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[Cancer Research 61, 7399-7403, October 15, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

DNA Polymerase ß Mediates Protection of Mammalian Cells against Ganciclovir-induced Cytotoxicity and DNA Breakage1

Maja T. Tomicic, Rudolf Thust, Robert W. Sobol and Bernd Kaina2

Division of Applied Toxicology, Institute of Toxicology, University of Mainz, D-55131 Mainz, Germany [M. T. T., B. K.]; Institute for Antiviral Chemotherapy, Friedrich Schiller University, D-07745 Jena, Germany [R. T.]; and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 [R. W. S.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
The efficacy of suicide herpes simplex virus-1 thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy is often limited by intrinsic resistance of tumor cells. Here we show that repair of GCV incorporated in DNA is a factor involved in GCV resistance. A protective role of DNA repair in GCV-induced cell killing is supported by the following findings: (a) GCV-exposed Chinese hamster ovary-HSVtk cells exhibited both reduced repair of GCV and cloning efficiency in the presence of a specific polymerase ß (ß-pol) inhibitor, prunasin; (b) DNA ß-pol-deficient mouse fibroblasts were more sensitive to the cytotoxic, apoptosis-inducing, and genotoxic (DNA breakage and chromosomal aberration-inducing) effects of GCV as compared with wild-type and ß-pol-complemented cell lines; (c) methoxyamine, an inhibitor of ß-pol-dependent short-patch base excision repair, sensitized wild-type and complemented ß-pol cells to GCV, whereas it had no effect on the sensitivity of ß-pol-null cells to GCV. Because methoxyamine-mediated sensitization of ß-pol wild-type and ß-pol-complemented cells to GCV did not reach the level of null cells, we suggest that both ß-pol-dependent short- and long-patch base excision repair are involved in protection of cells to GCV. Some implications for HSVtk/GCV gene therapy are being discussed.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Suicide gene therapy, which is based on transduction of tumor cells with HSVtk3 as a "suicide gene" and systemic administration of GCV, was initially considered to be a revolutionary anticancer protocol (1) by which target tumor cells are triggered to die by apoptosis (2) . After promising results obtained in several animal models (3) , notably by means of stably HSVtk-transfected cells using the "bystander effect" (4) , once at the clinical stage the therapy showed various limitations such as low target specificity and low transduction efficacy of target cells. In addition to the limitations of the therapy mentioned above, tumor cells may exhibit intrinsic resistance. However, this should not be mistaken for resistance to GCV because of loss of functional thymidine kinase in HSVtk-transduced tumor cells (5) . Here we suggest that intrinsic resistance of tumor cells can result from repair of the incorporated antiviral drug. GCV incorporated in DNA causes a stable and potent cytotoxic and genotoxic lesion exhibiting delayed effects, which require at least one postexposure round of DNA replication to be expressed (6, 7, 8) . By using cell lines proficient, deficient, and complemented for ß-pol and by down-modulation of ß-pol activity in CHO cells expressing HSVtk, we provide evidence that the repair enzyme ß-pol exerts protection of cells against the cytotoxic and genotoxic effects of GCV. Moreover, data obtained indicate that protection of cells against GCV involves ß-pol-dependent single-nucleotide and ß-pol-dependent long-patch BER. This is the first report showing that GCV incorporated in DNA is subject to repair.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Chemicals and Radioisotopes.
GCV (Cymevene) and [3H]GCV (20 Ci/mmol) were purchased from Syntex Arzneimittel GmbH (Aachen, Germany) and Moravek Biochemicals (Brea, CA), respectively. Prunasin, MX, and geneticin (G418 sulfate) were products of Sigma-Aldrich (München, Germany). Hygromycin B was purchased from Calbiochem (Bad Soden, Germany). Mouse monoclonal anti-ß-pol antibody was purchased from NeoMarkers (Fremont, CA).

Cell Culture.
ß-Pol cells [proficient (ß-pol+/+; wild-type Mb16tsA, clone 1B5) and deficient (ß-pol-/-; knockout Mb19tsA, clone 2B2) in ß-pol activity] are mouse embryonic fibroblasts routinely cultivated and selected with hygromycin B (400 µg/ml) as described (9) . The mouse embryonic clone (19/729.E5) exhibiting a partially reverted ß-pol phenotype after stable transfection of ß-pol-null cells with a ß-pol expression vector was described previously (10) . CHO-HSVtk cells are stable transfectants of CHO cells expressing HSV-1 thymidine kinase. They were cultured in the presence of G418 (1.5 mg/ml) as described previously (7) .

Incorporation of [3H]GCV and Isolation of Cellular DNA.
For a dose-dependent experiment, CHO-HSVtk cells were exposed to different concentrations of [3H]GCV for one cell cycle (14 h) and collected for isolation of genomic DNA. For kinetic experiments, the cells were exposed to [3H]GCV for 14 h, and the genomic DNA was isolated after different postexposure times. The concentration of the radioisotope used was 0.2 µM, a dose shown not to affect cell proliferation. For blocking of ß-pol activity, prunasin (0.5 mM) was added during exposure and postexposure period. For isolation of cellular DNA, Blood and Body Fluid Protocol (QIAamp Blood kit; Qiagen) was used according to the manufacturer’s instructions. Radioactivity in an aliquot of DNA was determined by scintillation counting.

Clonogenic Survival and Determination of Apoptosis.
Exponentially growing cells were seeded at a density of 500-1000 cells/6-cm dish and 17 h later treated with GCV, as described for clonogenic survival (7) . Treatment with GCV was chronic or lasted for one cell cycle (14 h/CHO cells and 18 h/ß-pol cells). Apoptosis was quantified after double staining with annexin V and propidium iodide by flow cytometric analysis using FACSort (Becton Dickinson, Heidelberg, Germany) as described (7 , 11) .

Prunasin and MX Treatment.
CHO-HSVtk cells were preincubated with prunasin for 3 h and then GCV was added to the medium. Colony formation as a measure of cell survival after the combined chronic exposure to prunasin and GCV was determined after 7–10 days when visible clones were grown. Colony-forming assays were also conducted in the presence of MX. A stock solution (1 M of MX in PBS) was freshly prepared before use, and immediately after addition of MX, the cell medium was neutralized using NaOH. MX was applied to ß-pol cells at a concentration of 15 mM during the last 10 h of the 18-h GCV exposure. Thereafter, the medium was changed, and cells were incubated until colonies became detectable.

SCGE.
The alkaline SCGE (comet assay) was performed basically as described (12 , 13) . In brief, after post-treatment incubation, cells were harvested by trypsinization and cooled down on ice. Cells (1 x 104/10 µl) were embedded in 120 µl of low-melting point agarose (0.5% in dH2O at 37°C) onto agarose-coated (1.5% in PBS) and dried slides that were submersed for 1 h in precooled lysis buffer [2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, and 1% Na-laurylsarcosine (pH 7.5); 1 h before use 1 ml Triton X-100 and 10 ml DMSO/100 ml were added]. Slides were alkali-denatured for 25 min at 4°C in electrophoresis buffer [300 mM NaOH and 1 mM EDTA (pH > 13)] and electrophoresed at 25 V (300 mA) for 15 min at 4°C. After neutralization [0.4 M Tris (pH 7.5)], slides were fixed in ethanol, dried, and stained with ethidium bromide (20 µg/ml). Slides were analyzed using a microscope (Olympus BX50), and "olive tail moment" was determined by measuring the fluorescence intensity using Kinetic Imaging Komet 4.02 software (BFI Optilas, Puchheim, Germany).

Clastogenicity Assay.
Asynchronously growing cells were seeded at a density of 3 x 105 cells/25-cm2 flask. One day later, the cultures were exposed to GCV for one cell cycle (18 h), carefully rinsed, and chromosome preparations were made after 36 h of postexposure, (a time period that had shown maximum aberration rates in background experiments) in the usual manner after trypsinization of the cultures. For metaphase arrest, colcemid (0.05 µg/ml) was added during the last 3 h before preparation. Clastogenicity was evaluated in 100 metaphases/sample of preparations stained by conventional Giemsa stain. The aberration types scored were chromatid and isochromatid gaps, chromatid and isochromatid breaks, translocations, dicentric and ring chromosomes, and premature chromosome condensations.

Preparation of Cell Extracts and immunoblotting.
Whole-cell extracts were prepared by lysis in ice-cold sample buffer [25 mM Tris-HCl (pH 6.8), 5% glycerol, 2.5% 2-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride] followed by sonification (Branson Sonifier; 30 kHz; 3 x 10 s) on ice. Fractions of 20 µg of protein were separated by 12% SDS-PAGE and electroblotted onto nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) using the transfer buffer (25 mM Tris-HCl, 100 mM glycine, and 25% methanol). The membrane was preincubated for 3 h with 5% nonfat dry milk and 0.1% Tween PBS, incubated with anti-ß-pol antibody, and diluted (1:500) in 5% nonfat dry milk and 0.2% Tween PBS overnight at 4°C. After extensive washing, the membrane was incubated for 1 h with horseradish peroxidase-conjugated antimouse IgG (Amersham) and diluted (1:3000) as above. Protein-antibody complexes were visualized by enhanced luminol reagent (NEN Life Science Products, Boston, MA).


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Repair of GCV Incorporated in DNA.
In a previous study we showed that GCV is efficiently metabolized and incorporated in the DNA of HSVtk-transfected CHO cells, triggering excessive apoptosis (7) . The dose-dependent incorporation of radioactively labeled GCV (concentration range 0.1–1.0 µM) into the cellular DNA of metabolically competent CHO cells expressing HSVtk (designated as CHO-HSVtk) is shown in Fig. 1ACitation . In this experiment, cellular DNA was isolated after one cell cycle (i.e., the period of 14 h) of exposure to GCV. To determine whether GCV incorporated into DNA is processed by a repair mechanism, we isolated cellular DNA from CHO-HSVtk cells immediately after incorporation of [3H]GCV for the duration of one cell cycle and after recovery times of 14, 28, 42, and 56 h (time after exposure). Parallel to this, experiments with nonradioactive GCV (0.2 µM) were conducted to prove that with the concentration used cell growth was not affected (data not shown). As shown in Fig. 1BCitation , during the GCV exposure cycle the drug became clearly incorporated into DNA (0 h postexposure value). After 14 h of postincubation (i.e., within the first postexposure cell cycle), we determined an additional (~2.5-fold) increase of radioactivity in DNA (~1100 cpm/µg DNA), which indicates that phosphorylated GCV was still present in the cells that became incorporated into DNA. However, after additional postexposure incubation (28–56 h) a significant decrease in radioactivity was observed, indicating that the incorporated drug was removed from DNA. Because our background experiments indicated an involvement of ß-pol in the repair of GCV-incorporated in DNA, we cotreated the cells with a specific ß-pol inhibitor, prunasin (14) , and determined the radioactivity in DNA extracted from cells exposed to [3H]GCV. Prunasin treatment resulted in a significant elevation of the overall radioactivity incorporated in DNA, as monitored 28–56 h after treatment with GCV (Fig. 1B)Citation , suggesting a role for ß-pol in the repair of GCV.



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  		Fig. 1. GCV incorporation and reproductive cell death after modulation of ß-pol activity in CHO-HSVtk cells. A, dose-dependent incorporation of GCV into the cellular DNA of CHO-HSVtk cells. GCV incorporation was determined by exposing cells for 14 h to [3H]GCV and expressed as radioactivity per µg of DNA. B, incorporation kinetics of GCV into DNA in the presence and absence of ß-pol inhibitor, prunasin (0.5 mM). GCV incorporation was determined as radioactivity fraction (cpm/µg DNA) after treatment with 0.2 µM [3H]GCV for the period of one cell cycle (14 h) and after different periods of postexposure. 0 h values: DNA extracted immediately after GCV exposure; bars, ± SE. C, clonogenic cell survival after exposure of CHO-HSVtk cells to 0.5 µM GCV in the absence and presence of prunasin, and of ß-pol-/- cells exposed to 40 µM of GCV with or without prunasin. Average data from three independent experiments are shown; bars, ± SE.

 
Sensitization of CHO-HSVtk Cells by Down-Modulation of ß-Pol Activity.
On the basis of the finding that GCV incorporated in DNA is subject to repair, we were interested to see whether inhibition of ß-pol activity influences clonogenic cell survival after exposure to GCV. For this purpose, CHO-HSVtk cells were chronically exposed to GCV together with prunasin. Prunasin itself (0.5 mM) affected neither the viability of the cells in the clonogenic survival assays nor the frequency of apoptosis (data not shown). A combination of the inhibitor (0.3 or 0.5 mM of prunasin) and the drug (0.5 µM of GCV) resulted in increased clonogenic cell death (Fig. 1C)Citation . To confirm that this is attributable to a specific effect of prunasin on DNA, cells deficient in ß-pol were cotreated with GCV (40 µM) and prunasin (0.5 mM). As shown in Fig. 1CCitation , prunasin had no effect on GCV-induced cytotoxicity in ß-pol-/- cells. On the basis of the data we conclude that inhibition of ß-pol by prunasin results in increased cytotoxicity in CHO-HSVtk cells, which is attributable to impaired ß-pol-dependent repair of GCV incorporated in DNA.

Hypersensitivity of ß-Pol-deficient Cells to GCV.
To verify this conclusion, we investigated the role of ß-pol in GCV-mediated cytotoxicity in more detail using mouse embryonic fibroblasts proficient (ß-pol+/+), deficient (null cells; ß-pol-/-), and complemented (null cells transfected with ß-pol designated as ß-pol/compl) for ß-pol activity. The ß-pol protein expression in the cell lines examined is shown in the top panel of Fig. 2ACitation . Whereas ß-pol-/- cells did not display any ß-pol protein, ß-pol+/+ and complemented cells were clearly positive for ß-pol expression. The complemented clone exhibited ß-pol activity was shown previously to be protected against MMS-induced apoptosis (10) . As determined in colony-forming experiments, ß-pol-/- cells were more sensitive than ß-pol+/+ and complemented ß-pol-/- cells after chronic exposure to 25–75 µM of GCV (Fig. 2A)Citation . Thus, at a concentration of 50 µM GCV cell survival in ß-pol-/- cells was reduced to 0.7%, whereas the viability of ß-pol+/+ and ß-pol/compl cells decreased to only 17 and 11%, respectively. To find out whether ß-pol-/- cells, similarly to HSVtk-expressing cells (7) , undergo apoptosis after exposure to GCV the cells were analyzed by flow cytometry using annexin V/propidium iodide double staining. Exponentially growing cells were exposed to 0.1–1 mM of GCV for the duration of one cell cycle (18 h) and 96 h later collected for analysis. We should note that ß-pol cells were not HSVtk transfected and, therefore, did not express HSVTK. Therefore, it was necessary to treat the cells with higher concentrations of GCV, compared with experiments with HSVtk-transfected cells, to obtain significant induction of genotoxicity and apoptosis (15) . As shown in Fig. 2Citation , induction of apoptosis was dependent on the GCV concentration (Fig. 2B)Citation and the duration of postexposure incubation (Fig. 2C)Citation . With a dose of 1 mM of GCV, induction of apoptosis in ß-pol-/- cells was ~80%, whereas <40% of ß-pol+/+ and ~50% of ß-pol/compl cells were apoptotic. Significant induction of apoptosis was observed 48–96 h after GCV exposure, whereas induction of necrosis remained <10%. Thus, the major route of GCV-induced cell killing appears to be apoptosis, which is a late response after treatment. For all of the doses and postexposure time points tested ß-pol-/- cells proved to be more sensitive than repair competent cells. To disprove the possibility that differences in cytotoxicity are attributable to differential incorporation of the drug into the DNA of ß-pol cells, we measured the incorporation of [3H]GCV in DNA and found it to be the same in the cell lines analyzed (data not shown).



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  		Fig. 2. Expression of ß-pol protein, clonogenic survival, and induction of apoptosis by GCV in ß-pol-proficient and -deficient cells. A, top panel, expression of the ß-pol protein as determined by Western blot analysis in wild-type (ß-pol+/+), null (ß-pol-/-), and complemented (ß-pol/compl) cells. Protein loading was checked by Poencaue R staining (not shown). Bottom panel, reproductive cell death, as measured by loss of colony-forming ability, after chronic treatment of ß-pol+/+, ß-pol-/-, and ß-pol/compl cells with GCV. Data are the mean of three independent experiments; bars, ± SD. B, frequency of apoptosis and necrosis determined 96 h after treatment with different doses of GCV. C, frequency of apoptosis and necrosis as a function of time after exposure to 1 mM of GCV. Data are the mean of two independent experiments.

 
Cells Deficient in ß-Pol Are Hypersensitive to the Genotoxic Effects of GCV.
GCV incorporated in the cellular DNA was shown previously to exert clastogenic effects (7 , 8) . If GCV is repaired at a lower rate in a given cell type, one would expect increase of genotoxicity. To prove this, we determined the rate of DNA strand breaks in the ß-pol cell lines after exposure to GCV. Cells were exposed to 0.5 mM of GCV for one cell cycle (18 h) and afterward, with the indicated times of postexposure, they were assayed for the presence of DNA breaks by alkaline SCGE. As shown in Fig. 3ACitation , with a postexposure time of 24–42 h after GCV treatment, the mean value of DNA breaks (expressed as olive tail moment) in ß-pol-/- cells was ~30, whereas significantly fewer DNA breaks were detected in ß-pol+/+ and ß-pol-complemented cells. Thus, ß-pol-/- cells are clearly hypersensitive as to the DNA breakage-inducing effect of GCV, indicating nonrepaired GCV as the cause of DNA breaks.



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  		Fig. 3. Induction of DNA breaks and chromosomal aberrations after exposure of ß-pol-proficient and -deficient cells to GCV. A, induction of DNA breaks (expressed as olive tail moment) as a function of postexposure time after 18-h treatment of cells with 0.5 mM of GCV. DNA breaks were determined by alkaline SCGE. Data are the mean of two or three independent experiments. B, frequency (%) of chromosomal aberrations determined at 36 h after treatment of cells with different GCV concentrations for 14 h. C, number of chromosomal aberrations per cell determined at 36-h postexposure to GCV.

 
To elucidate whether ß-pol cells exhibit differential sensitivity to the clastogenic effect of GCV, the cells were treated with the drug for 18 h and, after two postexposure cell cycles (i.e., 36 h, required for the expression of replication-dependent cyto- and genotoxic effects of the drug; Ref. 8 ), cells were harvested and chromosomal spreads prepared. As shown in Fig. 3BCitation , at a concentration of 0.33 mM of GCV, ß-pol-/- cells exhibited a chromosomal aberration frequency of ~90%, as compared with ß-pol+/+ and ß-pol/compl cells exhibiting 50 and 68% aberrations, respectively. Also, the absolute number of induced aberrations per cell was lower in ß-pol+/+ and ß-pol/compl cells than in ß-pol-/- cells (Fig. 3C)Citation . The results show that deficiency in ß-pol significantly affects chromosomal stability in GCV-exposed cells.

Sensitization of ß-Pol Cells via MX.
To examine a possible role of ß-pol-dependent long-patch BER in sensitivity to GCV, we cotreated cells with MX to block single-nucleotide BER at the time of exposure to GCV. This approach was used previously to demonstrate involvement of long-patch BER in the protection of cells against simple alkylating agents (16) . Cotreatment of ß-pol+/+ and ß-pol/compl cells with MX and GCV indeed sensitized the cells to GCV. MX had no influence on the sensitivity of ß-pol-/- cells to GCV (Fig. 4)Citation . From this we conclude that the modulation of sensitivity of wild-type and complemented cells by MX is attributable to inhibition of ß-pol-dependent single-nucleotide BER and, therefore, this repair pathway appears to be involved in repairing GCV-induced DNA lesions. However, although ß-pol-expressing cells were sensitized to GCV under conditions of inhibited single-nucleotide BER, ß-pol+/+ and ß-pol/compl cells became not as sensitive to GCV as ß-pol-/- cells, which may indicate that ß-pol-dependent long-patch BER could also be involved in the protection of cells against GCV.



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  		Fig. 4. Clonogenic survival of ß-pol-proficient and -deficient cells to GCV in the absence and presence of MX. Cells were treated with GCV for 8 h and additionally exposed (or not exposed) to 15 mM of MX for an additional 10 h. After the medium was changed, cells were incubated for 10 days until colonies appeared. Data are the mean of up to three independent experiments.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Treatment of HSVtk-expressing CHO cells with [3H]GCV followed by determination of radioactivity in DNA after the indicated time periods of postexposure revealed that GCV is efficiently incorporated into the cellular DNA (this paper and Ref. 7 ). In contrast to GCV, the metabolization product GCV-triphosphate cannot freely cross the cell membrane. Because of long intracellular half-life of 12–18 h (17) , GCV-triphosphate is supposed to be intracellularly available not only in the exposure period but also in the postexposure cell cycle(s) for additional incorporation. Indeed, incorporation of GCV into DNA was found to occur not only during the drug exposure cell cycle but also in the postexposure cycle. However, after additional postincubation times the radioactivity detected in DNA significantly decreased (by ~4-fold) indicating that the incorporated drug was removed from DNA by a repair process.

To elucidate which repair pathway could be involved, we initially analyzed the sensitivity of cells deficient in NER. Cells deficient in ERCC1 or ERCC3 and, therefore, impaired in NER were not more sensitive to the killing and clastogenic effects of GCV (preliminary data not shown) indicating that the NER pathway does not significantly contribute to the repair of GCV incorporated in DNA.

A possible involvement of BER in the repair of GCV adducts was shown by experiments in which ß-pol activity was inhibited with prunasin in metabolically competent (HSVTK expressing) CHO cells. Inhibition of ß-pol by prunasin was reported not to affect other DNA polymerases such as {alpha}, {delta}, and {epsilon} (14) . At nontoxic concentration, prunasin caused an increase of radioactivity remaining in DNA. It also clearly increased the cytotoxic effect of GCV, indicating ß-pol to play a role in the repair of GCV. A control experiment demonstrated that prunasin acted specifically on ß-pol-expressing cells, because exposure to prunasin did not increase GCV cytotoxicity in ß-pol-/- cells.

To provide more direct evidence for an involvement of BER in the repair of GCV-induced DNA damage, ß-pol-deficient mouse fibroblasts (derived from ß-pol knockout mice) were analyzed for GCV-induced cytotoxicity and genotoxicity. We demonstrate ß-pol-deficient cells to be more sensitive to the cytotoxic and apoptosis-inducing effect of GCV than ß-pol wild-type and ß-pol-/- cells that were complemented by retransfection of the ß-pol gene. Interestingly, ß-pol-/- cells also displayed a higher frequency of DNA single-strand breaks (as detected by alkaline SCGE) and chromosomal aberrations on GCV treatment. This finding indicates that incompletely repaired GCV results in DNA breaks that eventually become expressed as aberrations. DNA double-strand breaks have been shown previously to trigger apoptosis (18) . Therefore, it is reasonable to assume that accumulation of nonrepaired DNA breaks and/or chromosomal aberrations and a final trigger of GCV-induced apoptosis. This supposition is consistent with the time course of apoptotic cells, which appeared after DNA breaks and aberrations became detectable (7 ; and data not shown).

GCV incorporated in DNA is a stable lesion causing a change in the DNA secondary structure (19 , 20) . This probably signals recognition by BER enzymes. Cotreatment of ß-pol-expressing cells with MX sensitized them to GCV. MX reacts with the aldehydic C1 atom of the acyclic sugar residue (which likely also occurs in the incorporated GCV-MP because the C2 atom is missing) yielding a stable sugar adduct, which is refractory to ß-elimination by deoxyribose phosphate lyase activity of ß-pol. On the basis of this mechanism, MX is able to block single nucleotide BER, thus sensitizing cells to simple alkylating agents (16) . Although wild-type and complemented ß-pol cells were significantly sensitized to GCV in the presence of MX, i.e., when single-nucleotide BER was blocked, ß-pol-/- cells were still more sensitive to GCV than the wild-type and ß-pol complemented cells. This suggests that, in addition to ß-pol-dependent single-nucleotide BER, ß-pol-dependent long-patch BER is involved in GCV lesion repair. We should note that evidence is available to show that both short- and long-patch BER, regardless of the nature of the apurinic site, is initiated by ß-pol, which adds the first nucleotide into the repair gap (21) .

Altogether, the results presented here show that the nucleoside analogue GCV incorporated in DNA is subject to repair via the BER pathway and that the repair enzyme ß-pol mediates protection of cells against cytotoxicity and genotoxicity of GCV. The data bear implications for the utilization of GCV both as an antiviral drug and in suicide gene tumor therapy. Thus it has been shown that tumor cells express a variable amount of ß-pol (22) , which could have impact on their response to GCV. Also, intrinsic resistance of tumor cells might be diminished by systemic administration of GCV in combination with a specific ß-pol inhibitor after transduction of the suicide gene into target cells.


   ACKNOWLEDGMENTS
 
We thank Markus Christmann for critical reading of the manuscript.


   FOOTNOTES
 
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.

1 Supported by the Deutsche Forschungsgemeinschaft Grants KA 724/7-1 and 7-3 and TH 670/1-1 and 1-3. Back

2 To whom requests for reprints should addressed, at Division of Applied Toxicology, Institute of Toxicology, University of Mainz, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany. Phone: 49-6131-393-3246; Fax: 49-6131-393-3421; E-mail: kaina{at}mail.uni-mainz.de Back

3 The abbreviations used are: HSVtk, herpes simplex virus-1 thymidine kinase gene; GCV, ganciclovir; ß-pol, polymerase ß; CHO, Chinese hamster ovary; MX, methoxyamine; prunasin, D-mandelonitrile-ß-D-glucoside; SCGE, single-cell gel electrophoresis; NER, nucleotide excision repair. Back

Received 5/29/01. Accepted 8/27/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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