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[Cancer Research 59, 5233-5238, October 1, 1999]
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[Cancer Research 59, 5233-5238, October 15, 1999]
© 1999 American Association for Cancer Research

Experimental Therapeutics

Enzyme Prodrug Gene Therapy: Synergistic Use of the Herpes Simplex Virus-Cellular Thymidine Kinase/Ganciclovir System and Thymidylate Synthase Inhibitors for the Treatment of Colon Cancer

Oliver Wildner1, R. Michael Blaese and Fabio Candotti

Clinical Gene Therapy Branch/National Human Genome Research Institute, NIH, Bethesda, Maryland 20892-1851


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The goal of this study was to improve the therapeutic index of the herpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) system by the addition of thymidylate synthase (TS) inhibitors. For this, we assessed the potential of GCV to synergistically interact with 5-fluorouracil (5-FU), ZD1694 (Tomudex), and (E)-5-(2-bromovinyl)-2'-deoxyuridine in HSV-tk-expressing murine MC38 STK and human HT-29 STK colon carcinoma cell lines. Synergistic cell killing was observed in a clonogenic assay over most of the cytotoxic dose range by the median-effect principle of Chou and Talalay (T. C. Chou and P. Talalay, Adv. Enzyme Regul., 22: 27–55, 1984). In a s.c. HT-29 STK xenograft tumor model, we demonstrated that the combination of GCV and 5-FU resulted in statistically significant enhanced animal survival over single-agent treatment. Furthermore, we showed that the combination of GCV and ZD1694 in association with the HSV-tk/GCV system was at least as effective as GCV/5-FU in vitro and in vivo. The mechanism for the observed synergy is most likely attributable to the increased GCV phosphorylation in the presence of the tested TS inhibitors. Our data suggest that the HSV-tk/GCV metabolic suicide gene transfer system could serve as an adjuvant of the presently used TS inhibitors, thus potentially improving the efficacy of present cancer gene therapy approaches.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Suicide gene therapy is the approach whereby genetically altering a cell makes it susceptible to an otherwise nontoxic prodrug. The most widely studied gene-directed enzyme prodrug system is the HSV-tk/GCV2 paradigm (1) . This is based on the selective phosphorylation of GCV by the vector-encoded HSV-tk to the level of the monophosphate (GCV-MP). Cellular kinases further phosphorylate GCV-MP to di- and triphosphate metabolites (Fig. 1)Citation . GCV-TP is the active form of the drug (2) . Incorporation of GCV-TP into macromolecular DNA results in chain termination (3) , chromosomal aberrations, and sister chromatid exchange (4) , ultimately leading to cell death (5) .



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  		Fig. 1. Direct and indirect TS inhibitors: sites of action of ZD1694, 5-FU, and BVdU. UP, uridine phosphorylase; PRT, phosphoribosyl transferase; FdUMP, 5-fluoro-dUMP; FH2, dihydrofolate; UDP, uridine diphosphate; GCV-DP, GCV diphosphate; Td, thymidine.

 
Complete results of two early HSV-tk/GCV cancer gene therapy trials in patients with brain tumors and pleural mesotheliomas have been published recently (6 , 7) . In both trials, the number of tumor responses were low or of short duration. The administered GCV dose in these trials was 5 mg/kg twice daily for 14 days, which is the recommended dose for the treatment of cytomegalovirus retinitis (8) . The low response rate observed in these clinical trials might be attributable to failure in achieving adequate transduction efficiency and/or prodrug concentration within the tumor. Higher doses of GCV, however, may not be tolerated because of its myelosuppression and central nervous system side effects (9) . Simply increasing the intracellular HSV-tk enzyme level above a certain threshold is also not expected to augment the efficacy of this system (10) . Therefore, other strategies are needed to enhance therapeutic responses. Possible approaches include pharmacological augmentation of the "bystander effect" (11) , the use of more active HSV-tk mutants (12) , the utilization of replication-competent vectors (13, 14, 15) , and the development of new prodrugs with higher affinities for HSV-tk. Recently, it has been shown that the use of a dual prodrug activating system, which consists of the Escherichia coli CD/5-FC (16 , 17) and HSV-tk/GCV, results in enhanced cytotoxicity when compared with either system alone (18, 19, 20) .

These studies prompted us to examine whether the efficacy of the HSV-tk/GCV system could be enhanced when combined with TS inhibitors. TS converts dUMP into TMP, as schematically depicted in Fig. 1Citation . Other enzymes subsequently phosphorylate TMP to dTTP. TS inhibitors affect critical cell functions, such as DNA synthesis and repair. We tested the effects of 5-FU (21) , which is activated when metabolized to FdUMP, ZD1694 which directly inhibits TS (22) , and BVdU, which was shown to be cytostatic in HSV-tk-expressing murine mammary carcinoma cells in vitro by inhibiting TS through its metabolite BVdU-MP (Fig. 1Citation ; Refs. 23 and 24 ).

To assess whether the TS inhibitors BVdU, 5-FU, or ZD1694 may improve the efficacy of the HSV-tk/GCV system, we tested these in association with GCV in the HSV-tk-expressing murine MC38 STK and human HT-29 STK colon carcinoma cell lines in vitro and in vivo. Clonogenic assays and xenograft tumor models demonstrated a synergistic tumor cell killing of TS inhibitors in combination with GCV when compared with single-agent treatment.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Drugs.
The human cell lines HT-29 (ATCC HTB-38), HeLa (ATCC CCL-2), A375 (ATCC CRL-1619), and MDAH 2774 (ATCC CRL-10303) were purchased from the ATCC (Manassas, VA). The 3-methylcholanthrene-induced murine colon adenocarcinoma MC38 was a gift of S. A. Rosenberg (25) . All cell lines were propagated in D-10 medium, consisting of DMEM supplemented with 10% heat-inactivated fetal bovine serum and 50 µg/ml gentamicin. Tissue culture medium and supplements were purchased from Life Technologies, Inc. (Grand Island, NY). Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. HT-29 STK, HeLa STK, A375 STK, MDAH 2774 STK, and MC38 STK (26) were generated by transduction of the parental cell lines with supernatant from PA317-STK cells (27) and subsequent G418 selection (Geneticin; Life Technologies, Inc.; 1 mg/ml for 3 weeks), followed by subcloning by limiting dilution. Cells were in logarithmic phase of growth at the time of use.

The following drugs were used in the study: GCV (Cytovene-IV, obtained from Hoffman La Roche Laboratories, Nutley, NJ), 5-FU (Adrucil, purchased from Pharmacia Inc., Kalamazoo, MI), ZD1694 (Tomudex, kindly provided by Zeneca Pharmaceuticals, Macclesfield, Cheshire, United Kingdom), and BVdU (Sigma Chemical Co., St. Louis, MO).

Clonogenic Assay.
The colony-forming ability of HT-29 STK or MC38 STK cells was assessed after single- or double-agent treatment. Replicates of 106 cells were plated in 25-cm2 tissue culture flasks. After overnight incubation, serial dilutions of GCV and/or TS inhibitors were added to triplicate flasks at indicated concentrations for 24 h, after which 500 viable cells from each flask were plated in 100-mm-diameter tissue culture dishes. Untreated cells served as a control. After 14 days, plates were washed with PBS, fixed, and stained with 1% crystal violet/formaldehyde, and the numbers of colonies (>=1 mm in diameter) were counted. This procedure typically yielded about 400 colonies in the control plates and 50–300 colonies in the treated plates. The colony-forming ability (fraction of cells unaffected, Fu) was calculated by dividing the mean number of colonies present in drug-treated plates by the mean number of colonies present in the untreated control dishes. For each cell line, concentration-effect curves were generated for single- and double-agent treatment and presented as the fraction affected (Fa = 1 - Fu) versus concentration. From the same concentration-effect curves, the ED50 concentrations were derived.

Analysis of Combined Drug Effects.
Cells were treated with serial dilutions of each drug individually or with a fixed ratio of both drugs simultaneously at doses that corresponded to 1/2, 5/8, 3/4, 7/8, 1 and 1.5 times the respective ED50. When the two drugs were administered at a fixed ratio, the dose of the combination required to produce fractional survival (f) could be separated into the components (D)1 and (D)2 of drugs 1 and 2, respectively. For each level of cytotoxicity (f = 0.95, 0.90, ..., 0.05), a parameter called the CI was calculated using the software CalcuSyn (28) , according to the Chou and Talalay equation (29 , 30) :

A CI < 1 indicates synergism, whereas a CI = 1 shows an additive effect, and CI > 1 indicates antagonism (29 , 30) . In this equation, the dose reduction index (DRI) defines the extent of drug dose reduction possible in a combination for a given degree of effect as compared with the dose of each drug alone (30 , 31) . When the concentration-effect relationship follows the principle of mass action, the median-effect plot should be linear. Linear correlation coefficient (r) was generated for each curve to determine the applicability of the data to this method of analysis. In all synergy experiments, r was >0.9.

Animal Experiments.
All experimental protocols were approved by the Animal Care and Use Committee of the National Human Genome Research Institute in compliance with the Guide for the Care and Use of Laboratory Animals (NIH No. 85–23, revised 1985).

Female athymic nu/nu mice, 6–8 weeks of age, were obtained from the Frederick Cancer Research and Development Center of the National Cancer Institute (Frederick, MD). Mice were housed five per cage and allowed access to food and water ad libitum. For tumor cell implantation, cells were enzymatically detached from culture flasks and counted. Ten million viable cells were resuspended in 100 µl of serum-free DMEM with 10% Matrigel (Collaborative Products, Bedford, MA) and injected s.c. into the right flank of the mice. Bidimensional tumor measurements were performed at least once a week with calipers, and tumor volume was determined using the simplified formula of a rotational ellipse (l x w2 x 0.5; Ref. 32 ). GCV, 5-FU, and ZD1694 were administered i.p. in 1 ml of 0.9% NaCl solution for injection. In the first in vivo study, animals received 10 mg/kg GCV and/or 15 mg/kg 5-FU once a day. In the successive experiment, the mice received 5 mg/kg GCV alone and in combination with 6 mg/kg 5-FU or 10 mg/kg ZD1694 twice a day. Animals were euthanized by CO2 if their tumors exceeded 10% body weight or if they appeared to be in distress.

Radiolabeled Nucleotide Incorporation Assays.
Five million MC38 STK or HT-29 STK cells were seeded into 25-cm2 tissue culture flasks. Twelve h later, [8-3H]GCV (Moravek, Brea, CA) alone or in combination with 5-FU or ZD1694 was added at their respective 10 x ED50 concentration, as listed in Table 1Citation . After 24 h, cells were trypsinized, counted, and subsequently boiled in 1 ml of 60% methanol for 3 min. Cell pellets were then washed three times with cold 60% methanol and resuspended in 1 ml of distilled water (24) . [8-3H]GCV incorporation into the genomic DNA was determined by scintillation counting. The incorporation of radiolabeled GCV under each condition was normalized to cell count.


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  		Table 1 Drug doses necessary to inhibit colony formation in a clonogenic assay by 50% (ED50), 75% (ED75), and 90% (ED90)

 
Statistical Methods.
Survival analysis using the Cox-Mantel test, cumulative survival plots, and Mann-Whitney U test were performed using the software package STATISTICA (version 5.5 for Windows; StatSoft, Inc., Tulsa, OK).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Colony-forming Assay.
To assess whether the efficacy of the HSV-tk/GCV system could be enhanced when combined with TS inhibitors we first performed in vitro studies. The clonogenic potential of MC38 STK and HT-29 STK cells exposed to GCV alone and in combination with TS inhibitors are shown in Fig. 2Citation . From these dose-effect curves, the ED50 values (fraction of affected cells = 0.5) were determined for each drug (Table 1)Citation and provided the basis for the appropriate GCV/TS inhibitor constant ratio that was maintained when the drugs were studied in combination. The interaction of the drugs was analyzed with the computer program CalcuSyn (28) . The graphical output of this software is shown in Fig. 3Citation . In these CI plots, points above 1 indicate antagonistic drug effects, whereas those below 1 are synergistic. A CI of 1 represents additive effects of both drugs. The results are presented in Table 2Citation and demonstrated synergism between GCV and the tested TS inhibitors. For example, in the murine MC38 STK cells exposed to GCV and 5-FU, the concentration of GCV and 5-FU needed to achieve a 90% reduction in colony formation (ED90) was reduced ~3- and ~4-fold, respectively, when compared with single-agent treatment. In the same cells treated with GCV and ZD1694, the ED90 was more than 2-fold lower than that observed for GCV and ZD1694 alone. More striking results were observed in the human HT-29 STK cells, where the combination of GCV and 5-FU or ZD1694 showed a ~13-fold reduction of the ED90 for 5-FU and a more than 8-fold lower ED90 of ZD1694 (Table 2)Citation .



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  		Fig. 2. Concentration-effect curves for GCV, 5-FU, ZD1694, and BVdU alone or in combination in MC38 STK (A–C) and HT-29 STK (D and E) cells. The cells were exposed in vitro for 24 h to GCV and/or the TS inhibitors, and their ability to form colonies was assessed. For combination treatments, the final drug concentration plotted represents the additive concentrations of GCV and the indicated TS inhibitor.

 


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  		Fig. 3. To quantitate the interaction between the HSV-tk/GCV system and 5-FU, ZD1694, or BVdU in MC38 STK (A) and 5-FU or ZD1694 in HT-29 STK (B) colon cancer cells, CI versus cytotoxicity plots were generated from the median-effect plots (25) . For these curves, CI < 1 defines a synergistic interaction, and CI > 1 defines antagonistic drug effects. The straight line at CI = 1 represents the additive effects of both drugs.

 

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  		Table 2 Effect of drug combination on ED concentrations

 
For unclear reasons, BVdU treatment at concentrations up to 100 µM showed no detectable cytotoxic effects in colony formation assays on the HSV-tk-expressing human cell lines HT-29 STK, A375 STK, HeLa STK, and MDAH 2774 STK. For this reason, BVdU was not considered for additional studies.

In Vivo Studies.
To determine whether the synergy observed in the colony formation assays would also result in an enhanced in vivo treatment efficacy, we used a HT-29 STK xenograft model. It should be noted, that HT-29 human colon cancer cells expressing HSV-tk are known to be relatively resistant to GCV (33) .

The goal of the first animal experiment was to determine whether the combination of GCV and 5-FU would result in improved survival of the animals when compared with single-agent treatment. When the tumors reached a volume of 200–250 mm3, the mice were randomly assigned to treatment groups. As shown in Fig. 4Citation , untreated animals had a median survival of 30 days. Animals treated with GCV or 5-FU had a significantly improved survival with a median survival time of 72 and 65 days, respectively (P <= 0.004). However, the addition of both drugs resulted in further improved animal survival, with a median survival of 114 days when compared with single-agent treatments (P = 0.008). At the end of the observation period (day 114), none of the untreated animals were alive, whereas in the GCV, 5-FU, and GCV/5-FU treatment groups, 2, 3, and 9 animals, respectively, had survived. Of note, among surviving animals, there was one mouse in each of the single-agent treatment groups tumor free, whereas in the GCV/5-FU treatment group, five animals were disease free.



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  		Fig. 4. Kaplan-Meier survival analysis of HT-29 STK xenografted nude mice. The animals were treated once a day for 5 days with i.p. injections of GCV (10 mg/kg), 5-FU (15 mg/kg), and with a combination of both drugs at the same dose level as the single drug treatment.

 
Because HT-29 cells were shown to be more sensitive to ZD1694 than 5-FU (Fig. 2Citation and Table 1Citation ), in a successive animal experiment we assessed whether the combination of GCV and ZD1694 would be more efficacious than GCV + 5-FU. To stress the system, in this study the mice were randomly assigned to treatment groups when the tumors reached a volume of ~350 mm3. Mice received either GCV (5 mg/kg) alone or in combination with 5-FU (6 mg/kg) or ZD1694 (10 mg/kg) for 5 days twice daily. To improve the treatment efficacy, because at any given time only a small fraction of the tumor cells are in cycle and thus susceptible to the therapy, the animals in this second study received the drugs twice daily. The doses of the drugs were significantly lower than those used in a previously published study treating animals with HT-29 xenografts with 30 mg/kg 5-FU and 50 mg/kg ZD1694 daily for 5 days in combination with fractionated radiation therapy (34) . To detect small differences in the treatment efficacy of the xenografts treated with GCV alone or in combination with 5-FU or ZD1694, we adopted tumor growth curves as readout of this experiment (Fig. 5)Citation . Animals treated with GCV alone had a significant reduction in tumor size when compared with control animals (median tumor volume on day 43 was 550 mm3 versus 1912.5 mm3; P < 0.001). By day 43, all animals in the control group had to be euthanized because of their tumor size. On day 64, the last observation day in this study, GCV-treated animals had a median tumor size of 1912.5 mm3. Animals receiving GCV in combination with 5-FU or ZD1694 had a median tumor volume of 1009.2 mm3 and 448.4 mm3, respectively. Mann-Whitney U test revealed that animals receiving double agent treatment had a statistically significant reduced tumor volume when compared with animals receiving only GCV (P <= 0.0003). Tumors of animals treated with GCV + ZD1694 were somewhat smaller than those observed in GCV + 5-FU-treated mice. However, this difference was not statistically significant (P = 0.19). At the dose level used, neither 5-FU nor ZD1694 alone were more efficacious than GCV alone (data not shown). It is worthwhile to note that most of the animals, which received ZD1694 in combination with GCV, showed signs of cachexia at the end of the treatment period. However, all animals recovered within 1 week after the end of the drug treatment. In contrast, animals receiving GCV and 5-FU had no significant weight loss.



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  		Fig. 5. Treatment of established s.c. HT-29 STK tumors in nude mice with 5 mg/kg GCV twice daily for 5 days alone and in combination with 6 mg/kg 5-FU twice daily or 10 mg/kg ZD1694 twice daily for 5 days. At the dose level used in our study, neither 5-FU nor ZD1694 alone were more efficacious than GCV alone (data not shown). Horizontal bar, treatment period. Points, mean tumor size; bars, SD. All groups were composed of 10 animals.

 
[8-3H]GCV Incorporation.
In an attempt to determine the mechanism of the synergism between the HSV-tk/GCV system and TS inhibitors, we analyzed the incorporation of [8-3H]GCV into cellular DNA in HSV-tk-expressing cells in the presence and absence of 5-FU or ZD1694. In MC38 STK and HT-29 STK cells, the normalized incorporation of [8-3H]GCV into genomic DNA was significantly enhanced at 24 h in the presence of 5-FU or ZD1694 when compared with GCV alone (P <= 0.001; Fig. 6Citation ). Furthermore, in both cell lines the exposure to GCV and ZD1694 resulted in a higher [8-3H]GCV incorporation into genomic DNA than that observed in GCV + 5-FU-treated cells (P <= 0.01).



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  		Fig. 6. HSV-tk-expressing MC38 STK and HT-29 STK cells were exposed in five or six replicates to [8-3H]GCV, GCV and 5-FU, or ZD1694 at 10 times their respective ED50s for 24 h. Cellular extracts were then assayed for [8-3H]GCV-TP incorporated into macromolecular DNA as described in "Materials and Methods."

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we quantitatively analyzed the interactions between the HSV-tk/GCV system and TS inhibitors. In murine MC38 STK and human HT-29 STK colorectal carcinoma cell lines engineered to express HSV-tk, we demonstrated synergistic cytotoxicity of 5-FU and ZD1694 when combined with GCV using a clonogenic assay. Although BVdU was effective as a single cytotoxic agent and synergistically enhanced GCV-mediated killing of murine MC-38 STK cells, we could not detect cytotoxic effects of BVdU on a series of human cancer cell lines expressing HSV-tk. The reasons underlying the lack of sensitivity of these cells to BVdU are not clear at the moment, but these findings indicate that more studies are warranted to verify the applicability of BVdU for human cancer gene therapy studies. In our in vivo studies, we observed that the combination of GCV and TS inhibitors resulted in improved survival and enhanced treatment efficacy when compared with single-agent treatment. To assure that transduction efficiency and different levels of HSV-tk expression will not confound the interpretation of the results, we chose to perform our studies with monoclonal populations of HSV-tk-expressing cells rather than using an in vivo gene delivery system. Although our findings will have to be confirmed using more clinically relevant models, based for example on adenovirus-mediated in vivo gene transfer into preestablished tumors, our data indicate that the examined use of GCV and TS inhibitors improves the elimination of HSV-tk-expressing cells. The potentiation of the HSV-tk/GCV system by TS inhibitors in vitro and in vivo is most likely due to the increased GCV-TP incorporation into cellular DNA, because increased levels of GCV-TP incorporation are associated with enhanced cytotoxicity.3 These findings are in concert with a previous report demonstrating that the combination of the HSV-tk/GCV and CD/5-FC systems results in enhanced GCV phosphorylation (19) . Kinetic studies have demonstrated that in clinical settings the GCV treatment generates peak plasma concentrations of 45 µM (9) . This serum level assumedly corresponds to intracellular concentrations below the reported Km of GCV for HSV-tk (Km = 100 µM; Ref. 35 ). In contrast, the typical intracellular thymidine concentration (1–20 µM; Ref. 36 ) is well above the Michaelis-Menten constant of thymidine for HSV-tk (Km = 0.22 µM); consequently, thymidine is expected to normally outcompete GCV for phosphorylation by HSV-tk. Because phosphorylation of GCV by HSV-tk is the rate-limiting step in GCV-mediated cytotoxicity (37 , 38) , the intracellular thymidine pool represents a competitive regulator of the HSV-tk system. Previous studies demonstrated that drugs that reduce intracellular pools of thymidine will result in potentiation of the anti-herpetic effects of the drug acyclovir, which is similar in structure to GCV (36 , 39) . Because TS is the only source for de novo synthesis of thymidine, cells treated with TS inhibitors, such as 5-FU, may suffer from up to 90% lower intracellular dTTP concentrations (40) . Because of a lack of feedback inhibition, low dTTP levels will increase cellular tk activity (41) , which mediates salvage synthesis of dTMP from thymidine. Consequently, it is conceivable that the fraction of HSV-tk sites occupied by cellular thymidine is reduced, thus freeing up more HSV-tk active sites for GCV binding. In this situation, phosphorylation of GCV by HSV-tk is increased, leading to higher GCV-mediated cytotoxicity. This series of events could explain the synergism of TS inhibitors and the HSV-tk/GCV system observed in these studies. In addition, it is possible that TS inhibitors enhance the cytotoxicity of the HSV-tk/GCV system by inhibition of DNA repair because of a lack of dTTP.

Our findings may lead to important clinical applications. First, the combination of HSV-tk/GCV and TS inhibitors can be used with all presently available HSV-tk-expressing vector systems and might be especially of interest for clinically tested and approved vectors. In addition, 5-FU and ZD1694 are approved for treatment of colon cancer, although the latter one is presently available only in some European countries. By combining TS inhibitors with HSV-tk/GCV, it would be possible to test an experimental gene therapy approach without withholding a clinically accepted treatment, and this would potentially permit the treatment of patients at an earlier stage of disease that would have a better chance of beneficial outcome. This approach should be ethically acceptable because there are no reasons to believe that the HSV-tk/GCV system would interfere with the efficacy of the chemotherapeutic agents. On the contrary, the combination of TS inhibitors with efficient adenovirus-mediated HSV-tk gene transfer and GCV treatment could improve the overall efficacy by mechanisms described here and by in situ tumor vaccination derived by xenogenization of the transduced tumor cells (42, 43, 44, 45, 46, 47) . Furthermore, a combined treatment may diminish the risk of acquired drug resistance that is likely in single agent-based treatments. In particular, 5-FU resistance can result from altered binding affinity of TS for FdUMP and from amplification of the TS gene (48) , whereas GCV resistance can arise from changed affinity of DNA polymerase to GCV-TP (49) .

The recently described CD/5-FC, HSV-tk/GCV dual suicide/prodrug system (18) showed promising increased cytotoxicity when compared with single-agent treatment and has inspired the development of the present study. As in all in vivo gene delivery approaches, transduction efficiency will likely to be the major obstacle to this approach because the spread of vectors is restricted to the close proximity of the injection site because of the fundamental physics of diffusion of vector particles in tissue spaces and the high density of viral receptors in tissue. Only the transduced cells will be subjected to the toxic effects of the activated prodrugs. We have now demonstrated that TS inhibitors can be used directly in combination with GCV to improve efficacy of the HSV-tk/GCV suicide gene transfer system. Our approach includes the use of generally accepted antineoplastic agents and may therefore have the advantage of achieving gene transfer-independent antitumor effects. Prior to clinical testing, further in vivo toxicological studies and in vitro efficacy studies in a broader spectrum of cell lines will be necessary to validate this approach and to determine the potential additional toxicity of this combined drug treatment.


   ACKNOWLEDGMENTS
 
We thank Drs. John C. Morris and William Jay Ramsey for stimulating discussions. Furthermore, we thank Dr. Morris for providing the HT-29 STK, MDAH 2774 STK, and HeLa STK cell lines. We also acknowledge the efforts of Drs. Alfred Fallavollita, Jr. and James A. Zwiebel from the Cancer Therapy Evaluation Program at National Cancer Institute, NIH, and Kim Vaughan, Zeneca Pharmaceuticals, Macclesfield, Cheshire, United Kingdom, to receive Tomudex.


   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 To whom requests for reprints should be addressed, at NIH, 10 Center Drive, Building 10, Room 10C103, Bethesda, MD 20892-1851. Phone: (301) 402-1833; Fax: (301) 496-7184; E-mail: owildner{at}nhgri.nih.gov Back

2 The abbreviations used are: HSV-tk/GCV, herpes simplex virus-thymidine kinase/ganciclovir; MP, monophosphate; TP, triphosphate; CD/5-FC, cytosine deaminase/5-fluorocytosine; TS, thymidylate synthase; 5-FU, 5-fluorouracil; BVdU, (E)-5-(2-bromovinyl)-2'-deoxyuridine; ATCC, American Type Culture Collection; CI, combination index. Back

3 J. C. Morris, R. Agbaria, H. J. Ford, J. S. Scheel, R. M. Blaese, and O. Wildner. The importance of pro-drug dose intensity in HSV-tk/GCV cancer gene therapy, manuscript in preparation. Back

Received 5/26/99. Accepted 8/18/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Moolten F. L. Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res., 46: 5276-5281, 1986.[Abstract/Free Full Text]
  2. Matthews T., Boehme R. Antiviral activity and mechanism of action of ganciclovir. Rev. Infect. Dis., 10 (Suppl. 3): S490-S494, 1988.
  3. Ilsley D. D., Lee S. H., Miller W. H., Kuchta R. D. Acyclic guanosine analogs inhibit DNA polymerases alpha, delta, and epsilon with very different potencies and have unique mechanisms of action. Biochemistry, 34: 2504-2510, 1995.[Medline]
  4. Thust R., Schacke M., Wutzler P. Cytogenetic genotoxicity of antiherpes virostatics in Chinese hamster V79-E cells. I. Purine nucleoside analogues. Antiviral Res., 31: 105-113, 1996.[Medline]
  5. Haynes P., Lambert T. R., Mitchell I. D. Comparative in-vivo genotoxicity of antiviral nucleoside analogues: penciclovir, acyclovir, ganciclovir and the xanthine analogue, caffeine, in the mouse bone marrow micronucleus assay. Mutat. Res., 369: 65-74, 1996.[Medline]
  6. Ram Z., Culver K. W., Oshiro E. M., Viola J. J., DeVroom H. L., Otto E., Long Z., Chiang Y., McGarrity G. J., Muul L. M., Katz D., Blaese R. M., Oldfield E. H. Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells. Nat. Med., 3: 1354-1361, 1997.[Medline]
  7. Sterman D. H., Treat J., Litzky L. A., Amin K. M., Coonrod L., Molnar-Kimber K., Recio A., Knox L., Wilson J. M., Albelda S. M., Kaiser L. R. Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a Phase I clinical trial in malignant mesothelioma. Hum. Gene Ther., 9: 1083-1092, 1998.[Medline]
  8. Spector S. A., Weingeist T., Pollard R. B., Dieterich D. T., Samo T., Benson C. A., Busch D. F., Freeman W. R., Montague P., Kaplan H. J. A randomized, controlled study of intravenous ganciclovir therapy for cytomegalovirus peripheral retinitis in patients with AIDS. AIDS Clinical Trials Group and Cytomegalovirus Cooperative Study Group. J. Infect. Dis., 168: 557-563, 1993.[Medline]
  9. Markham A., Faulds D. Ganciclovir. An update of its therapeutic use in cytomegalovirus infection. Drugs, 48: 455-484, 1994.[Medline]
  10. Elshami A. A., Cook J. W., Amin K. M., Choi H., Park J. Y., Coonrod L., Sun J., Molnar-Kimber K., Wilson J. M., Kaiser L. R., Albelda S. M. The effect of promoter strength in adenoviral vectors containing herpes simplex virus thymidine kinase on cancer gene therapy in vitro and in vivo. Cancer Gene Ther., 4: 213-221, 1997.[Medline]
  11. Touraine R. L., Vahanian N., Ramsey W. J., Blaese R. M. Enhancement of the herpes simplex virus thymidine kinase/ganciclovir bystander effect and its antitumor efficacy in vivo by pharmacologic manipulation of gap junctions. Hum. Gene Ther., 9: 2385-2391, 1998.[Medline]
  12. Black M. E., Newcomb T. G., Wilson H. M., Loeb L. A. Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy. Proc. Natl. Acad. Sci. USA, 93: 3525-3529, 1996.[Abstract/Free Full Text]
  13. Miyatake S., Martuza R. L., Rabkin S. D. Defective herpes simplex virus vectors expressing thymidine kinase for the treatment of malignant glioma. Cancer Gene Ther., 4: 222-228, 1997.[Medline]
  14. Freytag S. O., Rogulski K. R., Paielli D. L., Gilbert J. D., Kim J. H. A novel three-pronged approach to kill cancer cells selectively: concomitant viral, double suicide gene, and radiotherapy. Hum. Gene Ther., 9: 1323-1333, 1998.[Medline]
  15. Wildner O., Morris J. C., Vahanian N. N., Ford H. J., Ramsey W. J., Blaese R. M. Adenoviral vectors capable of replication improve the efficacy of HSVtk/GCV suicide gene therapy of cancer. Gene Ther., 6: 57-62, 1999.[Medline]
  16. Mullen C. A., Kilstrup M., Blaese R. M. Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system. Proc. Natl. Acad. Sci. USA, 89: 33-37, 1992.[Abstract/Free Full Text]
  17. Huber B. E., Austin E. A., Good S. S., Knick V. C., Tibbels S., Richards C. A. In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase. Cancer Res., 53: 4619-4626, 1993.[Abstract/Free Full Text]
  18. Rogulski K. R., Kim J. H., Kim S. H., Freytag S. O. Glioma cells transduced with an Escherichia coli CD/HSV-1 TK fusion gene exhibit enhanced metabolic suicide and radiosensitivity. Hum. Gene Ther., 8: 73-85, 1997.[Medline]
  19. Aghi M., Kramm C. M., Chou T. C., Breakefield X. O., Chiocca E. A. Synergistic anticancer effects of ganciclovir/thymidine kinase and 5-fluorocytosine/cytosine deaminase gene therapies. J. Natl. Cancer Inst., 90: 370-380, 1998.[Abstract/Free Full Text]
  20. Uckert W., Kammertons T., Haack K., Qin Z., Gebert J., Schendel D. J., Blankenstein T. Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo. Hum. Gene Ther., 9: 855-865, 1998.[Medline]
  21. Heidelberger C. Fluorinated pyrimidines. Prog. Nucleic Acid Res. Mol. Biol., 4: 1-50, 1965.[Medline]
  22. Jackman A. L., Taylor G. A., Gibson W., Kimbell R., Brown M., Calvert A. H., Judson I. R., Hughes L. R. ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study. Cancer Res., 51: 5579-5586, 1991.[Abstract/Free Full Text]
  23. Balzarini J., De Clercq E., Verbruggen A., Ayusawa D., Shimizu K., Seno T. Thymidylate synthase is the principal target enzyme for the cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine against murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 or type 2 thymidine kinase gene. Mol. Pharmacol., 32: 410-416, 1987.[Abstract]
  24. Balzarini J., Bohman C., De Clercq E. Differential mechanism of cytostatic effect of (E)-5-(2-bromovinyl)-2'- deoxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, and other antiherpetic drugs on tumor cells transfected by the thymidine kinase gene of herpes simplex virus type 1 or type 2. J. Biol. Chem., 268: 6332-6337, 1993.[Abstract/Free Full Text]
  25. Restifo N. P., Esquivel F., Asher A. L., Stotter H., Barth R. J., Bennink J. R., Mule J. J., Yewdell J. W., Rosenberg S. A. Defective presentation of endogenous antigens by a murine sarcoma. Implications for the failure of an anti-tumor immune response. J. Immunol., 147: 1453-1459, 1991.[Abstract]
  26. Ishii-Morita H., Agbaria R., Mullen C. A., Hirano H., Koeplin D. A., Ram Z., Oldfield E. H., Johns D. G., Blaese R. M. Mechanism of "bystander effect" killing in the herpes simplex thymidine kinase gene therapy model of cancer treatment. Gene Ther., 4: 244-251, 1997.[Medline]
  27. Moolten F. L., Wells J. M. Curability of tumors bearing herpes thymidine kinase genes transferred by retroviral vectors. J. Natl. Cancer Inst., 82: 297-300, 1990.[Abstract/Free Full Text]
  28. Chou T. C., Hayball M. P. CalcuSyn. Windows software for dose effect analysis (1.1) Biosoft Cambridge, England 1996.
  29. Chou T. C., Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul., 22: 27-55, 1984.[Medline]
  30. Chou T. C. The median-effect principle and the combination index for quantitation of synergism and antagonism Chou T. C. Rideout D. C. eds. . Synergism and Antagonism in Chemotherapy, : 61-102, Academic Press New York 1991.
  31. Chou J. Quantitation of synergism and antagonism of two or more drugs by computerized analysis Chou T. C. Rideout D. C. eds. . Synergism and Antagonism in Chemotherapy, : 223-244, Academic Press New York 1991.
  32. Dethlefsen L. A., Prewitt J. M., Mendelsohn M. L. Analysis of tumor growth curves. J. Natl. Cancer Inst., 40: 389-405, 1968.
  33. Boucher P. D., Ruch R. J., Shewach D. S. Differential ganciclovir-mediated cytotoxicity and bystander killing in human colon carcinoma cell lines expressing herpes simplex virus thymidine kinase. Hum. Gene Ther., 9: 801-814, 1998.[Medline]
  34. Teicher B. A., Ara G., Chen Y. N., Recht A., Coleman C. N. Interaction of Tomudex with radiation in vitro and in vivo. Int. J. Oncol., 13: 437-442, 1998.[Medline]
  35. Chen M. S., Walker J., Prussia W. H. Kinetic studies of herpes simplex virus type 1-encoded thymidine and thymidylate kinase, a multifunctional enzyme. J. Biol. Chem., 254: 10747-10753, 1979.[Free Full Text]
  36. Harmenberg J. Intracellular pools of thymidine reduce the antiviral action of acyclovir. Intervirology, 20: 48-51, 1983.[Medline]
  37. Biron K. K., Stanat S. C., Sorrell J. B., Fyfe J. A., Keller P. M., Lambe C. U., Nelson D. J. Metabolic activation of the nucleoside analog 9-[(2-hydroxy-1-(hydroxymethyl)ethoxy] methyl)guanine in human diploid fibroblasts infected with human cytomegalovirus. Proc. Natl. Acad. Sci. USA, 82: 2473-2477, 1985.[Abstract/Free Full Text]
  38. Smee D. F., Boehme R., Chernow M., Binko B. P., Matthews T. R. Intracellular metabolism and enzymatic phosphorylation of 9-(1,3-dihydroxy-2-propoxymethyl)guanine and acyclovir in herpes simplex virus-infected and uninfected cells. Biochem. Pharmacol., 34: 1049-1056, 1985.[Medline]
  39. Prichard M. N., Prichard L. E., Shipman C. J. Inhibitors of thymidylate synthase and dihydrofolate reductase potentiate the antiviral effect of acyclovir. Antiviral Res., 20: 249-259, 1993.[Medline]
  40. Tattersall M. H., Harrap K. R. Changes in the deoxyribonucleoside triphosphate pools of mouse 5178Y lymphoma cells following exposure to methotrexate or 5-fluorouracil. Cancer Res., 33: 3086-3090, 1973.[Abstract/Free Full Text]
  41. Ives D. H., Morse P. A., Potter V. R. Feedback inhibition of thymidine kinase by thymidine triphosphate. J. Biol. Chem., 238: 1467-1474, 1963.[Free Full Text]
  42. Klein G., Klein E. Immune surveillance against virus-induced tumors and nonrejectability of spontaneous tumors: contrasting consequences of host versus tumor evolution. Proc. Natl. Acad. Sci. USA, 74: 2121-2125, 1977.[Abstract/Free Full Text]
  43. Barba D., Hardin J., Sadelain M., Gage F. H. Development of anti-tumor immunity following thymidine kinase-mediated killing of experimental brain tumors. Proc. Natl. Acad. Sci. USA, 91: 4348-4352, 1994.[Abstract/Free Full Text]
  44. Chen S. H., Chen X. H., Wang Y., Kosai K., Finegold M. J., Rich S. S., Woo S. L. Combination gene therapy for liver metastasis of colon carcinoma in vivo. Proc. Natl. Acad. Sci. USA, 92: 2577-2581, 1995.[Abstract/Free Full Text]
  45. Yarovoi S. V., Mouawad R., Colbere-Garapin F., Soubrane C., Khayat D., Rixe O. In vitro sensitization of the B16 murine melanoma cells to ganciclovir by different RNA and plasmid DNA constructions encoding HSVtk. Gene Ther., 3: 913-918, 1996.[Medline]
  46. Melcher A., Todryk S., Hardwick N., Ford M., Jacobson M., Vile R. G. Tumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Nat. Med., 4: 581-587, 1998.[Medline]
  47. Toda M., Rabkin S. D., Kojima H., Martuza R. L. Herpes simplex virus as an in situ cancer vaccine for the induction of specific anti-tumor immunity. Hum. Gene Ther., 10: 385-393, 1999.[Medline]
  48. Mader R. M., Muller M., Steger G. G. Resistance to 5-fluorouracil. Gen. Pharmacol., 31: 661-666, 1998.[Medline]
  49. Faulds D., Heel R. C. Ganciclovir. A review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy in cytomegalovirus infections. Drugs, 39: 597-638, 1990.[Medline]



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