Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Cisplatin (cis-diamminedichloroplatinum II) is one of the most frequently used anticancer drugs. It is used as a single agent and in combination regimens to treat a variety of solid tumor types, including head and neck, ovarian, and non-small-cell lung (NSCLC) cancers (Cooley et al., 1994; Highley and Calvert, 2000). The therapeutic efficacy of cisplatin derives from its ability to form complexes with DNA (Cohen and Lippard, 2001), where it binds the N7 reactive center on purine residues to form both monofunctional and bifunctional DNA adducts. The 1,2-intrastrand GG cross-link (65%) and the 1,2-intrastrand AG cross-link (25%) are the predominant lesions; minor lesions include the 1,3-intrastrand GNG cross-link (6%), the interstrand GG cross-link (1-3%), monoadducts, and protein-DNA cross-links (Eastman, 1986). Cisplatin-DNA adducts are suggested to exert their cytotoxicity by directly inhibiting DNA and RNA synthesis and inducing apoptosis (Sorrenson et al., 1990; Meyn et al., 1995).

A major clinical problem associated with cisplatin therapy is that although many tumors may be initially responsive to platinum therapy, these often relapse and become refractory to further platinum agent treatment (Perez, 1998). Several mechanisms of cisplatin resistance have been identified, including reduced intracellular drug accumulation, elevated levels of cellular thiols, increased tolerance to cisplatin DNA damage, and enhanced DNA repair (Masuda et al., 1990; Perez et al., 1993; Akiyama et al., 1999). The increased capacity of cells to repair and/or tolerate cisplatin-induced DNA damage has been proposed as a major mechanism of acquired resistance to cisplatin (Reed, 1998). Several studies have shown that cisplatin resistance is associated with the increased capacity of cells to repair drug-induced DNA damage both in the overall genome (Johnson et al., 1994) and in active genes (Zhen et al., 1992). Furthermore, elevated levels of DNA repair proteins have also been shown both in cell lines and in patient tumors resistant to cisplatin (Reed, 1998). Inhibition of DNA repair pathways may therefore increase the sensitivity of a tumor to cisplatin therapy.

Gemcitabine (2',2'-difluorodeoxycytidine) is a nucleoside analog with clinical activity against various solid tumors including ovarian, NSCLC, head and neck cancer, and pancreatic cancers (Sandler and Ettinger, 1999; Storniolo et al., 1999). Upon entering the cell, gemcitabine is anabolized to its triphosphate form by deoxynucleoside salvage pathways. The triphosphate can then become incorporated into DNA, where it blocks further DNA synthesis by inhibiting DNA polymerase activity (Plunkett et al., 1995). In vitro studies have shown that the cytotoxicity of gemcitabine correlates directly with the level of incorporation of the analog into cellular DNA (Kufe et al., 1980; Huang et al., 1990, 1991). Gemcitabine also inhibits ribonucleotide reductase, hence depleting the deoxynucleotide pools required for DNA repair and replication, thereby potentiating its incorporation into newly synthesized DNA (Tseng et al., 1982; Heinemann et al., 1990).

Cisplatin and gemcitabine are ideal candidates for use in combination regimens because of their different but complementary mechanisms of action, similar antitumor activity profiles, and nonoverlapping side effect profiles (Braakhuis et al., 1995; Carmichael, 1998). Although the clinical utility of platinum agents and gemcitabine combinations has been demonstrated (Carmichael, 1998; Stewart, 1998), the molecular basis for this interaction has yet to be defined.

Previous studies have shown that the cytotoxic interaction between the nucleoside analog, fludarabine and cisplatin is accompanied by an inhibition of repair of cisplatin-induced interstrand cross-links (Yang et al., 1995). Because gemcitabine is readily incorporated into newly synthesized DNA, the treatment of cells with cisplatin in combination with gemcitabine may potentially inhibit the repair of cisplatin DNA damage, resulting in the persistence of DNA damage and increased cytotoxicity. Indeed, previous studies have shown that the nucleoside analog fludarabine synergizes with cisplatin in vitro.

The aim of this study was to investigate the cytotoxic interaction between cisplatin and gemcitabine in cisplatin-sensitive and -resistant ovarian cancer cell lines. A synergistic interaction between the drugs was demonstrated in each of the cell lines. Gemcitabine was shown to inhibit the repair of both inter- and intrastrand cross-links induced by cisplatin and to potentiate the accumulation of these lesions in ovarian cells. These results suggest that the synergistic interaction between these drugs may be because of the inhibitory effect of gemcitabine on the repair of the major cytotoxic lesions induced by cisplatin.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Drugs and Chemicals

Gemcitabine was kindly supplied by Eli Lilly Inc. (Indianapolis, IN) and was dissolved in phosphate-buffered saline to a concentration of 10 mM. Cisplatin was obtained from the Institute of Drug Technology (Melbourne, Australia) and was dissolved in phosphate-buffered saline to a concentration of 5 mM. All other chemicals were of analytical grade and commercially available.

Cell Culture The 2008 human ovarian adenocarcinoma cell line and the cisplatin-resistant subline C13*5.25 (2008/R) (Professor S Howell, Cancer Center, University of California, San Diego, CA) were maintained in RPMI 1640 medium containing 10% fetal bovine serum and 20 µg/ml gentamicin. The A2780 human ovarian carcinoma cell line (Professor L. R. Kelland, Institute of Cancer Research, Sutton, UK) and the resistant subline CP70 (Dr. V. A. Bohr, National Institute on Aging, National Institutes of Health, Baltimore, MD) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 20 µg/ml gentamicin and nonessential amino acids. All cells were maintained in a humidified incubator with 5% CO₂ in air at 37°C and were routinely tested for mycoplasma. All cells were grown as monolayers in tissue culture flasks and were passaged twice weekly.

Cytotoxicity Assay

The cytotoxicity of cisplatin and gemcitabine as single agents and in combination was evaluated in the four ovarian cell lines by a clonogenic assay. On day one, 2 × 10⁵ cells were seeded in 25-cm² flasks containing 10 ml of growth medium. On day 3, the cells were exposed to drugs alone or in combination for 24 h and then washed twice with phosphate-buffered saline, harvested, and plated into 60-mm dishes at various cell densities such that 50 to 100 colonies/dish would be obtained after 8 days of incubation. The plates were then fixed in neutral formalin (40% formaldehyde, 30 mM NaH₂PO₄, and 45 mM Na₂HPO₄, pH 7) and stained with 0.01% (w/v) crystal violet. Colonies consisting of 50 cells or more were counted in four replicate plates. The survival fractions were calculated after setting the plating efficiency of untreated control cells at 100%.

To study the cytotoxic effects of the two drugs in combination, the cells were treated with increasing concentrations of cisplatin (0.23, 0.3, and 0.45 µM for 2008 and A2780 cells; 5.63, 7.5, and 11.25 µM for C13 and CP70 cells) or gemcitabine (11.25, 15, and 22.5 nM for 2008 and C13 cells; 2.25, 3, and 4.5 nM for A2780 and CP70 cells) as single agents or with a combination of the two agents at concentrations in a fixed molar ratio (molar ratio of cisplatin to gemcitabine of 20:1 for 2008 cells, 100:1 for A2780 cells, 500:1 for C13 cells, and 2500:1 for CP70 cells). The cytotoxic interactions between the two drugs in each cell line was determined using the median effect method of Chou and Talalay (1984). A drug concentration dependence plot was generated for each drug alone and multiple dilutions of a fixed ratio combination of the drugs. The mutually nonexclusive combination index (CI) was then determined using a computer program developed by Chou and Chou (1988) (Biosoft, Cambridge, UK). The CI is defined as the ratio of the combination drug concentration to the sum of the single-agent concentrations at an equitoxic level. A CI <1 indicates synergy, CI >1 indicates antagonism, and CI = 1 indicates additivity.

The dose-reduction index (DRI) (Chou and Chou, 1988; Yang et al., 1995), which represents the -fold cisplatin concentration reduction in the combination, compared with the drug as a single agent at given level of effect (x), was calculated using the equation DRI = (D_x)/(D). In the equation, D_x is the concentration of cisplatin as a single agent required for x% effect, and D is the concentration of cisplatin that, in combination with gemcitabine, is required to cause x% effect.

Detection of Cisplatin-DNA Damage

DNA Probes. All probes were genomic inserts labeled by random priming. The 1.8-kb EcoRI fragment containing exons I and II of the DHFR gene was isolated from pBH31R1.8 plasmid probe (Dr. V. A. Bohr, National Institute on Aging, National Institutes of Health, Baltimore, MD), and labeled using [-³²P]dATP. The probe was used to detect a 22-kb fragment of the DHFR gene, including the 5' end of the gene. The -globin probe (Dr. V. A. Bohr) was used to detect the entire 18 kb gene.

Detection of Gene-Specific Cisplatin Intrastrand Adducts. Cells were seeded in 15-cm dishes at a density of 5 × 10⁶ cells/dish for 16 h before the experiment to ensure exponential growth of the cells at the time of drug treatment. 2008 and C13 cells were incubated in the absence or presence of 1 µM gemcitabine for 2 h before exposure to increasing concentrations of cisplatin for 7 h. Cells were subsequently washed twice with phosphate-buffered saline, trypsinized, and pelleted.

Detection of Gene-Specific Cisplatin Interstrand Cross-Links. To study the effect of gemcitabine on the accumulation of cisplatin-induced ICL in specific DNA genes, DNA (6 µg) from treated cells was restricted with HindIII (to release the 22-kb DHFR fragment and the 18 kb -globin gene) for 2 h at 37°C. The DNA was extracted once with phenol and once with chloroform and precipitated with ethanol. The pellet was resuspended in 15 µl of Tris/EDTA buffer and denatured by the addition of an equal volume of 100 mM NaOH and incubated for 20 min at 37°C. Loading buffer (4 µl of 10× buffer: 10 mM EDTA, 26% Ficoll, 0.25% bromocresol green) was added to each sample and the DNA was then electrophoresed through a 0.5% agarose gel in Tris-acetate/EDTA buffer at 27 V overnight. The DNA was transferred to nylon (Hybond N⁺; Amersham Biosciences, Piscataway, NJ), fixed to the membrane by baking for 2 h at 80°C, and probed for the gene fragment of interest. Band visualization and quantitation were performed by PhosphorImager analysis. The Poisson distribution was used to calculate the number of cross-links in the gene fragment and this value was normalized to cross-links/10 kb.

Detection of Gene-Specific Repair of Cisplatin Interstrand and Intrastrand Cross-Links. Cells were treated with cisplatin (200 µM in 2008, A2780; 400 µM in CP70) for 5 h in the absence or presence of gemcitabine, followed by treatment with 0.1 M thiourea for 1 h to block the conversion of monoadducts to ICL during the post-treatment incubation. The cells were then incubated for times up to 8 h in drug-free medium or in 1 µM gemcitabine-treated medium. Cells were harvested, and the DNA was extracted and analyzed for total adducts and ICLs as described above. The repair efficiency was expressed as the percentage of initial cross-links that remained at that time point.

Statistical Analysis. Statistical significance was determined using Student's t test calculated with SigmaStat software (SPSS Science, Chicago, IL).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Cytotoxic Synergy between Cisplatin and Gemcitabine. Clonogenic survival assays were used to investigate the sensitivity of two matched pairs of cisplatin-sensitive and -resistant cell lines to gemcitabine and cisplatin. The IC₅₀ for each drug after a 24-h exposure was determined in each line. The C13*5.25 (IC₅₀ = 7.5 µM) and CP70 (IC₅₀ = 5.5 µM) cell lines demonstrated 19- and 13-fold resistance to cisplatin compared with their 2008 (IC₅₀ = 0.4 µM) and A2780 (IC₅₀ = 0.4 µM) parental lines, respectively. The 2008/C13 (IC₅₀ = 15 nM) and A2780/CP70 (IC₅₀ = 3 nM) pairs had the same sensitivity to gemcitabine.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Cytotoxic interactions between cisplatin and gemcitabine. A, the survival of 2008 cells was assessed by clonogenic assay after cells were treated with cisplatin () (0.23, 0.3, and 0.45 µM) or gemcitabine () (11.25, 15, and 22.5 nM) as single agents, or with both agents in combination () at concentrations in a fixed molar ratio of 20:1 for 24 h (cisplatin, 75, 112.5, 150, 225, and 450 nM; gemcitabine, 3.75, 5.63, 7.5, 11.25, and 22.5 nM). The median effect plot was constructed using the data from the survival curves, where Fa is the affected fraction, Fu is the unaffected fraction, and D is the drug concentration. B, the fraction-affected CI plot was constructed by computer analysis of the data in A using the method of Chou and Talalay (1984).

Effects of Gemcitabine on the Accumulation of Cisplatin-Induced DNA Intrastrand Adducts in the DHFR Gene. Subsequent studies sought to investigate the mechanistic basis of the synergy between cisplatin and gemcitabine in combination, specifically the effect of gemcitabine on the accumulation and removal of cisplatin DNA damage. A quantitative PCR (QPCR) method was first employed to investigate the effect of gemcitabine on the formation of cisplatin-induced IA in the ovarian cell lines. This method exploits the ability of drug-DNA lesions to block the progression of Taq polymerase, thereby inhibiting the amplification of the damaged DNA template (Ponti et al., 1991).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Effects of gemcitabine on the cisplatin-induced DNA intrastrand adducts in the DHFR gene. 2008 cells were incubated in absence or presence of 1 µM gemcitabine for 2 h before exposure to increasing concentrations of cisplatin (as shown) for 7 h. Total genomic DNA was isolated and restricted with Hind III and an 1858-bp fragment in the DHFR gene was subjected to QPCR amplification. A, representative image of QPCR amplification products resolved on a 1% agarose gel. Lane C represents control DNA from untreated cells, and lane C/2 contains half the DNA concentration in lane C. `Control' lanes represent cells treated with increasing concentrations of cisplatin in the absence of gemcitabine; and `+ gemcitabine' lanes represent cells treated in the presence of 1 µM gemcitabine. Band quantitation was performed using a Molecular Imager FX. B, The Poisson distribution was used to calculate the number of DNA adducts in the DNA fragment from cells incubated in the absence () or presence () of gemcitabine. These values were normalized to lesions/2 kb and expressed as the mean ± S.E. and plotted as a function of cisplatin concentration.

Effects of Gemcitabine on the Accumulation of Cisplatin-Induced DNA Interstrand Cross-Links in the DHFR Gene. We next sought to investigate the effects of gemcitabine on the accumulation of the other important cisplatin lesion, the interstrand cross-link. DNA from cells treated as described for QPCR was analyzed for the presence of the ICLs in a 22-kb fragment of the DHFR gene. As shown in Fig. 3a, DNA from control cells incubated in the absence of any drugs was completely denatured under the conditions employed and migrated as single-strand DNA. DNA from cells treated with cisplatin showed a concentration-dependent increase in double-strand cross-linked DNA. The bands were quantitated and the results summarized in Fig. 3b. ICL levels increased linearly as a function of drug concentration. Incubation of cells with cisplatin in the presence of gemcitabine enhanced ICL levels by 1.4-fold (P < 0.05) at 120 µM. Similar results were obtained after analysis of ICL levels in the transcriptionally inactive -globin gene (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Effects of gemcitabine on the cisplatin-induced DNA interstrand cross-links in the DHFR gene. DNA from cells treated as described in Fig. 2 was isolated and analyzed for ICL formation using a denaturation/renaturation assay as described. A, representation of HindIII-digested DNA denatured and electrophoresed through a 0.5% agarose gel, before being transferred to nylon and probed for a 22-kb DHFR gene fragment. DS is double-strand DNA; SS is single-strand DNA. The data shown were based on at least three independent experiments and DNA samples from each biological experiment were subjected to gel electrophoresis and Southern analysis at least twice. B, band quantitation was performed using a PhosphorImager. The Poisson distribution was used to calculate the number of cross-links in the DHFR gene and this value was normalized to cross-links/10 kb in the absence () or presence () of 1 µM gemcitabine, expressed as the mean ± S.E. and plotted as a function of cisplatin concentration.

Effects of Gemcitabine on the Removal of Cisplatin-Induced Intrastrand Adducts in the DHFR Gene. To investigate the possibility that gemcitabine potentiates the accumulation of cisplatin damage by suppressing the repair of cisplatin-induced DNA lesions, the effect of gemcitabine on the repair of cisplatin ICL and IAs was directly examined. The QPCR assay was used to investigate the effect of gemcitabine on the removal of cisplatin-induced IA in the DHFR gene. Cells were incubated with cisplatin in the absence or presence of gemcitabine to induce damage, followed by treatment with thiourea (to prevent the slow conversion of monoadducts to cross-links) before the cells were allowed to repair the damage. The DNA was then isolated and analyzed for the presence of IAs in the DHFR gene and the results are summarized in Table 3.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effects of gemcitabine on the repair of cisplatin-induced DNA intrastrand cross-links in the DHFR gene. 2008 cells were incubated with 100 µM cisplatin in absence or presence of 1 µM gemcitabine for 5 h. The cells were then incubated with thiourea before incubation up to 8 h (as shown) in drug-free medium or in 1 µM gemcitabine-treated medium. A, representative image of amplification products resolved on a 1% agarose gel. B, quantitation of cisplatin-induced IA removal from the DHFR gene fragment. The Poisson distribution was used to calculate the number of adducts in the 1858-bp fragment of the DHFR gene in the absence () or presence () of gemcitabine, and this value was normalized to lesions/2 kb, expressed as percentage repair, and plotted as a function of repair time.

Effects of Gemcitabine on the Removal of Cisplatin-Induced DNA Interstrand Cross-Links in the DHFR Gene. The effect of gemcitabine on the repair of cisplatin ICL was also examined. DNA from cells treated as described above was analyzed for ICL in the DHFR gene and the results are shown in Fig. 5. In both the control (cisplatin alone) and gemcitabine-treated cells, ICLs were removed rapidly, with few cross-links remaining after 8 h. Quantitation revealed that 2008 cells efficiently repaired cisplatin ICL with 72% of damage removed within 8 h. In the presence of gemcitabine, however, the extent of repair was reduced to 60%. Similar results were observed in the A2780 and CP70 cell lines (Table 4). Analysis of ICL repair in the DHFR gene in A2780 and CP70 again revealed a greater extent of repair in the CP70 cell line. These combined data suggest that gemcitabine suppresses the repair of cisplatin-induced ICL in gene-specific DNA sequences.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Effects of gemcitabine on the repair of cisplatin-induced DNA interstrand cross-links in the DHFR gene. 2008 cells were incubated with 100 µM cisplatin in the absence or presence of 1 µM gemcitabine for 5 h. DNA from cells allowed to repair for up to 8 h was analyzed for ICL remaining. A, representative ICL blot probed for the 22-kb DHFR gene fragment. B, quantitation of cisplatin ICLs remaining in the DHFR gene as a function of repair time. The Poisson distribution was used to calculate the number of cross-links remaining after repair in the absence () or presence () of gemcitabine, and was expressed as a percentage of initial ICL levels.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The aim of this study was to explore the mechanistic basis of the synergistic cytotoxic interaction between gemcitabine and cisplatin in cisplatin sensitive and resistant ovarian cancer cell lines. Synergistic interactions between gemcitabine and cisplatin have been described previously in ovarian cell lines using growth inhibition assays (Bergman et al., 1996; van Moorsel et al., 1999). Because such assays reflect the short-term growth inhibitory effects of a drug or drug combination and not necessarily cytotoxicity, we used clonogenic survival assays to ensure a rigorous analysis of the nature of the interaction between gemcitabine and cisplatin in the cell lines studied. A synergistic cytotoxic interaction was revealed between cisplatin and gemcitabine in both the cisplatin-sensitive and -resistant ovarian cell lines.

The greatest cytotoxic synergy, as determined by the DRI, and the maximum inhibition of cisplatin IA and ICL repair by gemcitabine were observed in the cisplatin-resistant CP70 cells. One of the major mechanisms of cisplatin resistance identified in this cell line is enhanced DNA repair (Li et al., 1998; Ferry et al., 2000). In a recent study by Yang et al. (2000), cellular inactivation of the ERCC1 repair gene was shown to abrogate the synergistic cytotoxic interaction between gemcitabine and cisplatin. Furthermore, in NSCLC patients treated with gemcitabine and cisplatin, survival was shown to correlate with the level of expression of the ERCC1 repair gene (Lord et al., 2002). Our finding is therefore consistent with the proposal that inhibition of repair of cisplatin DNA damage by gemcitabine is critical to the cytotoxic synergy observed between the drugs.

Using an in vitro repair synthesis assay, Yang et al. (2000) also demonstrated that gemcitabine triphosphate, the active metabolite of gemcitabine, inhibits the repair of plasmid DNA containing cisplatin damage. To define the role of gemcitabine in the repair of specific cisplatin lesions in cells, gene-specific assays were used in the current study. Because DNA repair is heterogeneous throughout the genome and repair in transcriptionally active genes is a critical determinant in cell survival after DNA damage (Jones et al., 1991), cisplatin damage in the transcriptionally active DHFR gene was analyzed.

Because more than 90% of DNA lesions induced by cisplatin are IAs, the results from the QPCR analysis primarily reflect the presence of these lesions (Talarico et al., 2001). As observed previously by Zhen et al. (1992) using the Escherichia coli ATP-binding cassette excinuclease, cisplatin IAs are rapidly removed from the DHFR gene. However, in the presence of gemcitabine, the repair of these lesions was reduced, consistent with an inhibitory effect of the nucleoside analog on cellular repair of these lesions.

Repair of cisplatin IAs occurs through the concerted activity of over 30 proteins comprising the nucleotide excision repair pathway. After lesion recognition, the DNA is incised on either side of the lesion, a ~29-base oligomer containing the DNA lesion is excised, and the DNA strand is resynthesized and ligated (de Laat et al., 1999). Gemcitabine is suggested to mediate its cytotoxic effects by inhibiting the repair synthesis step of this pathway. Through its actions as a ribonucleotide reductase inhibitor, gemcitabine depletes the intracellular deoxynucleotide pools, thereby enhancing the potential for its own incorporation into newly synthesized DNA. Once incorporated into DNA, the analog causes termination of DNA synthesis and is resistant to removal by exonucleases, resulting in DNA strand breaks (Plunkett et al., 1995). Incorporation of gemcitabine into a cisplatin IA repair patch may therefore lead to a persistence of DNA damage, which leads to cell death.

The results of the current study demonstrated that gemcitabine also inhibited the cellular repair of interstrand cross-links, the other major cytotoxic lesion induced by cisplatin. Repair of interstrand cross-links in mammalian cells is not well understood but is suggested to involve recombination and aspects of the nucleotide excision repair pathway. In a recently proposed model of cross-link repair in replicating DNA, the replication fork stalls upon encountering a cross-link, resulting in a double-strand DNA break. The DNA is then incised, the cross-link unhooked, and the resulting gap in the DNA strand is repaired by recombination. The second strand is then incised on either side of the damage, the DNA fragment containing the cross-link is released, and the resultant gap is resynthesised and ligated (McHugh et al., 2001).

The denaturation/renaturation assay used to detect cisplatin ICL in the current study has been used previously by Yang et al. (1995) to investigate the effects of the nucleoside analog, fludarabine on the repair of cisplatin ICL. As observed with gemcitabine in the current study, fludarabine inhibited the repair of cisplatin damage in specific gene fragments. Because this cross-linking assay detects an early event in the repair of cross-links (the unhooking of an ICL), the results therefore indicate that the nucleoside analogs are able to block this step of ICL repair.

Because the nucleoside analogs are expected to exert their inhibitory effects on cross-link repair at the level of DNA resynthesis, these results suggest that the steps in the ICL pathway are tightly coupled. This hypothesis is supported by results that involve the nucleotide excision repair pathway. Using an in vitro reconstituted human DNA repair excision nuclease, Svoboda et al. (1993) demonstrated that incision of damaged templates is greatly inhibited in the absence of dNTPs required for repair synthesis. In that study, it was suggested that the complex of proteins comprising the human excision nuclease remains stalled on the DNA after incision and requires displacement by the proteins involved in repair replication. Incorporation of gemcitabine into a repair patch may therefore result in the sequestering of the repair replication proteins at that site because of incomplete repair. The reduction of free repair replication proteins may result in the reduced displacement of other repair complexes from damaged DNA and inhibition at all steps of the ICL repair pathway.

The results of the current study directly demonstrate that gemcitabine inhibits the repair of both IA and ICL in ovarian cancer cell lines. These findings strongly support a role for inhibition of nucleotide excision repair and ICL repair pathways in the mechanism of interaction between gemcitabine and cisplatin in these cell lines. These results, together with other findings that gemcitabine produces synergistic cytotoxicities with cisplatin, suggest that gemcitabine may serve as a repair modulator to improve antitumor efficacy when combined with DNA-damaging agents that induce cellular repair.