD18, hREV1, hREV3, and hREV7. hREV1 is highly distributive and catalyzes the insertion of a deoxycytidine when it encounters a guanine, a guanine bearing a large chemical adduct, or an abasic site, but it is unable to bypass UV photoproducts (Zhang et al., 2002). In addition to hREV1, higher eukaryotic cells have at least three other DNA polymerases in the Y family, including Pol , Pol , and Pol . These enzymes are characterized by their low fidelity when copying undamaged templates and their ability to bypass lesions that block DNA polymerases belonging to other families (Friedberg et al., 2002). Mouse REV1 has been shown to bind to Pol , Pol , and Pol , and all the interactions occur at the same site on REV1 protein (Guo et al., 2003). This suggests that in mammalian cells, hREV1 may have a role in supporting translesional synthesis carried out by multiple This work was supported in part by National Institutes of Health grant CA78648 and by a grant from the Foundation for Medical Research. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.020446. ABBREVIATIONS: Pol, polymerase; DDP, cisplatin (cis-diamminedichloroplatinium II); PCR, polymerase chain reaction. 0026-895X/06/6905-1748–1754$20.00 MOLECULAR PHARMACOLOGY Vol. 69, No. 5 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 20446/3112891 Mol Pharmacol 69:1748–1754, 2006 Printed in U.S.A. 1748 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from DNA polymerases. However, the relative contribution of most of these enzymes to DNA damage-induced mutagenesis in genomically unstable tumor cells in vivo, if any, is unknown. The clinical effectiveness of DDP is limited by the rapid development of resistance. The most abundant lesions that DDP produces are intrastrand cross-links, and these are believed to account for the majority of both the cytotoxic and mutagenic effects of the drug. Its mutagenic potential has been well documented in both bacterial (Yarema et al., 1994, 1995) and mammalian cells (Turnbull et al., 1979; Johnson et al., 1980; Cariello et al., 1992; Lin and Howell, 1999). We have reported that many of the mutations induced by DDP adducts seem to result from error-prone translesional DNA synthesis mediated by DNA polymerase and/or hREV1 (Wu et al., 2004; Okuda et al., 2005). Several investigators have reported previously that a loss of hREV1 function markedly reduces UV-induced hypoxanthine guanine phosphoribosyl transferase mutations in human cells engineered to contain reduced levels of hREV1 mRNA through the expression of an hREV1 antisense RNA (Gibbs et al., 2000) or a ribozyme that cleaves endogenous hREV1 mRNA (Clark et al., 2003). It has also been demonstrated that inactivation of the REV1 gene in chicken DT40 cells renders them hypersensitive to a wide variety of mutagens, including DDP (Simpson and Sale, 2003). We demonstrated recently that suppression of hREV1 expression by a short hairpin RNA targeted to hREV1 mRNA in human ovarian carcinoma cells, whereas it moderately increases DDP sensitivity and markedly reduces its mutagenicity and the rate at which human ovarian carcinoma cells acquire resistance to DDP during repeated cycles of exposure (Okuda et al., 2005). This indicates an important role for hREV1 in DDP-induced mutagenesis and identifies hREV1 as being of particular interest with respect to the mechanism underlying the emergence of the multidrug-resistant phenotype that so frequently accompanies the development of DDP resistance. To further explore the role of hREV1 in the genesis of resistance to DDP, we have molecularly engineered human ovarian carcinoma 2008 cells to express increased amounts of hREV1. We report here that such forced expression of hREV1 results in increased resistance to DDP and a significant elevation in DDP-induced mutagenicity and that these phenotypes are associated with an enhanced hREV1-mediated capacity to express a gene containing DDP adducts. Materials and Methods Drugs. DDP was a gift from Bristol-Myers Squibb (Princeton, NJ). A stock solution of 1 mM cisplatin in 0.9% NaCl was stored in the dark at room temperature. 6TG was purchased from Sigma Chemical Co. (St. Louis, MO) and dissolved in 0.2 N sodium hydroxide to form a 20 mM stock solution and stored at 20°C. Vector Construction. The 3.8-kilobase, full-length human REV1 cDNA was removed from the yeast expression vector pEGLh6hREV1 (Lin et al., 1999) with SalI and was inserted into the SalI site of pBluescript II SK( ) (Fermentas Inc., Hanover, MD). The insert was then removed from pBluescript II SK( ) by digesting with NotI and ApaI and cloned into pcDNA3.1( ) (Invitrogen, Carlsbad, CA) by sticky-end ligation. The resulting vector containing full-length hREV1 cDNA and expressing a geneticin resistance marker was sequence-verified and designated pcDNA3.1-hREV1. Cell Lines, Transfection, and Selection. The human ovarian carcinoma cell line 2008 was grown in RPMI 1640 supplemented with 5% fetal bovine serum. Cells were transfected with pcDNA3.1hREV1 using Fugene 6 (Roche, Indianapolis, IN) according to the manufacturer’s recommendations. Transfected cells were then selected by continuous exposure to 400 g/ml geneticin. Geneticinresistant colonies were isolated, expanded, and screened for hREV1 mRNA expression level by real-time PCR and for hREV1 protein level by Western blotting. Four clones, designated 2008-hREV1-C1, -C7, -C8, and -C9, in which the steady-state hREV1 protein level was at least 2-fold increased, were chosen for all subsequent experiments. Quantification of hREV1 mRNA by Reverse-Transcriptase PCR. Total RNA was extracted with TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) and random primers. Real-time PCR was performed using the Bio-Rad iCycler iQ detection system in the presence of SYBR-Green I dye (Bio-Rad, Valencia, CA). For the hREV1 gene expression, the forward (5 -AAGGCTGATGCAATCG3 ) and reverse (5 -CCACCTGGACATTGTCAAGAATAA-3 ) primers were used for amplification with an iCycler protocol consisting of a denaturation program (95°C for 3 min) and amplification and quantification program repeated 40 times (95°C for 10 s and 55°C for 45 s) and melting curve analysis. A melting-curve analysis immediately followed amplification and was executed using 95°C for 1 min then 55°C for 1 min, followed by 80 repeats of heating for 10 s starting at 55°C with 0.5°C increments. The data were analyzed by using the comparative Ct method, where Ct is the cycle number at which fluorescence first exceeds the threshold. The Ct values from each cell line were obtained by subtracting the values for 18S Ct from the sample Ct. A 1-unit difference in Ct value represents a 2-fold difference in the level of mRNA. Western Blot Analysis. The nuc
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