Resistance to cisplatin (DDP) is often accompanied by impaired accumulation in mammalian cells. The mechanism of impaired DDP accumulation is unknown, but copper uptake is diminished as well. We investigated the ability of the copper transporter CTR1 to control the accumulation of DDP in Saccharomyces cerevisiae. Parallel studies of copper and DDP cellular pharmacokinetics were carried out using an isogenic pair of wild-type CTR1 and ctr1 knockout S. cerevisiae strains. Both copper and platinum accumulation increased linearly as a function of time and drug concentration in the parental cells. Deletion of CTR1 resulted in a 16-fold reduction in the uptake of copper and an 8-fold reduction in the uptake of DDP measured at 1 h. The CTR1-deficient cells accumulated 2.3-fold (p Ͻ 0.05) less platinum in their DNA and were 1.9-fold more resistant to the cytotoxic effect of DDP than the CTR1-replete cells. The kinetics of cellular copper accumulation were similar to those of DDP. Based on measurements of accumulation at 1 h, the K m for copper influx was 128.8 M, and the V max was 169.5 ng/mg of protein/min; for DDP, the K m was 140.2 M and the V max was 76.9 ng/mg of protein/min. DDP blocked the uptake of copper into the parental cells but not ctr1-deficient cells. CTR1-deficient cells also demonstrated impaired accumulation of the DDP analogs carboplatin, oxaliplatin, and ZD0473 [cis-amminedichloro(2-methylpyridine) platinum (II)]. These results indicate that CTR1 function markedly influences the uptake of all of the clinically used platinum-containing drugs and suggest that this copper transporter may also transport DDP.The effectiveness of cell killing by cisplatin (DDP) is generally acknowledged to be a function of how much drug gets into the cell, how much of it enters the nucleus and actually reacts with DNA, how tolerant the cell is of lesions in its DNA, and how effectively it removes these adducts (Andrews and Howell, 1990). Intracellular detoxification of DDP through mechanisms that involve binding to thiols may contribute to resistance (reviewed in Perez et al., 1993). Both defects in the ability of the cell to recognize adducts in DNA (reviewed in Fink et al., 1998) and enhanced repair of and tolerance to adducts (Johnson et al., 1997) have been identified as contributing to resistance in some cell types. However, impaired uptake of DDP is the most consistently identified characteristic of cells selected for DDP resistance both in vitro and in vivo (reviewed in Andrews and Howell, 1990;Gately and Howell, 1993).The mechanism underlying impaired DDP accumulation in resistant cells is unknown; in fact, the mechanism by which DDP enters or exits cells remains poorly defined. DDP accumulates in cells relatively slowly compared to many other classes of anticancer agents, and earlier evidence suggested that at least one component of DDP uptake is mediated by a transport mechanism or channel (Andrews and Albright 1991; . In fact, the behavior of DDP is similar in many ways to that of transition metals such ...

DNA polymerase participates in translesional bypass replication. Here we show that reduced expression of the catalytic subunit hREV3 renders human fibroblasts more sensitive to the cytotoxic effect of cisplatin, reduces their sensitivity to the ability of cisplatin exposure to generate drug resistant variants in the surviving population, and reduces the rate of emergence of resistance to cisplatin at the population level. Reduction of REV3 mRNA did not alter the rate of cisplatin adduct removal but did impair both spontaneous and cisplatin-induced extrachromosomal homologous recombination and attenuated bypass replication as reflected by reduced ability to express luciferase from a platinated plasmid. Cisplatin induced a concentration-and time-dependent increase in hREV3 mRNA. The results indicate that, following formation of cisplatin adducts in DNA, REV3 mRNA levels increase, and polymerase functions to promote both cell survival and the generation of drug-resistant variants in the surviving population. We conclude that when cisplatin adducts are present in the DNA, polymerase is an important contributor to cisplatin-induced genomic instability and the subsequent emergence of resistance to this chemotherapeutic agent.

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Endometrial cancers exhibit a different mechanism of tumorigenesis and progression depending on histopathological and clinical types. The most frequently altered gene in estrogen-dependent endometrioid endometrial carcinoma tumors is PTEN. Microsatellite instability is another important genetic event in this type of tumor. In contrast, p53 mutations or Her2/neu overexpression are more frequent in non-endometrioid tumors. On the other hand, it is possible that the clear cell type may arise from a unique pathway which appears similar to the ovarian clear cell carcinoma. K-ras mutations are detected in approximately 15%–30% of endometrioid carcinomas, are unrelated to the existence of endometrial hyperplasia. A β-catenin mutation was detected in about 20% of endometrioid carcinomas, but is rare in serous carcinoma. Telomere shortening is another important type of genomic instability observed in endometrial cancer. Only non-endometrioid endometrial carcinoma tumors were significantly associated with critical telomere shortening in the adjacent morphologically normal epithelium. Lynch syndrome, which is an autosomal dominantly inherited disorder of cancer susceptibility and is characterized by a MSH2/MSH6 protein complex deficiency, is associated with the development of non-endometrioid carcinomas.

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.

Endometriosis shares some features characteristic of malignancy; however, it remains unclear whether endometriosis is a precursor to malignant disease. The objective is to determine the genetic relationship between endometriosis and ovarian clear cell carcinoma (OCCA). Among 37 Japanese patients with OCCA who underwent primary surgery at Showa University Hospital between 1987 and 1999, K-ras mutations were detected in 6. Three of these patients had ectopic endometrial tissue adjacent to the site of carcinoma. These cases demonstrated areas of endometriosis and areas of OCCA bordered by atypical endometriosis. We retrieved cells from regions of endometriosis and atypical endometriosis, as well as OCCA cells, by laser microdissection in each case. K-ras mutations were analyzed in each specimen dissected. DNA analysis of each region revealed that K-ras mutations were detectable in OCCA but not in endometriosis or atypical endometriosis. It is thought that a number of genetic alterations are involved in malignant transformation. It is possible that K-ras mutations are associated with malignant transformation of atypical endometriosis into OCCA, although further research is needed to define this mechanism.

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.