Combined suicide gene therapy for human colon cancer cells using adenovirus-mediated transfer of escherichia coli cytosine deaminase gene and Escherichia coli uracil phosphoribosyltransferase gene with 5-fluorocytosine

The virus-directed enzyme/prodrug system using the Escherichia coli cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) suffers from a sensitivity limitation in many tumor cells. The E. coil uracil phosphoribosyltransferase (UPRT), which is a pyrimidine salvage enzyme, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine monophosphate at the first step of its activating pathway. To improve the antitumoral effect of the CD/5-FC system, we investigated a combined suicide gene transduction therapy for human colon cancer cells using two separate adenovirus vectors expressing the E. coli CD and E. coli UPRT genes and systemic 5-FC administration (the CD, UPRT/5-FC system). The present study demonstrates that the CD, UPRT/5-FC system generates a co-operative effect of CD and UPRT, resulting in dramatic increases in both RNA- and DNA-directed active forms, including 5-fluorouridine triphosphate incorporated into RNA, 5-fluorodeoxyuridine monophosphate, and the thymidylate synthase inhibition rate, compared with the CD/5-FC system. Furthermore a significant increase in the 5-FC sensitivity of colon cancer cells was demonstrated in the CD, UPRT/5-FC system compared with the CD/5-FC system in vitro and in vivo. These results suggest that the CD, UPRT/5-FC system is a powerful approach in gene therapy for colorectal cancer.     

Yeast cytosine deaminase improves radiosensitization and bystander effect by 5-fluorocytosine of human colorectal cancer xenografts

The efficacy of cancer gene therapy using bacterial cytosine deaminase (bCD)/5-fluorocytosine (5-FC) enzyme/prodrug strategy is limited by the inefficiency of cytosine deaminase (CD)-catalyzed conversion of 5-FC into 5-fluorouracil (5-FU). We have shown previously that yeast CD (yCD) is more efficient at the conversion of 5-FC than bCD. In the current study, we hypothesized that the increased production of 5-FU by yCD would enhance the efficacy of the CD/5-FC treatment strategy by increasing the bystander effect as well as the efficacy of radiotherapy because of the radiosensitizing capacity of 5-FU. To test this hypothesis, we generated stable HT29 human colon cancer cell lines expressing either bCD (HT29/bCD) or yCD (HT29/yCD). The amount of 5-FU produced in HT29/yCD tumors after a single injection of 5-FC (1000 mg/kg, i.p.) was 15-fold higher than that produced in HT29/bCD tumors. In tumor-bearing nude mice, the average minimum relative tumor size (compared with pretreatment values) of HT29/bCD tumors treated with 5-FC and radiation (500 mg/kg i.p. and 3 Gy, 5 days a week for 2 weeks) was 0.55+/-0.1, compared with 0.01+/-0.01 in HT29/yCD tumors (P = 0.002). Moreover, an increased cytotoxic and radiosensitizing effect of 5-FC on bystander cells was observed in vitro and in vivo when yCD was expressed in HT29 cells instead of bCD. In mice bearing HT29 tumors containing 10% HT29/yCD cells, the combined treatment resulted in a minimum tumor size of 0.20+/-0.07 compared with 0.60+/-0.1 in 10% HT29/bCD cells (P < 0.001). These results demonstrate that the use of yCD in the CD/5-FC strategy has a high potential to improve the therapeutic outcome of combined gene therapy and radiotherapy in cancer patients.     

Superiority of yeast over bacterial cytosine deaminase for enzyme/prodrug gene therapy in colon cancer xenografts

The enzyme/prodrug strategy using bacterial cytosine deaminase (bCD) and 5-fluorocytosine (5-FC) is currently under investigation for cancer gene therapy. A major limitation for the use of bCD is that it is inefficient in the conversion of 5-FC into 5-fluorouracil. In the present study, we show that the K(m) of yeast cytosine deaminase (yCD) for 5-FC was 22-fold lower when compared with that of bCD. HT29 human colon cancer cells transduced with yCD (HT29/yCD) were significantly more sensitive to 5-FC in vitro than HT29 cells transduced with bCD (HT29/bCD). In tumor-bearing nude mice, complete tumor regression was observed in 6 of 13 HT29/yCD tumors in response to 5-FC treatment (500 mg/kg i.p. daily, 5 days a week for 2 weeks), whereas 0 of 10 HT29/bCD tumors were cured. Our study demonstrates an improved efficacy of the CD/5-FC treatment strategy when yCD was used. This enzyme has, therefore, a high potential to increase the therapeutic outcome of the enzyme/prodrug strategy in cancer patients.     

High and selective expression of yeast cytosine deaminase under a carcinoembryonic antigen promoter-enhancer

Yeast cytosine deaminase (yCD)-based gene therapy offers the potential for selective production of the cytotoxic and radiosensitizing drug 5-fluorouracil (5-FU) from the benign prodrug 5-fluorocytosine within colorectal cancers. Although previous attempts to target therapy to colorectal cancer using the carcinoembryonic antigen (CEA) promoter have demonstrated specificity, this has been achieved at the cost of 10- to 300-fold loss in activity compared with strong but nonspecific rous sarcoma virus (RSV) or cytomegalovirus promoters. We developed a highly specific and active gene transfer method for colorectal cancer using CEA under control of a promoter-enhancer. We compared the RSV promoter-derived with the CEA promoter-enhancer-derived transgene expression in 10 different cell lines with differing CEA status. We found that the transgene expression resulting from both transient transfection and adenoviral infection with the CEA promoter-enhancer was as strong as the RSV promoter while maintaining specificity for CEA-producing cell lines. For instance, when we compared yCD expression between LoVo (CEA+) and human fibroblast (CEA-), we found a 30-fold-increased yCD expression in LoVo cells from CEA-enhancer adenovirus although there was no difference in the yCD expression between the cell lines when infected with RSV/yCD virus. This specificity was also achieved while maintaining a higher yCD enzyme activity than we obtained with RSV/yCD adenovirus in an HT-29 intrahepatic tumor model. We then compared the response of HT-29 xenografts to treatment with 5-fluorocytosine and yCD adenovirus driven by either the RSV or the CEA promoter-enhancer and found similar tumor growth inhibition. These findings suggest that the CEA promoter-enhancer strategy confers specificity while preserving activity and is worth exploring in additional animal and, potentially, clinical trials.     

Liposomal formulation of 5-fluorocytosine in suicide gene therapy with cytosine deaminase--for colorectal cancer

It is generally accepted that successful gene therapy depends on two major factors: tumor-specific expression of a therapeutic gene and the efficient transfer of a therapeutic gene to tumor cells. For gene-directed enzyme prodrug therapy (GDEPT) involving Escherichia coli cytosine deaminase (CD) and 5-fluorocytosine (5-FC), several tumor-specific promoters and virus-based vectors were used. No attention whatsoever was paid to the way of 5-FC delivery to solid tumors, despite the fact that the delivery of drugs to such tumors is generally low because of their insufficient transfer from the blood. To compare the effectiveness of GDEPT with free and liposomal 5-FC, the prodrug was encapsulated in liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (1:1). When the liposomal form of 5-FC was administered i.v., mice treated with a dose of 5mg of liposomal 5-FC/kg body weight for 10 days, showed complete regression of transplanted tumors and complete cure was observed, whereas in animals treated with the same amounts of the free prodrug, 50% tumor regression and only insignificantly prolonged median survival were found. In summary, these results showed a remarkable enhancement of the antitumor effects of the liposomal form of 5-FC in comparison with the free prodrug. Therapy with liposomal 5-FC thus represents a new approach to achieving a high local concentration of the prodrug for suicide gene therapy using E. coli CD.     

Selective in vivo radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells transduced with the E. coli cytosine deaminase (CD) gene

Purpose: The E. coli cytosine deaminase (CD) gene encodes an enzyme capable of converting the nontoxic prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a known radiosensitizer. Having previously shown that combined CD suicide gene therapy and radiation (RT) results in pronounced radiosensitization in vitro, we progressed to in vivo studies of combined therapy.Methods and Materials: WiDr human colon cancer cells were transduced in vitro with the CD gene and cells expressing CD were selected for use as xenografts in a nude mouse model. After administration of 5-FC, tumors received 10–30 Gy local field radiation (RT) and tumor growth delay was compared to control animals receiving either 5-FU, 5-FC, or RT alone.Results: Maximal growth delay was seen in mice treated with 5-FC for 6 consecutive days prior to RT. Combined treatment with 15 Gy radiation resulted in a dose-modifying factor (DMF) of 1.50, and a greater DMF was observed with higher doses of radiation. There was no appreciable toxicity using this new approach. In contrast, a similar treatment of combined 5-FU and radiation resulted in considerable toxicity and no appreciable radiosensitization.Conclusion: The present results show that combined suicide gene therapy and RT results in pronounced antitumor effect without any notable toxicity. This indicates that the CD gene may be useful in the development of novel treatment strategies combining radiation and gene therapy in the treatment of locally advanced cancers.     

Adenoviral-mediated transfer of Escherichia coli uracil phosphoribosyltransferase (UPRT) gene to modulate the sensitivity of the human colon cancer cells to 5-fluorouracil

5-Fluorouracil (5-FU) has been used as a chemotherapeutic drug for colorectal cancer. Escherichia coli uracil phosphoribosyltransferase (UPRT), a pyrimidine salvage enzyme, converts 5-FU into 5-fluorouridine monophosphate (5-FUMP) at the initial step of 5-FU activation. We investigated the effects of adenoviral-mediated transfer of the E. coli UPRT gene into human colon cancer cells on 5-FU metabolism and 5-FU chemosensitivity. Three cell lines were used (HT29, KM12 and SW1116). The intracellular levels of 5-fluorodeoxyuridine monophosphate (5-FdUMP) and 5-FU incorporated into RNA after 5-FU treatment in cells infected with adenovirus containing the UPRT gene (AdCA-UPRT) were significantly higher than those of non-infected cells. This was accompanied by marked inhibition of thymidylate synthase (TS) in all cell lines. Furthermore, HT29, KM12 and SW1116 infected with AdCA-UPRT were, respectively, 13.1-, 30.2- and 70.5-fold more sensitive to 5-FU than non-infected cells. Most importantly, treatment with AdCA-UPRT and 5-FU effectively inhibited the growth of HT29-xenografted subcutaneous tumours in nude mice. Therefore, AdCA-UPRT/5-FU treatment had the potential to enhance the actions of 5-FU at both the DNA and RNA levels. Treatment augmented the sensitivity of human colon cancer cells to 5-FU both in vitro and in vivo. We conclude that adenoviral-mediated transfer of the E. coli UPRT gene into colon cancer cells can achieve biochemical modulation of 5-FU and this provides a new approach in the treatment of colorectal cancer.     

Modified vaccinia virus Ankara as a vector for suicide gene therapy

Modified vaccinia virus Ankara (MVA) has been used successfully to express various antigens for the development of vaccines. Here we show that MVA can also be used as an efficient vector for the transfer of suicide genes to cancer cells. We have generated a new and highly potent suicide gene, FCU1, which encodes a fusion protein derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. We now describe the therapeutic benefit of using MVA to deliver and express the FCU1 gene in cancer cells. MVA-mediated transfer of the FCU1 gene to various human tumor cells results in the production of a bifunctional intracellular enzyme, such that exposure to the prodrug 5-FC suppresses the growth of the tumor cells both in vitro and in vivo. Moreover, we report a more potent tumor growth delay at lower doses of 5-FC using MVA-FCU1 in comparison to adenovirus encoding FCU1. Prolonged therapeutic levels of cytotoxic 5-FU were detected in tumors in mice treated with both MVA-FCU1 and 5-FC while no detectable 5-FU was found in the circulation. This original combination between MVA and FCU1 represents a potentially safe and attractive therapeutic option to test in man.     

Management of malignant pleural effusion by suicide gene therapy in advanced stage lung cancer: a case series and literature review

Gene therapy can be defined as the transfer of genetic material into a cell for therapeutic purposes. Cytosine deaminase (CD) transferred into tumor cells by an adenoviral vector (Ad.CD), can convert the antifungal drug fluorocytosine (5-FC) to the antimetabolite 5-fluorouracil (5-FU), which kills not only the transfected tumor cells but also their neighbors by the so-called 'bystander effect'. After testing a protocol for Ad.CD transfer and lung tumor burden control in a Lewis mouse model, we used this technique in the management of lung cancer patients with malignant pleural effusion (MPE): two cases are presented investigating the possible enhancement of anticancer effect in both non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) by local activation of the pro-drug 5-FC. Results were discussed in parallel to a literature review on the topic. 5-FC and Ad.CD were administered intratumorally to Lewis mouse lung carcinoma and the effect was monitored by tumor size and electromicroscopy. Two patients with advanced stage lung cancer (1SCLC, 1NSCLC), which developed MPE during first-line treatment were administered 10(12) plaque-forming unit (pfu) Ad.CD by intrapleural instillation, in two doses (day1 and day7). Instillation was performed when the pleural fluid was 200?ml. In addition, they received 5-FC 500?mg four times daily for 14 days. Lung tumor regression and successful transfer of adenoviral particles were observed in treated animals. Patients presented complete regression of pleural effusion as monitored by computerized tomography scan. Neutrapenia and anemia were the most severe adverse effect presented (grade III/grade IV 100%). The increased toxicity followed by the intrapleural gene therapy indicates the augmentation of anticancer effect of transformed pro-drug 5-FC to active 5-FU. The obtained data indicate that intrapleural gene therapy may be a useful tool, adjunct to chemotherapy, in the management of MPE related to lung cancer.     

Efficacy of adenovirus-mediated CD/5-FC and HSV-1 thymidine kinase/ganciclovir suicide gene therapies concomitant with p53 gene therapy.

Recent evidence has suggested that tumor cells having a wild-type p53 status are more sensitive to chemotherapeutic agents and radiation than cells that lack functional p53. The heightened sensitivity of wild-type p53 cells is thought to be attributable to their propensity to undergo p53-mediated apoptosis after insult. Given that suicide gene therapy is essentially tumor-targeted chemotherapy, we examined the hypothesis that coexpression of wild-type p53 could enhance the efficacy of adenovirus-mediated suicide gene therapy. Human Hep3B and SK-OV-3 cells, which are null for p53, were infected with a pair of replication-deficient adenoviruses that expressed a cytosine deaminase/herpes simplex virus thymidine kinase (CD/HSV-1 TK) fusion gene without (fusion gene nonreplicative adenovirus, FGNR) or with (FGNRp53) the wild-type human p53 gene. The sensitivity of cells to the CD/5-fluorocytosine (CD/5-FC) and HSV-1 TK/ ganciclovir (GCV) enzyme/prodrug systems was determined in vitro and in vivo. Coexpression of p53 did not enhance the cytotoxicity of either the CD/5-FC or HSV-1 TK/GCV system in vitro. The failure to observe an effect of p53 could not be explained on the basis of insufficient or transient p53 expression, because FGNRp53-infected cells growth arrested in G1, induced Bax, and underwent apoptosis at an increased rate after prodrug treatment, particularly when the adenovirus E1A protein was present. Intratumoral injection of FGNRp53 concomitant with single or double pro-drug therapy resulted in a tumor growth delay that was equal to or less than that observed with the FGNR virus. Our results indicate that coexpression of p53 may not necessarily improve the efficacy of adenovirus-mediated CD/ 5-FC and HSV-1 TK/GCV suicide gene therapies in vivo.     

Tumor-specific intravenous gene delivery using oncolytic adenoviruses

In this report, we describe a vector system that specifically delivers transgene products to tumors following intravenous (i.v.) administration. The Escherichia coli cytosine deaminase (CD) gene was placed in the E3B region of the tumor-selective, replication-competent adenovirus ONYX-411, under the control of endogenous viral late gene regulatory elements. Thus, CD expression was directly coupled to the tumor-selective replication of the viral vector. In vitro, CD was expressed efficiently in various human cancer cell lines tested but not in cultured normal human cells, including human hepatocytes. Following i.v. administration into nude mice carrying human tumor xenografts, robust CD activity was detected only in tumors but not in liver or other normal tissues. Levels of CD activity in the tumors increased progressively following i.v. virus administration, correlating closely with virus replication in vivo. Subsequent administration of 5-fluorocytosine (5-FC) demonstrated a trend to improve the antitumor efficacy of these viruses in a mouse xenograft model, presumably due to the intratumoral conversion of 5-FC to the chemotherapeutic drug 5-fluorouracil. We show that the combination of a highly selective oncolytic virus, ONYX-411, with the strategic use of the viral E3B region for transgene insertion provides a powerful platform that allows for tumor-specific, persistent and robust transgene expression after i.v. administration. This technology provides an opportunity to enhance greatly both safety and efficacy of cancer gene therapy.     

Gene therapy for ovarian cancer using adenovirus-mediated transfer of cytosine deaminase gene and uracil phosphoribosyltransferase gene directed by MDR1 promoter

OBJECTIVE: To evaluate the specific killing effects of the adenoviral vector in which the CD::UPP genes were directed by the MDR1 promoter on Taxol-resistance ovarian cancer cells in vitro and in vivo. METHODS: Taxol-resistance (A2780/Taxol, SKOV3/Taxol) ovarian cancer cells and Taxol-sensitive (A2780, SKOV3) ovarian cancer cells were infected with adenovirus vector carrying the CD::UPP gene driven by the MDR1 promoter, followed with 5-fluorocytosine administration. Sensitivity to 5-fluorocytosine (5-FC) was analyzed. The AdMDR1-CD::UPP was subcutaneously injected into the xenografts of the nude mice and 5-FC intraperitoneally. The overall survival and anti-tumor effects were observed. RESULTS: In vitro, AdMDR1-CD::UPP showed a stronger cytotoxicity in A2780/Taxol cells and SKOV3/Taxol cells than that in A2780 cells and SKOV3 cells. Subcutaneous injection of AdMDR1-CD::UPP into the xenografts of mice bearing tumors of A2780/Taxol cells could significantly suppress the tumor growth and prolong survival as compared with the group of A2780 cells. CONCLUSION: AdMDR1-CD::UPP in combination with 5-FC is an effective approach to suppress the growth of Taxol-resistant ovarian cancer.     

Combination of cytosine deaminase suicide gene expression with DR5 antibody treatment increases cancer cell cytotoxicity

Combined treatment using adenoviral-directed enzyme/prodrug therapy and immunotherapy has the potential to become a powerful alternative method of cancer therapy. We have developed adenoviral vectors encoding the cytosine deaminase gene (Ad-CD) and cytosine deaminase:uracil phosphoribosyltransferase fusion gene (Ad-CD:UPRT). A monoclonal antibody, TRA-8, specifically binds to death receptor 5, one of two death receptors bound by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of Ad-CD:UPRT and TRA-8 against human pancreatic cancer and glioma cell lines. The present study demonstrates that Ad-CD:UPRT infection resulted in increased 5-FC-mediated cell killing, compared with Ad-CD. Furthermore, a significant increase of cytotoxicity following Ad-CD:UPRT/5-FC and TRA-8 treatment of cancer cells in vitro was demonstrated. Animal studies showed significant inhibition of tumor growth of MIA PaCa-2 pancreatic and D54MG glioma xenografts by the combination of Ad-CD:UPRT/5-FC plus TRA-8 as compared with either agent alone or no treatment. The results suggest that the combination of Ad-CD:UPRT/5-FC with TRA-8 produces an additive cytotoxic effect in cancer cells in vitro and in vivo. These data indicate that combined treatment with enzyme/prodrug therapy and TRAIL immunotherapy provides a promising approach for cancer therapy.     

Intratumoral 5-fluorouracil produced by cytosine deaminase/5-fluorocytosine gene therapy is effective for experimental human glioblastomas

5-Fluorouracil (5-FU) is a potent antimetabolite used for chemotherapy of gastrointestinal (GI), breast, and head and neck malignancies. Although clinical trials have been conducted, the poor therapeutic index of 5-FU has precluded its clinical use for a number of other tumor types. It is unclear whether this lack of utility is due to problems with drug delivery or inherent insensitivity. Adenovirus (Ad) vector-mediated cytosine deaminase (CD)/5-fluorocytosine (5-FC) gene therapy has the potential to overcome pharmacokinetic issues associated with systemic 5-FU and is particularly well suited to use with tumors in which local control is paramount, such as recurrent, localized prostate cancer and malignant gliomas. In this study, the in vitro response by a panel of human tumor cell lines derived from both GI (colon, pancreas) and non-GI (prostate, glioma) tumors to 5-FU and to AdCMVCD (an Ad encoding Escherichia coli CD)/5-FC was examined. Whereas the sensitivity (IC(50)) of individual cell lines to these agents varied, no significant difference in median IC(50) for either 5-FU or AdCMVCD/5-FC was evident for the four tumor types tested (P > 0.1). The relevant contributions of Ad gene transfer efficiency and inherent 5-FU sensitivity in determining response to AdCMVCD/5-FC were then assessed. Multiple linear regression analysis revealed that whereas both factors significantly contribute to the response, inherent 5-FU sensitivity was substantially more important (beta= 0.78 versus 0.48; P < 0.001). Finally, the therapeutic efficacy of a single intratumoral injection of AdCMVCD followed by systemic 5-FC was assessed in three intracranial C.B17 severe combined immunodeficient mouse models of human glioma. AdCMVCD/5-FC efficacy was specific, virus dose-dependent, and closely paralleled in vitro 5-FU and CD/5-FC sensitivity in two of three models tested. These results reveal that glioma cells are as sensitive as GI tumor cells to the antineoplastic effects of 5-FU, identify inherent 5-FU sensitivity as an important factor in determining CD/5-FC efficacy, and confirm previous findings in rat models that demonstrate the potential clinical utility of AdCMVCD/5-FC gene therapy for gliomas.     

Suppression of the growth of human colorectal cancer cells by therapeutic stem cells expressing cytosine deaminase and interferon-β via their tumor-tropic effect in cellular and xenograft mouse models

Genetically engineered stem cells (GESTECs) exhibit a potent therapeutic efficacy via their strong tumor tropism toward cancer cells. In this study, we introduced the human parental neural stem cells, HB1.F3, with the human interferon beta (IFN-β) gene which is a typical cytokine gene that has an antitumor effect and the cytosine deaminase (CD) gene from Escherichia coli (E. coli) that could convert the non-toxic prodrug, 5-fluorocytosine (5-FC), to a toxic metabolite, 5-fluorouracil (5-FU). Two types of stem cells expressing the CD gene (HB1.F3.CD cells) and both the CD and human IFN-β genes (HB1.F3.CD.IFN-β) were generated. The present study was performed to examine the migratory and therapeutic effects of these GESTECs against the colorectal cancer cell line, HT-29. When co-cultured with colorectal cancer cells in the presence of 5-FC, HB1.F3.CD and HB1.F3.CD.IFN-β cells exhibited the cytotoxicity on HT-29 cells via the bystander effect. In particular, HB1.F3.CD.IFN-β cells showed the synergistic cytotoxic activity of 5-FU and IFN-β. We also confirmed the migration ability of HB1.F3.CD and HB1.F3.CD.IFN-β cells toward HT-29 cells by a modified migration assay in vitro, where chemoattractant factors secreted by HT-29 cells attracted the GESTECs. In a xenograft mouse model, the volume of tumor mass was decreased up to 56% in HB1.F3.CD injected mice while the tumor mass was greatly inhibited about 76% in HB1.F3.CD.IFN-β injected mice. The therapeutic treatment by these GESTECs is a novel strategy where the combination of the migration capacity of stem cells as a vector for therapeutic genes towards colorectal cancer and a synergistic antitumor effect of CD and IFN-β genes can selectively target this type of cancer.     

Gene transfer into human prostate adenocarcinoma cells with an adenoviral vector: Hyperthermia enhances a double suicide gene expression, cytotoxicity and radiotoxicity

We have previously developed a recombinant adenovirus containing a fusion gene of Escherichia coli cytosine deaminase (CD) and herpes simplex virus type 1 thymidine kinase (HSV-1 TK) controlled by a cytomegalovirus (CMV) enhancer-promoter. This replication-incompetent adenovirus effectively transduced the CD-TK gene into human prostate adenocarcinoma DU-145 or PC-3 cells. Interestingly, heat shock at 41 degrees C for 4 hours elevated the level of CD-TK by approximately 5- to 20-fold at a multiplicity of infection (MOI) of 1. Heat-enhanced expression of CD-TK promoted cytotoxicity by 23-, 9-, or 47-fold in the presence of 50 microg/mL ganciclovir (GCV), 500 microg/mL 5-fluorocytosine (5-FC), or 50 microg/mL GCV+500 microg/mL 5-FC, respectively, at an MOI of 1. Moreover, there was an increase in radiosensitivity when adenovirus-infected cells were heated at 41 degrees C for 4 hours followed by irradiation in the presence of the prodrugs. Virus+heat+1 microg/mL GCV treatment increased radiosensitivity by a dose-modifying factor (DMF) of 2.2, whereas virus+heat+10 microg/mL 5-FC exposure resulted in a DMF of 2.3. Radiosensitization was clearly enhanced as a result of combined prodrug exposure (DMF=4.4). Our results suggest that the efficiency in expression of suicide genes from an adenoviral vector used for cytotoxic anticancer therapy could be improved by combining heat treatment with radiation therapy.     

In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene

Direct transfer of prodrug activation systems into tumors was demonstrated to be an attractive method for the selective in vivo elimination of tumor cells. However, most current suicide gene therapy strategies are still handicapped by a poor efficiency of in vivo gene transfer and a limited bystander cell killing effect. In this study, we describe a novel and highly potent suicide gene derived from the Saccharomyces cerevisiae cytosine deaminase (FCY1) and uracil phosphoribosyltransferase genes (FUR1). This suicide gene, designated FCU1, encodes a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direct conversion of 5-FC, a nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine-5'monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Unexpectedly, although the uracil phosphoribosyltransferase activity of FCU1 was equivalent to that encoded by FUR1, its cytosine deaminase activity was 100-fold higher than the one encoded by FCY1. As a consequence, tumor cells transduced with an adenovirus expressing FCU1 (Ad-FCU1) were sensitive to concentrations of 5-FC 1000-fold lower than the ones used for cells transduced with a vector expressing FCY1 (Ad-FCY1). Furthermore, bystander cell killing was also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination. Finally, intratumoral injections of Ad-FCU1 into allo- or xenogeneic tumors implanted s.c. into mice, with concomitant systemic administration of 5-FC, led to substantial delays in tumor growth. These unique properties make of the FCU1/5-FC prodrug activation system a novel and powerful candidate for cancer gene therapy strategies.