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Molecular Cancer Research 4:257-265 (2006)
© 2006 American Association for Cancer Research

DNA Damage and Cellular Stress Responses

A Novel Role of DNA Polymerase {eta} in Modulating Cellular Sensitivity to Chemotherapeutic Agents

Yih-wen Chen1, James E. Cleaver3, Fumio Hanaoka4, Ching-fang Chang2 and Kai-ming Chou2

1 Cancer Research Institute and 2 Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, Alabama; 3 Auerback Melanoma Laboratory, University of California at San Francisco Cancer Center, University of California, San Francisco, California; and 4 Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan

Requests for reprints: Kai-ming Chou, Department of Cell Biology and Neuroscience, University of South Alabama, 307 North University Boulevard, MSB 2350, Mobile, AL 36688. Phone: 251-460-6604; Fax: 251-460-6771. E-mail: kchou{at}usouthal.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 
Genetic defects in polymerase {eta} (pol {eta}; hRad30a gene) result in xeroderma pigmentosum variant syndrome (XP-V), and XP-V patients are sensitive to sunlight and highly prone to cancer development. Here, we show that pol {eta} plays a significant role in modulating cellular sensitivity to DNA-targeting anticancer agents. When compared with normal human fibroblast cells, pol {eta}–deficient cells derived from XP-V patients were 3-fold more sensitive to ß-D-arabinofuranosylcytosine, gemcitabine, or cis-diamminedichloroplatinum (cisplatin) single-agent treatments and at least 10-fold more sensitive to the gemcitabine/cisplatin combination treatment, a commonly used clinical regimen for treating a wide spectrum of cancers. Cellular and biochemical analyses strongly suggested that the higher sensitivity of XP-V cells to these agents was due to the inability of pol {eta}–deficient cells to help resume the DNA replication process paused by the gemcitabine/cisplatin-introduced DNA lesions. These results indicated that pol {eta} can play an important role in determining the cellular sensitivity to therapeutic agents. The findings not only illuminate pol {eta} as a potential pharmacologic target for developing new anticancer agents but also provide new directions for improving future chemotherapy regimen design considering the use of nucleoside analogues and cisplatin derivatives. (Mol Cancer Res 2006;4(4):257–65)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 
DNA polymerase {eta} (pol {eta}) is a recently discovered enzyme with the ability to perform translesion synthesis across cys-syn pyrimidine dimers (1), the primary lesion induced by UV radiation, which blocks replicative DNA polymerases (2). Genetic defects in the pol {eta} gene result in xeroderma pigmentosum variant (XP-V) syndrome (1), and XP-V patients are highly sensitive to sunlight and prone to skin cancer development (2). In addition to cys-syn pyrimidine dimers, pol {eta} also replicates across other DNA lesions, including 8-oxoguanine and thymine glycol (3). Structural analysis of the pol {eta} homologue from Saccharomyces cerevisiae revealed a unique mechanism of action, which allows the polymerase to tolerate abnormal DNA lesion structures (4). This special translesion synthesis ability of pol {eta} might accommodate the unusual structures introduced by DNA-targeting anticancer compounds.

DNA-targeting compounds, such as antimetabolite nucleoside analogues, cis-diamminedichloroplatinum (cisplatin), alkylating agents, and anthracyclines, represent an important class of anticancer therapeutic agents. Among these compounds, nucleoside analogue ß-D-arabinofuranosylcytosine (AraC, cytarabine) is used for the treatment of leukemia and ß-D-2',2'-difluorodeoxycytidine (dFdC, gemcitabine) is used for the treatment of breast, ovarian, and non–small cell lung cancers (5). Once inside the cell, AraC and gemcitabine are converted to their monophosphate, diphosphate, and triphosphate metabolites by cellular enzymes and the triphosphates then serve as substrates for replicative DNA polymerases. Therefore, enzymes involved in the metabolism of these compounds are essential in determining the therapeutic activity. For instance, drug resistance to AraC and gemcitabine is frequently observed in clinics and several cellular enzymes, including deoxycytidine kinase and ribonucleotide reductase, have been shown to contribute to drug resistance (5). Once incorporated into DNA, AraC and gemcitabine have been shown to act primarily as inhibitors of replicative DNA polymerases by blocking further extension of their 3' termini (6, 7) and the cytotoxicity of these compounds is correlated with their incorporation level into DNA (6, 7). It stands to reason that DNA polymerases would play an important role in determining the therapeutic activity of nucleoside analogues.

Cisplatin and its derivatives represent another important category of DNA-targeting antitumor agents and are used for the treatments of a wide spectrum of cancers (8). Cisplatin exerts its activity mainly by forming intrastrand cross-linked DNA adducts, which block DNA replication (8). The major clinical complication of using cisplatin is the development of drug resistance (8) and studies have shown that the major mechanism of resistance is increased tolerance of cisplatin-induced DNA damages and enhanced DNA repair capacity (8). To achieve more profound therapeutic responses, combination therapy using gemcitabine with cisplatin has been adopted, and promising results were observed in clinical settings (9, 10), although the results from different studies were not consistent. The biological basis for the efficacy of combination therapy is still not well understood, although DNA repair processes have been suggested to contribute to the observed cytotoxicity. Complicating our understanding of this process is the observation that the presence of gemcitabine does not affect the cellular accumulation of cisplatin and its DNA adducts (11), suggesting that mechanism(s) other than DNA repair plays an important role in the cytotoxic effects obtained with these two compounds. For example, studies have shown that pol {eta} efficiently bypasses cisplatin cross-linked DNA (12, 13) and pol {eta} was shown to participate in cisplatin-induced translesion replication in normal cells (14).

Although AraC and gemcitabine exert their activity mainly by inhibiting DNA replication, both compounds are not true DNA chain terminators because they have an available 3'-hydroxyl group on the sugar moiety. Rather, their ability to inhibit most DNA polymerase is likely inherent to their unnatural structures. Because pol {eta} exhibits better tolerance of abnormal DNA structures and interactions among pol {eta}, AraC, or gemcitabine were not well studied, the effect of pol {eta} on the cellular sensitivity to AraC, gemcitabine, cisplatin, and gemcitabine/cisplatin combination treatments was explored.


   Results and Discussion
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
Materials and Methods
 Acknowledgements
 References
 
Pol {eta} and Cellular Sensitivity to Therapeutic Agents
Pol {eta} is a key enzyme that replicates across the bulky structure of pyrimidine dimers in cells. In addition to the established biological function, in vitro studies have shown that pol {eta} efficiently replicates across cisplatin intrastrand cross-linked DNA (12). The observation that pyrimidine dimers and cisplatin are DNA lesions that block replicative DNA polymerases, a property that is shared by several chemotherapeutic DNA-damaging agents, suggests the involvement of pol {eta} in the processing of DNA-targeting anticancer agents. Previous studies have shown that both AraC and gemcitabine treatments result in DNA fragmentation, and both compounds stop the elongation process carried out by replicative DNA polymerases (6). To test whether pol {eta} affected the in vivo activity of AraC, gemcitabine, or cisplatin, cytotoxicity studies were conducted using SV40-transformed human pol {eta}–deficient XP30RO and the wild-type GM637 fibroblast cells (15). The XP-V cells used in this study were more sensitive to UV damage than wild-type GM637 cells; the LC50 were 5.0 and 11.7 J/m2, respectively (Fig. 1A ; Table 1 ). This degree of sensitivity represents the contribution of pol {eta} to the repair of its primary target lesion and is the standard of comparison against which we determined the effect of a series of anticancer agents. To measure the LC50s, different concentrations of each tested compound were added to either wild-type or XP30RO cells for 24 hours. The cells were then washed and incubated for an additional 72 hours in drug-free medium. At the end of incubation, the cell viability was measured by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion assays. As shown in Fig. 1B and Table 1, the MTT assay results indicated that XP30RO cells were ~3-fold more sensitive to AraC compared with wild-type cells; LC50s were 0.53 and 1.53 µmol/L, respectively. The XP-V cells were 2-fold more sensitive to gemcitabine compared with wild-type cells (Fig. 1C; Tables 1 and 2 ).


Figure 1
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  		FIGURE 1. Cellular sensitivity to anticancer compounds. Cell survival curves of wild-type cells ({square}), wild-type cells stably transfected with a plasmid encoding siRNA against pol {eta} ({circ}), XP30RO cells ({blacktriangledown}), and XP30RO cells stably transfected with a plasmid encoding EGFP-pol {eta} cells ({blacktriangleup}). A. UV. B. AraC. C. Gemcitabine. D. Cisplatin.

 

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  		Table 1. Cell Viability to UV and Therapeutic Compounds

 

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  		Table 2. Cell Viability to Gemcitabine and Cisplatin

 
Previous studies suggest that translesion synthesis can be an important potential mechanism of resistance to cisplatin treatment (12). Recent studies have shown that the cisplatin-induced mutation frequency in XP-V cells is higher than in the normal cells (14). To evaluate whether pol {eta} affects the cellular sensitivity to cisplatin, the LC50s of both wild-type and XP30RO cells to cisplatin were also determined. As shown in Fig. 1D and Tables 1 and 2, the XP30RO cells were ~3-fold more sensitive to cisplatin treatment than the wild-type cells, which is consistent with previous studies (12, 16). In contrast, the results indicated that the wild-type and XP30RO cells have similar sensitivity to deoxycytidine (>100 µmol/L). The major mechanism of action of AraC, gemcitabine, and cisplatin is disruption of cellular replication due to the inhibition of the DNA elongation processes. To test whether pol {eta} also affected the activity of other DNA-targeting anticancer compounds with different mechanisms of action, the cellular sensitivity of both wild-type and XP-V cells to camptothecin was determined. Camptothecin is a DNA topoisomerase I inhibitor that exerts its anticancer activity by producing ssDNA breaks (17). As shown in Table 1, there is no significant difference between these two cell lines against camptothecin (data not shown). These results also indicate that the different cellular sensitivity between wild-type and XP-V cells to AraC, gemcitabine, and cisplatin is a specific cellular response to these agents. To confirm that the observed results are not due to the growth inhibition by these compounds, we also did the trypan blue exclusion assay at 72 hours after drug removal. As shown in Table 1, both trypan blue exclusion and MTT assays produced similar results.

To achieve better therapeutic effects, combination treatments using gemcitabine/cisplatin have been adopted because both drugs have a different mechanism of action, nonoverlapping toxicity, and similar antitumor activity profile. Preclinical and cellular studies have shown promising outcomes with ovary, head and neck, and colon carcinomas (18). Because we showed that the wild-type and the pol {eta}–deficient cells have different sensitivity to cisplatin or gemcitabine when added as a single treatment, it is possible that pol {eta} also has an effect on the cellular sensitivity to the gemcitabine/cisplatin combination treatments. Both the results from MTT and trypan blue exclusion assays indicated that the LC50 of XP-V cells to cisplatin was at least 10-fold lower than that of the wild-type cells in the presence of 2 nmol/L gemcitabine (Fig. 2A ), which suggested that pol {eta} has a significant effect on the cytotoxicity caused by gemcitabine/cisplatin combination treatment.


Figure 2
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  		FIGURE 2. Cellular sensitivity to gemcitabine and cisplatin combination treatments correlates with the expression levels of pol {eta}. A. Dose-response curves of wild-type cells ({square}), wild-type cells stably transfected with a plasmid encoding siRNA against pol {eta} ({circ}), and XP30RO cells ({blacktriangledown}) to cisplatin in the presence of 2 nmol/L gemcitabine. Western immunoblot analysis. Lane 1, wild-type; lane 2, XP30RO; lane 3, wild-type transfected with pol {eta} siRNA. B. Dose-response curves of XP30RO cells ({blacktriangledown}) and XP30RO cells stably transfected with a plasmid encoding EGFP-pol {eta} (XP30RO EGFP-pol {eta}; {blacktriangleup}) to cisplatin in the presence of 1 nmol/L gemcitabine. Western immunoblot analysis. Lane 1, XP30RO; lane 2, XP30RO transfected with a plasmid encoding EGFP-pol {eta}.

 
To confirm that the observed difference in sensitivity between wild-type and XP-V cells was directly related to the cellular abundance of pol {eta}, the GM637 wild-type cells were transfected with a small interfering RNA (siRNA) expression plasmid, which targeted exon 11 of the pol {eta} gene (19). Based on the Western blot analysis (Fig. 2A), the level of pol {eta} in the siRNA-expressing cells was 25% to 30% of the amount observed in the parental cells. Consistent with the observation made with XP-V cells, the suppression of pol {eta} expression in wild-type cells increased the cellular sensitivity to either cisplatin or gemcitabine single-agent treatments (Fig. 1C) as well as the cisplatin/gemcitabine combination treatment (Fig. 2A; Table 3 ). In a complementary approach, a recombinant enhanced green fluorescent protein (EGFP)-pol {eta} expression vector was transformed into the pol {eta}–deficient XP30RO cells as described previously (20), and cytotoxicity studies were conducted (Figs. 1D and 2B). Further confirming our previous observations, the EGFP-pol {eta}–transfected XP-V cells were rendered less sensitive than XP-V cells to either cisplatin or gemcitabine single-agent treatment or the cisplatin/gemcitabine combination treatment (Figs. 1D and 2B; Table 4 ). The expression of pol {eta} in the EGFP-pol {eta}–transfected XP-V cells was also monitored by Western blot analysis as shown in Fig. 2B. Together, these results establish a strong and direct link between the expression level of pol {eta} and cellular sensitivity to AraC, gemcitabine, cisplatin, and, notably, the combination of gemcitabine and cisplatin. This is the first demonstration that the lesion bypass enzyme DNA pol {eta} affects the cytotoxicity of anticancer nucleoside analogues AraC and gemcitabine. Most importantly, pol {eta} has a significant effect (≥10-fold) on the cellular sensitivity to gemcitabine/cisplatin combination treatment.


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  		Table 3. Down-Regulation of Pol {eta} Sensitizes Cells to Gemcitabine/Cisplatin Treatments

 

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  		Table 4. Relationship between Pol {eta} Expression and Cellular Sensitivity to Gemcitabine/Cisplatin Treatments

 
Intracellular Location of Pol {eta} and Drug Treatments
In XP-V cells which lack pol {eta}, an increased replication arrest frequency is observed and fork breakage ensues resulting in the accumulation of MRE11 and proliferating cell nuclear antigen (PCNA) proteins at the replication forks (20). Studies have shown that pol {eta} relocates and forms foci in the cell nucleus after UV irradiation and these foci colocalized with PCNA (20), suggesting that pol {eta} is recruited to the stalled replication forks blocked by DNA damages to perform translesion synthesis and help the resumption of the DNA replication process. Because cisplatin, AraC, and gemcitabine also introduce DNA lesions, which inhibit replicative DNA polymerases (6, 7), pol {eta} may affect the cell sensitivity by relocating to these damaged sites. In XP-V cells, cisplatin induces accumulation of short translesion products and delayed elongation of these products (14). Such results suggested that pol {eta} may relocate to the cisplatin-paused DNA replication sites to help resume DNA replication, which in turn may contribute to a decrease in cisplatin activity in normal cells. To test this hypothesis, the XP30RO cells transfected with the EGFP-pol {eta} expression vector were treated with AraC, gemcitabine, or cisplatin as single agents or in combination, and the cellular distribution patterns of pol {eta} were monitored before and after treatment using confocal laser microscopy (Fig. 3A ). A dispersed distribution pattern of pol {eta} was observed in the nucleus before treatment, but distinct foci formed within 4 hours of drug treatments (Fig. 3A). This is similar to reports studying UV irradiation (20). In contrast, only dispersed distribution patterns were observed when these cells were treated with natural deoxycytidine (data not shown). To further analyze the pol {eta} foci induced by AraC, gemcitabine, and cisplatin, the intracellular localization of PCNA was also determined by immunostaining with a monoclonal anti-PCNA antibody, and the colocalization between pol {eta} and PCNA was determined by confocal microscopy. As shown in Fig. 3A, a large amount of the pol {eta} foci colocalized with PCNA after the cells were treated with these therapeutic compounds. Therefore, it is highly likely that the replication-blocking abnormal DNA structures introduced by these therapeutic agents result in a cellular response similar to that produced by UV irradiation (i.e., recruitment of pol {eta} to the blocked DNA replication forks). It has been reported that UV irradiation results in PCNA monoubiquitination and that pol {eta} specifically interacts with monoubiquitinated PCNA (21). Therefore, the monoubiquitinated PCNA was suggested to play a crucial role in the recruitment of pol {eta} to the stalled DNA replication forks after UV irradiation (21). To test whether AraC, gemcitabine, and cisplatin also induce PCNA ubiquitination, the ubiquitination status of PCNA in the wild-type GM637 cells before and after drug treatment was examined. As shown in Fig. 3B, a higher molecular weight form of PCNA is also observed after UV irradiation (Fig. 3B, lane 2), which is consistent with the results published by others (21). Treatment with AraC, gemcitabine, and cisplatin also induced the formation of a higher molecular weight form of PCNA with the same mobility on the SDS-PAGE gel (Fig. 3B, lanes 3-5). These results suggest that AraC, gemcitabine, and cisplatin also induce PCNA monoubiquitination. To confirm the observed higher molecular protein band was indeed ubiquitinated PCNA, a monoclonal PCNA antibody was used to immunoprecipitated the intracellular PCNA from the cell lysate before and after UV irradiation. The immunoprecipitated complexes were then probed using an anti-ubiquitin monoclonal antibody. As shown in Fig. 3C, the anti-ubiquitin antibody recognized the same higher molecular weight band, and the amount of this ubiquitinated PCNA increased after UV irradiation. This is consistent with a previous study (21). In addition, AraC, gemcitabine, cisplatin, and UV irradiation also induced the formation of ubiquitinated PCNA in XP30RO cells (data not shown). These results suggest that, like the UV irradiation, therapeutic compounds may evoke a similar cellular response to recruit pol {eta} to the stalled DNA replication fork.


Figure 3
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  		FIGURE 3. Intracellular location of pol {eta} and PCNA ubiquitination. A. Intracellular location of pol {eta} and PCNA before and after treatments. A. XP30RO cells stably transfected with a plasmid encoding EGFP-pol {eta} were treated with UV irradiation (10 J/m2) or different anticancer agents (100 nmol/L AraC, 10 nmol/L gemcitabine, 5 µmol/L cisplatin, and combination of 5 nmol/L gemcitabine and 1 µmol/L cisplatin) for 4 hours at 37°C followed with confocal microscopy analysis. B. PCNA ubiquitination induced by UV irradiation, AraC, gemcitabine, and cisplatin. The XP30RO EGFP-pol {eta} cells were treated with different agents and PCNA from the cell extracts was detected using a monoclonal anti-PCNA antibody after Western blotting. Lane 1, control cells; lane 2, UV irradiation (10 J/m2); lane 3, AraC (5 µmol/L); lane 4, gemcitabine (10 nmol/L); lane 5, cisplatin (5 µmol/L). C. Immunoprecipitation of PCNA. PCNA from the cell extracts was immunoprecipitated using a monoclonal anti-PCNA antibody and the precipitated immunocomplexes were probed with a monoclonal anti-ubiquitin antibody on a Western blot. Lane 1, control cells; lane 2, UV irradiation (10 J/m2).

 
In vitro Biochemical Studies of Pol {eta} with Nucleoside Analogues in DNA
It is possible that the observed lower sensitivity of the normal cells compared with the XP-V cells is caused by the ability of pol {eta} to extend DNA terminated by nucleoside analogues once pol {eta} relocates to the stalled DNA replication forks caused by these compounds in cells. If so, such a reaction should facilitate the resumption of the replication process. To test this hypothesis, recessed dsDNA substrates with either AraC or dFdC at the 3' terminus were prepared (22) and the ability of pol {eta} to extend these DNA substrates was examined. As shown in Fig. 4 , pol {eta} efficiently extended both AraC and dFdC terminated DNA in the presence of deoxynucleotide triphosphates (Fig. 4, lanes 4-6). In contrast, replicative DNA polymerase {alpha} (pol {alpha}) could not extend DNA with AraC at the 3' terminus (Fig. 4, lane 8) and seemed to have a lower activity on DNA with gemcitabine at the 3' terminus compared with pol {eta} (Fig. 4, lane 9). These results are consistent with what has been shown for replicative DNA polymerases (6, 7).


Figure 4
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  		FIGURE 4. In vitro extension of nucleoside analogues AraC or gemcitabine terminated DNA by lesion bypass protein pol {eta} or replicative enzyme DNA pol {alpha}. DNA with dC, AraC, or gemcitabine incorporated at the 3' termini were extended by 1 nmol/L pol {eta} or pol {alpha}. The reaction conditions are described in Materials and Methods. Lanes 1 to 3, DNA substrates alone; lanes 4 to 6, in the presence of pol {eta}; lanes 7 to 9, in the presence of pol {alpha}.

 
Extension of AraC and dFdC by pol {eta} would result in internalization of these analogues into DNA, which then can serve as templates for the next round of DNA replication. It has been reported that internalized AraC or dFdC in DNA changes the structure and stability of DNA (23, 24) and inhibits DNA replication (25). It was also shown that AraC within DNA templates can block the Klenow fragment of DNA pol I and T4 DNA polymerase (26). We therefore tested the ability of pol {eta} to bypass an internal AraC or dFdC in template DNA in vitro. In the presence of all four deoxynucleotide triphosphates, pol {eta} was able to bypass both AraC and dFdC sites in template DNA (Fig. 4A). In contrast, the ability of replicative pol {alpha} to insert a nucleotide opposite the AraC or gemcitabine sites was very low and no further bypass products were detected. This suggests that pol {alpha} cannot bypass either AraC or dFdC sites in the DNA. Thus, both AraC and gemcitabine may exert their anticancer activity not only by inhibiting replicative polymerases from extension after their incorporation into DNA but also when they are present in the DNA template strand (Fig. 5 ).


Figure 5
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  		FIGURE 5. In vitro bypass reactions of pol {eta} or pol {alpha} across specific AraC or gemcitabine sites in DNA templates. Recessive DNA substrates containing regular dC or site-specific AraC or gemcitabine were incubated with 1 nmol/L pol {eta} or pol {alpha}. The reaction conditions are described in Materials and Methods. Lanes 1 to 3, DNA substrates alone; lanes 4 to 6, in the presence of pol {eta}; lanes 7 to 9, in the presence of pol {alpha}.

 
Studies have also shown that pol {eta} efficiently bypasses cisplatin cross-linked DNA (13), which might explain the observed increased sensitivity to cisplatin in XP-V cells. Together, the high efficiency of pol {eta} to extend AraC or gemcitabine terminated DNA or to replicate across the abnormal structures introduced by AraC, gemcitabine, or cisplatin provides an explanation of the observed higher sensitivity to the gemcitabine and cisplatin combination treatments in XP-V cells (Figs. 1 and 2). In contrast to this report, a previous study (27) has shown that pol {eta}–transfected XP-V cells exhibited similar cellular sensitivity as the nontransfected cells. It is likely that the discrepancy between our data and those reported earlier is a byproduct of the much shorter drug treatment time (1 hour) used in the earlier study (27) versus our study (24 hours). It has been shown that intracellular accumulation of cisplatin is linear within the first several hours of treatment and does not reach a steady-state level until ~24 hours (28), leading us to believe that the conditions under which our experiments were done are more appropriate to discern differences in cellular sensitivity between XP-V and wild-type cells to cisplatin treatment. Furthermore, a recent study also observed that XP-V cells transfected with a pol {eta}–expressing vector are less sensitive to cisplatin, which is consistent with the data presented in this study (16).

This is the first demonstration that pol {eta} is involved in the action of nucleoside analogues AraC and gemcitabine. Previous studies have shown that when AraC was used to inhibit the nucleotide excision repair (NER) pathway and thus accumulate single-strand breaks at sites of excision and resynthesis there was always a small but significantly higher break frequency in XP-V than normal cells (29). Similarly, the break frequency during NER in the absence of inhibitors was also slightly higher in XP-V than normal cells (30). Our current observations that pol {eta} can extend AraC terminated DNA suggest that pol {eta} may play a role in NER. These results would now be explicable if pol {eta} was required for a part of resynthesis during NER. XP-V cells would then be less effective in NER resynthesis resulting in higher break frequencies, and normal cells would be better able than XP-V cells to resist inhibition by AraC through the action of pol {eta}. A focused search for the mechanism of pol {eta} during NER would therefore be potentially important and integrate the XP-V complementation groups within the other NER groups.

Although the relationship between pol {eta} and XP-V has been established, the biological function of pol {eta} in tissues other than skin is not understood, given that pol {eta} is expressed in most tissues (20). Combination treatments using gemcitabine and cisplatin have shown promising results in clinical studies, and DNA repair pathways, such as NER and homologous recombination repair pathways, were reported to play roles in the observed cytotoxicity of these two compounds (9, 10). However, results from different studies were quite inconsistent (9). In this report, we showed that XP-V cells were more sensitive to AraC, gemcitabine, cisplatin, and particularly the gemcitabine/cisplatin combination treatment. In combination with intracellular localization studies and the biochemical analysis, we have revealed a new potential mechanism for the toxicity observed in clinics. However, this hypothesis needs to be further tested in different cancer cells, particularly the cancers for which cisplatin and gemcitabine are the main components in the therapeutic regimen. If similar results are observed, it would suggest that pol {eta} can be one of the important cellular modulators of DNA-targeting therapeutic agents. Furthermore, long-term management with these therapeutic agents may also affect the level of pol {eta} or the molecules that are involved in the regulation of pol {eta} activity in cells. The data presented in this study also imply that inhibitors of pol {eta} may augment the effectiveness of DNA-damaging chemotherapeutics. Further cellular, biophysical, and biochemical studies on the mechanism of action of pol {eta} and these therapeutic compounds are needed to facilitate the design of such drugs.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Culture
SV40-transformed human fibroblast cells that exhibited normal (GM637) or pol {eta}–deficient (XP30RO) were used in this report and have been described (15). GM637 pol {eta} siRNA stably expressing cells and the XP30RO transfected with EGFP-pol {eta} were generated and characterized as described (19, 20).

Cell Viability Assays
Exponentially growing cells were seeded in a 24-well plate for 24 hours before drug treatment in triplicate. The cells were treated with drugs for 24 hours followed by an additional 72-hour incubation at 37°C. At the end of the time course, 5 mg/mL MTT (100 µL) was added and incubated for 4 hours before determining the absorbance at 595 nm using a microtiter plate reader. For the trypan blue assay, the cells were treated as described for the MTT assay. At the end of 72-hour incubation, the cultured cells were then trypsinized, washed with PBS, and then incubated with a 0.4% trypan blue solution at room temperature for 10 minutes. Cells viability was quantified on a hemacytometer. For clonogenic assay, 600 cells were seeded on 10-cm plates and allowed to grow for 24 hours before UV irradiation or drug treatments. The cells were then treated with drugs for 24 hours. The cells were then washed with PBS and fresh medium was added for an additional 7-day incubation in a humidified 10% CO2 incubator at 37°C. After 7 days, the cultured cells were stained with 0.5% methylene blue in 50% ethanol to visualize the colonies. The number of colonies in each treatment group was counted, and the cell survival data were presented as percentage of the colony numbers of treated/untreated cells.

Western Blotting
Cell extracts were collected at the end of the cytotoxicity study and resolved on a 10% Tris-HCl SDS-polyacrylamide gel. The proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) by standard methods. Pol {eta} was identified with a rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and actin was identified with a mouse monoclonal antibody (Sigma, St. Louis, MO). For the PCNA ubiquitination experiment, the cells were treated with agents for 8 hours. The cells were lysed with lysis buffer [10 mmol/L Tris-HCl (pH 7.4), 4 mmol/L EDTA, 30 mmol/L KCl, 1% NP40, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L NaF, protease inhibitor cocktail (Sigma)] and aliquots of the cell extracts were collected and resolved on a 12% Tris-HCl SDS-polyacrylamide gel. The proteins were then transferred to nitrocellulose membranes (Bio-Rad) by standard methods. PCNA was detected using a monoclonal anti-PCNA antibody (PC10, Sigma). In addition, an anti-PCNA monoclonal antibody (C-20, Sigma) was added to the cell extracts and incubated at 4°C for 2 hours followed with the addition of protein G agarose for an additional hour. The immunocomplexes were precipitated by centrifugation and washed thrice with cold PBS. The immunocomplexes were then resolved on a 12% SDS-PAGE gel, and the proteins were transferred to a nitrocellulose membrane for detection of ubiquitinated PCNA using an anti-ubiquitin monoclonal antibody (Santa Cruz Biotechnology). Immune complexes were visualized by using SuperSignal West Dura (Pierce, Rockford, IL) Western blotting detection reagents. Quantification of the signal was achieved using a Fuji (Stanford, CT) LAS-1000 imaging system.

Confocal Microscopy
Exponentially growing cells were plated in chamber slides for 24 hours followed with 4-hour drug treatments at 37°C. The cells were fixed with cold 1% paraformaldehyde and permeabilized in an ice-cold mixture of 50:50 acetone/methanol. The permeabilized cells were blocked for 1 hour in 10% fetal bovine serum in PBS at 37°C, rinsed, and incubated with anti-PCNA antibody (PC10) for 1 hour at 37°C followed by the addition of a secondary anti-mouse IgG antibody conjugated with Alexa Fluor 555 (Invitrogen, Carlsbad, CA). The cells were incubated for an additional 1 hour at 37°C before slides were washed with PBS twice and mounted using Prolong Antifade (Molecular Probes, Eugene, OR). The cells were analyzed with a Leica (Exton, PA) laser scanning confocal system equipped with krypton/argon laser.

Enzymes and DNA Oligonucleotides
Human pol {eta} was overexpressed and purified as described (31). DNA pol {alpha} is a generous gift from Dr. Fred Perrino (Wake Forest University, NC; ref. 32). DNA oligonucleotide primers labeled with fluorophore Quasar 570 at the 5' ends and terminated with AraCMP or dFdCMP at the 3' end were generated and purified as previously described (22). The purified oligonucleotides with either AraC or gemcitabine at the 3' termini were annealed to a template oligonucleotide to form recessed DNA substrates for extension studies. The recessed dsDNA substrates are 5'-GTGGCGCGGAGACTTAGAGAX-3' (X = AraC or dFdC) and 3'-CACCGCGCCTCTGAATCTCTGTAAACCGCGCCCCTTAAGG-5'.

To make DNA templates with internal AraC or dFdC, DNA oligonucleotides with AraC or dFdC at the 3' termini were first annealed to template DNA to form recessive duplex DNA. The AraC or dFdC containing primers were extended by HIV reverse transcriptase (Amersham, Piscataway, NJ) at 37°C for 10 minutes in the presence of 500 µmol/L deoxynucleotide triphosphates. The extended oligonucleotides with internal AraC or dFdC were then purified by a denaturing gel electrophoresis and annealed with another oligonucleotide primer with fluorophore Quasar 670 at 5' end to be used as substrate for the bypass study. The DNA substrates used for the bypass reaction are 5'-GGAATTCCCCGCGCCAAAT-3' (X = AraC or dFdC) and 3'-CCTTAAGGGGCGCGGTTTAXAGAGATTCAGAGGCGCGGTG-5'.

Enzymatic Reactions
Standard polymerase reactions containing 100 nmol/L primer-template DNA, 100 µmol/L nucleoside analogue triphosphates, 5 mmol/L MgCl2, 10 mmol/L DTT, 0.1 mg/mL bovine serum albumin, and 1 nmol/L pol {eta} were incubated at 37°C for 10 minutes in 10 µL. The reactions were stopped by adding 5 µL formamide and heating at 95°C for 3 minutes. The reaction mixtures were loaded on a 15% denaturing gel for electrophoresis.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Drs. Zafer Hatahet and Richard Honkanen for valuable discussions.


   Notes
Top
 Notes
Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/ 2/05; revised 2/ 3/06; accepted 2/13/06.


   References
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 

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