Cytosine Deaminase Gene Research Articles

Apoptotic Induction with Bifunctional E.coli Cytosine Deaminase-Uracil Phosphoribosyltransferase Mediated Suicide Gene Therapy is Synergized by Curcumin Treatment In vitro

Development of novel suicide gene therapy vector with potential application in cancer treatment has a great impact on human health. Investigation to understand molecular mechanism of cell death is necessary to evaluate the therapeutic application of suicide vectors. For example, the bifunctional E.coli cytosine deaminase & uracil phosphoribosyltransferase fusion (CD-UPRT) gene expression is known to sensitize a wide range of cells toward nontoxic prodrug 5-flurocytosine (5-FC) by converting it to toxic compounds, but the exact pathway of cell death is yet to be defined. Herein, we investigated the mechanism of cell death by 5-FC/CD-UPRT suicide system in both cancer and non-cancer cells and found that the optimum 5-FC concentration led to programmed cell death in vitro. The CD-UPRT expression of transfected cells was measured by the RT-PCR analysis. Biochemical assays, such as mitochondrial activity (MTS) and lactate dehydrogenase (LDH) measurements exhibited cell death. Microscopic experiments showed characteristic onset of apoptosis which was further supported by internucleosomal DNA cleavage of BrdU labeled cellular DNA, appearance of characteristic laddering of chromosomal DNA and involvement of caspase pathway. Furthermore, the 5-FC/CD-UPRT-mediated apoptosis was potentiated with addition of a known anticancer agent curcumin. Our in vitro studies confirmed synergistic induction of apoptotic pathway in the combination treatment. Therefore, combination of 5-FC/CD-UPRT with curcumin could be a potential chemosensitization strategy for cancer treatment.

Oncolytic virotherapy for ovarian carcinomatosis using a replication-selective vaccinia virus armed with a yeast cytosine deaminase gene

In this study, we assessed the ability of a highly tumor-selective oncolytic vaccinia virus armed with a yeast cytosine deaminase gene to infect and lyse human and murine ovarian tumors both in vitro and in vivo. The virus vvDD-CD could infect, replicate in and effectively lyse both human and mouse ovarian cancer cells in vitro. In two different treatment schedules involving either murine MOSEC or human A2780 ovarian carcinomatosis models, regional delivery of vvDD-CD selectively targeted tumor cells and ovarian tissue, effectively delaying the development of either tumor or ascites and leading to significant survival advantages. Oncolytic virotherapy using vvDD-CD in combination with the prodrug 5-fluorocytosine conferred an additional long-term survival advantage upon tumor-bearing immunocompetent mice. These findings demonstrate that a tumor-selective oncolytic vaccinia combined with gene-directed enzyme prodrug therapy is a highly effective strategy for treating advanced ovarian cancers in both syngeneic mouse and human xenograft models. Given the biological safety, tumor selectivity and oncolytic potency of this armed oncolytic virus, this dual therapy merits further investigation as a promising new treatment for metastatic ovarian 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.

Treatment of colon cancer cells using the cytosine deaminase/5-fluorocytosine suicide system induces apoptosis, modulation of the proteome, and Hsp90β phosphorylation

The bacterial cytosine deaminase (CD) gene, associated with the 5-fluorocytosine (5FC) prodrug, is one of the most widely used suicide systems in gene therapy. Introduction of the CD gene within a tumor induces, after 5FC treatment of the animal, a local production of 5-fluorouracil resulting in intratumor chemotherapy. Destruction of the gene-modified tumor is then followed by the triggering of an antitumor immune reaction resulting in the regression of distant wild-type metastasis. The global effects of 5FC on colorectal adenocarcinoma cells expressing the CD gene were analyzed using the proteomic method. Application of 5FC induced apoptosis and 19 proteins showed a significant change in 5FC-treated cells compared with control cells. The up-regulated and down-regulated proteins include cytoskeletal proteins, chaperones, and proteins involved in protein synthesis, the antioxidative network, and detoxification. Most of these proteins are involved in resistance to anticancer drugs and resistance to apoptosis. In addition, we show that the heat shock protein Hsp90beta is phosphorylated on serine 254 upon 5FC treatment. Our results suggest that activation of Hsp90beta by phosphorylation might contribute to tumor regression and tumor immunogenicity. Our findings bring new insights into the mechanism of the anticancer effects induced by CD/5FC treatment.

Replication-competent Adenovirus-mediated Suicide Gene Therapy with Radiation in a Preclinical Model of Pancreatic Cancer

In preparation for a Phase I trial, we evaluated the efficacy and toxicity of replication-competent adenovirus-mediated suicide gene therapy in combination with radiation in a preclinical model of pancreatic cancer. Human MiaPaCa-2 and PANC-1 pancreatic adenocarcinoma cells were found to be sensitive to the oncolytic effects of the Ad5-yCD/mutTK(SR39)rep-ADP adenovirus and also to the cytotoxic effects of the yeast cytosine deaminase (yCD) and herpes simplex virus thymidine kinase (HSV-1 TK(SR39)) genes in vitro. Combining Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy with radiation significantly increased tumor control beyond that of either modality alone. Injection of Ad5-yCD/mutTK(SR39)rep-ADP in the dog pancreas at doses (10(12) virus particle (vp)) to be used in humans resulted in mild pancreatitis but not peritonitis or hepatotoxicity. Following administration of 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]-FHBG), a positron-emitting substrate of HSV-1 TK, Ad5-yCD/mutTK(SR39)rep-ADP activity could be monitored non-invasively by positron emission tomography (PET). [(18)F]-FHBG uptake was readily detected in the pancreas but not in other major abdominal organs, indicating that little of the injected adenovirus disseminates to collateral tissues. These results demonstrate that Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy has the potential to augment the effectiveness of pancreatic radiotherapy without resulting in excessive toxicity. Hence they provide the scientific basis for an ongoing Phase I trial in pancreatic cancer.

Highly Efficient Gene Delivery for Bladder Cancers by Intravesically Administered Replication-Competent Retroviral Vectors

In an attempt to improve viral delivery of potentially therapeutic genes via an intravesical route, we have recently developed murine leukemia virus-based replication-competent retrovirus (RCR) vectors. We evaluated the transduction efficiency of intravesically administered RCR vectors to bladder tumor using orthotopic animal models to determine their potential as delivery vectors for bladder cancer. The RCR vector containing green fluorescent protein (GFP) marker gene achieved efficient in vitro transmission of the GFP transgene. Murine bladder tumor-2 mouse bladder tumors exposed to intravesically administered RCR vectors exhibited 0%, 9.2 +/- 2.9%, and 30.0 +/- 6.2% of GFP expression at 9, 18, and 27 days after exposure in the orthotopic model, respectively. Orthotopic KU-19-19 human bladder tumors exposed to intravesically administered RCR vectors exhibited 3%, 85 +/- 1.0%, and 100% of GFP expression at 7, 21, and 35 days after exposure, respectively. GFP staining was observed only in the tumor cells in the bladder. No detectable PCR products of GFP gene could be observed in distant organs. Treatment with RCR vectors containing yeast cytosine deaminase (CD) gene plus 5-fluorocytosine (5-FC) dramatically inhibited the growth of preestablished murine bladder tumor-2 tumors. A single course of 5-FC treatment resulted in a 50% animal survival in mice exposed to RCR-CD compared with a 0% survival in all controls over a 70-day follow-up period. Intravesically administered RCR vectors can efficiently deliver genes to orthotopic bladder tumor without viral spread in distant organs. RCR-CD/5-FC suicide gene therapy promises to be a novel and potentially therapeutic modality for bladder cancer.

PD6-1-5: Dose-response relationships in extensive small cell lung cancer and implications for resistance mechanisms.

Copyright © 2007 by the International Association for the Study of Lung Cancer S426 Restoration of the tumor suppressor genes P53, Retinoblastoma, FHIT and FUS1 had only minimal effect on cell death, although the tumor suppressors are functional defective in >80 % of SCLC. Modulating the oncogene activity of Bcl-2 was attempted by introducing a truncated version of Bax, with increased apoptotic activity. Inhibition of Hsp70-mediated cancer survival was attempted by the introduction of Hsp40 J domain, a dominant negative gene inhibiting Hsp70 activity. Although the gene products activated cellular death pathways in transfected cells, no significant cell death was observed. In contrast suicide gene strategies showed great potential for SCLC gene therapy. The suicide gene system herpes simplex virus thymidine kinase (HSVtk) with the prodrug ganciclovir (GCV) had already been tested and implemented at the laboratory showing >50 % cell death in vitro and tumor regression in vivo. HSVtk efficacy was improved by the replacement of GCV with penciclovir (PCV), which demonstrated significant less toxicity to untransfected cells. Another suicide gene system was implemented for testing: the yeast cytosine deaminase (YCD)gene either alone or fused with the yeast uracil phosphoribosyltransferase (YUPRT) gene and combined with the prodrug 5-FC. The YCD/YUPRT fusion gene showed superior efficacy, mediating 90 % SCLC cell death. This dual suicide gene strategy targets in contrast to HSVtk/GCV or PCV, both DNA and RNA synthesis, also causing cell death to non-proliferating, dormant cancer cells. The strategy demonstrated significant bystander cytotoxicity due to the diffusion of toxic metabolites to nearby cancer cells, showing potential to overcome the low transfection efficiency currently characterizing in vivo gene therapy. An update will be presented.

Construction of double suicide genes system controlled by MDR1 promoter with targeted expression in drug-resistant glioma cells

The multiple drug resistance protein (MDR1) is frequently overexpressed in human glioma. The aim of this study is to clone the MDR1 promoter from C6/ADR, construct the double suicide genes expressive vector controlled by MDR1 promoter, and explore its targeted expression in C6/ADR cells. MDR1 promoter from C6/ADR genomic DNA, which was linked with T vector, was amplified by using Polymerase chain reaction (PCR). After cut by NdeI and HindIII, MDR1 promoter was cloned into pcDNA3-TK (thymidine kinase) plasmid. The cytosine deaminase (CD) gene from pcDNA3-CD-TK plasmid was directly cloned into the above vector to construct pcDNA3-MDR1-promoter-CD-TK vector. Then this vector was transfected into C6 and C6/ADR cells respectively by liposome. After selection by G418, the tumor cell lines were stably established. Then these cell lines were examined through PCR and RT-PCR to respectively detect the integration and expression of TK and CD genes. The results showed the length and sequence of MDR1 promoter amplified by PCR were confirmed by DNA sequencing. The pcDNA3-MDR1-promoter-CD-TK expression vectors were constructed successfully. PCR indicated the double suicide genes were integrated into C6 and C6/ADR cells. RT-PCR revealed that CD and TK genes expressed in C6/ADR/CD-TK cells, whereas not in C6/CD-TK cells. In conclusions, construction of expressive vector containing double suicide genes controlled by MDR1 promoter with targeted expression in C6/ADR will provide a sound basis for targeted gene therapy for multidrug resistance (MDR) glioma.

Cellular and molecular events associated with the antitumor response induced by the cytosine deaminase/5-fluorocytosine suicide gene therapy system in a rat liver metastasis model

The bacterial cytosine deaminase (CD) gene converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil. We have previously shown, in a rat liver metastasis model from colon carcinoma, that intratumoral injection of a CD-expressing plasmid into the animals followed by 5-FC treatment results in the regression of the treated tumor as well as distant uninjected tumors. The aim of this study was to further analyze the mechanisms associated with tumor regression induced upon application of suicide CD/5-FC strategy. Tumor regression was associated with an increased apoptosis, the recruitment of natural killer cells, CD4- and CD8 T lymphocytes within the tumors and an increased expression of several cytokines/chemokines mRNAs. These data indicate that the CD/5-FC suicide strategy is associated with the triggering of cellular and molecular events leading to an efficient antitumor immune response involving both innate and acquired immunity.

Therapeutic Efficacy of Replication-Competent Retrovirus Vector–Mediated Suicide Gene Therapy in a Multifocal Colorectal Cancer Metastasis Model

Replication-competent retrovirus (RCR) vectors are intrinsically incapable of infecting quiescent cells and have been shown to achieve highly efficient and tumor-restricted replicative spread and gene transfer in vivo after direct intratumoral injection in a variety of primary cancer models. However, i.v. delivery of RCR vectors expressing therapeutic genes has never previously been tested, particularly in an immunocompetent tumor model. Therefore, in the present study, we sought to test the therapeutic effect of an RCR vector (ACE-CD) carrying the yeast cytosine deaminase (CD) gene, which converts the nontoxic prodrug 5-fluorocytosine (5FC) into the chemotoxin 5-fluorouracil, after delivery by infusion into the locoregional circulation in a multifocal hepatic metastasis model of colon cancer. After confirmation of suicide gene cytotoxicity in vitro, multifocal hepatic tumors were established in syngeneic mice with murine CT26 colorectal cancer cells expressing firefly luciferase (CT26-Luc), and the ACE-CD vector was infused via intrasplenic injection into the portal circulation. Fourteen days after locoregional infusion, systemic administration of 5FC resulted in significant inhibition of bioluminescent signals in mice whose tumors had been infected with RCR but not in control mice. Notably, there was no detectable RCR vector spread to normal liver or bone marrow by quantitative PCR analysis. Our results thus show that locoregional delivery of a suicide gene by RCR vectors infused into the portal circulation results in progressive transduction of multiple tumor foci in the liver, without evidence of spread to adjacent normal parenchyma or extrahepatic tissues, and can achieve significant tumor growth inhibition.

Cytotoxic effect of a replication-incompetent adenoviral vector with cytosine deaminase gene driven by L-plastin promoter in hepatocellular carcinoma cells

Great expectations are set on gene therapy for the treatment of malignant hepatocellular carcinomas (HCC) in East Asia. Recombinant adenoviral vectors (AV) have been developed in which the L-plastin promoter (LP) regulates the expression of transgenes, in a tumor cell specific manner, resulting in an increase in the therapeutic index. The development of the AdLPCD vector, a replication-incompetent AV, containing a transcription unit of LP and E. coli cytosine deaminase (CD), was reported in our previous work. In the present study, the AdLPCD vector combined with 5-fluorocytosine (5-FC) administration was tested to see if it might have significant utility in the chemosensitization of L-plastin positive HCC. Four HCC cell lines (HepG2, Chang Liver, Huh-7 and SK-Hep-1 cells) were investigated for the expression of LacZ after infecting the cells with the AdLPLacZ vector containing a 2.4 kb fragment of LP and the LacZ gene. Relatively high levels of LP activity were detected in HepG2, followed by Chang Liver cells; whereas, no promoter activity was found in Huh-7 and SK-Hep-1 cells, as determined by AdLPLacZ infection followed by the beta-galactosidase assay. In addition, the results of RT-PCR assays for the detection of endogenous L-plastin mRNA in these cells lines correlated well with those of the beta-galactosidase activity after infection with AdLPLacZ. Based on these data, the cytotoxic effect of AdLPCD/5-FC was evaluated in HepG2 cells. These results indicate that the CD gene delivered by AV could sensitize HepG2 cells to the prodrug, 5-FC. However, the observed effects were insufficient to cause the death of most of cells. This suggests that the screening of patients for an AdLP/5-FC strategy based on AdLPLacZ data might not always guarantee a good therapeutic outcome.

Adenovirus-mediated hypoxia-targeting cytosine deaminase gene therapy enhances radiotherapy in tumour xenografts

Hypoxia is closely associated with the radioresistance of tumours; therefore, targeting hypoxic areas is very important for cancer therapy. The aim of this study is to establish such a targeting strategy by applying a bacterial cytosine deaminase (BCD)/5-fluorocytosine (5-FC) gene therapy system and to examine whether the strategy enhances the efficacy of radiotherapy in a tumour xenograft. The hypoxia-responsive promoter 5HREp, in which five copies of the hypoxia-response element (HRE) enhance transcription from a cytomegalovirus minimal promoter, was employed to induce the expression of BCD under hypoxic conditions. The adenoviral vector Ad/5HREp-BCD, encoding the gene 5HREp-BCD, robustly induced BCD expression under hypoxic conditions and this led to significant cytotoxicity in combination with 5-FC in vitro. Intratumoral Ad/5HREp-BCD administration resulted in the expression of BCD at the border between normoxic and necrotic regions. The BCD/5-FC gene therapy enhanced the therapeutic effects of both single (12.5 Gy) and fractionated (3 Gy × 5 days) radiotherapy with few side effects and significantly increased tumour growth doubling time by up to 2.4-fold (P<0.01) and 2.5-fold (P<0.05), respectively. All of these results suggest that the present BCD/5-FC gene therapy has the ability to specifically target hypoxic tumour cells and significantly improves the control of tumour growth after radiotherapy.

Human neural stem cells target and deliver therapeutic gene to experimental leptomeningeal medulloblastoma

Medulloblastomas are highly malignant neuroectodermal cerebellar tumors of children. One of the reasons for the difficulty for the treatment of medulloblastomas is their inherent tendency to metastasize through the cerebrospinal fluid (CSF) pathway leading to leptomeningeal dissemination. Recently, genetically modified neural stem cells (NSCs) were shown to have the capability of selectively migrating into glioma mass and delivering therapeutic agents with significant therapeutic benefits. In the present study, we applied the NSC strategy to target medulloblastomas, particularly their leptomeningeal dissemination. We used NSCs that were retrovirally transduced with the cytosine deaminase gene (CD-NSCs). In vitro studies demonstrated that CD-NSCs had sufficient migratory activity toward medulloblastoma cells and exerted a remarkable bystander effect on these cells following the application of 5-fluorocytosine (5-FC). It is noteworthy that neutralization of the hepatocyte growth factor blocked their migration In animal studies using our leptomeningeal dissemination model, CD-NSCs implanted directly into CSF space were shown to distribute diffusely within the disseminated tumor cells and could provide remarkable antitumor effect after intraperitoneal administration of 5-FC. Furthermore, CD-NSC treatment followed by 5-FC administration prolonged survival periods significantly in experimental animals. Our data suggest that the CD-NSC strategy can also be applied to target leptomeningeal dissemination of medulloblastomas.

Mutation of Escherichia coli cytosine deaminase significantly enhances molecular chemotherapy of human glioma

Combined treatment using adenoviral (Ad)-directed enzyme/prodrug therapy and radiation therapy has the potential to become a powerful method of cancer therapy. We have developed an Ad vector encoding a mutant bacterial cytosine deaminase (bCD) gene (AdbCD-D314A), which has a higher affinity for cytosine than wild-type bCD (bCDwt). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of AdbCD-D314A with the prodrug 5-fluorocytosine (5-FC) and ionizing radiation against human glioma. The present study demonstrates that AdbCD-D314A infection resulted in increased 5-FC-mediated cell killing, compared with AdbCDwt. Furthermore, a significant increase in cytotoxicity following AdbCD-D314A and radiation treatment of glioma cells in vitro was demonstrated as compared to AdbCDwt. Animal studies showed significant inhibition of subcutaneous or intracranial tumor growth of D54MG glioma xenografts by the combination of AdbCD-D314A/5-FC with ionizing radiation as compared with either agent alone, and with AdbCDwt/5-FC plus radiation. The results suggest that the combination of AdbCD-D314A/5-FC with radiation produces markedly increased cytotoxic effects in cancer cells in vitro and in vivo. These data indicate that combined treatment with this novel mutant enzyme/prodrug therapy and radiotherapy provides a promising approach for cancer therapy.

Strong Enhancement of Recombinant Cytosine Deaminase Activity inBifidobacterium longumfor Tumor-Targeting Enzyme/Prodrug Therapy

In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity. In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy.

Cytosine Deaminase/5-Fluorocytosine Exposure Induces Bystander and Radiosensitization Effects in Hypoxic Glioblastoma Cells in vitro

Treatment of glioblastoma (GBM) is limited by therapeutic ratio; therefore, successful therapy must be specifically cytotoxic to cancer cells. Hypoxic cells are ubiquitous in GBM, and resistant to radiation and chemotherapy, and, thus, are logical targets for gene therapy. In this study, we investigated whether cytosine deaminase (CD)/5-fluorocytosine (5-FC) enzyme/prodrug treatment induced a bystander effect (BE) and/or radiosensitization in hypoxic GBM cells. We stably transfected cells with a gene construct consisting of the SV40 minimal promoter, nine copies of a hypoxia-responsive element, and the yeast CD gene. During hypoxia, a hypoxia-responsive element regulates expression of the CD gene and facilitates the conversion of 5-FC to 5-fluorouracil, a highly toxic antimetabolite. We used colony-forming efficiency (CFE) and immunofluorescence assays to assess for BE in co-cultures of CD-expressing clone cells and parent, pNeo- or green fluorescent protein-stably transfected GBM cells. We also investigated the radiosensitivity of CD clone cells treated with 5-FC under hypoxic conditions, and we used flow cytometry to investigate treatment-induced cell cycle changes. Both a large BE and radiosensitization occurred in GBM cells under hypoxic conditions. The magnitude of the BE depended on the number of transfected cells producing CD, the functionality of the CD, the administered concentration of 5-FC, and the sensitivity of cell type to 5-fluorouracil. Hypoxia-inducible CD/5-FC therapy in combination with radiation therapy shows both a pronounced BE and a radiosensitizing effect under hypoxic conditions.

P22 The value of hypoxic agent, Tc-99m HL91, in gene therapy with the bacterial cytosine deaminase gene for the lewis lung cancer: a study in vitro

P22 The value of hypoxic agent, Tc-99m HL91, in gene therapy with the bacterial cytosine deaminase gene for the lewis lung cancer: a study in vitro

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.

Antitumor effects of cytosine deaminase and thymidine kinase fusion suicide gene under the control of mdr1 promoter in mdr1 positive leukemia cells

The multidrug resistance (mdr) mediated by P-glycoprotein (P-gp), the mdr1 gene product, is one of the major obstacles in leukemia treatment. The present study was designed to explore a suicide gene therapy approach targeting mdr1 for reversal of P-gp-mediated mdr in the mdr positive K562/A02 cells. To study targeted killing effects of cytosine deaminase (CD)-thymidine kinase (TK) fusion suicide gene on multi-drug resistant leukemia, the CD-TK fusion suicide gene expression vector driven by mdr1 promoter was constructed and transferred into K562 and K562/A02 cells using lipofectin™ 2000. RT-PCR was used to demonstrate that there were CD and TK genes expression in K562/A02 cells, but not in K562 cells. MTT analysis showed that, compared with that in K562/CDTK, the survival rate of K562/A02-CDTK cells decreased and at the same time the apoptotic rate increased after treatment with GCV and 5-FC (P < 0.05). In vivo studies showed that the tumor volume in the prodrug treated K562/A02-CDTK groups was significantly less than that in the NS-control and K562-CDTK groups (P < 0.05). These findings show that the CD and TK fusion suicide gene expression driven by mdr1 promoter is effective in killing multidrug resistant K562/A02 cells.

The Role of Enhanced Radiosensitivity and Tumor-specific Suicide Gene Vector in Genetherapy of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is one of the common malignant tumors in China. Radiotherapy and chemotherapy are the main therapy methods for NPC. To enhance the specific antitumor effect, a novel vector with radiosensitivity and tumor specificity was constructed in this study, which enables the reduction of dosage of radiation and chemotherapeutic drugs due to its double killing effect. Four DNA elements, Egr-1 promoter, Cytosine deaminase (CD) gene, hTERT promoter, Survivin antisense oligonucleotides were amplified and constructed in pcDNA3.1 vector. CD and Survivin gene expression in CNE-2 cells were detected by RT-PCR. High performance liquid chromatography (HPLC) was employed to determine the transformation from the prodrugs 5-FC to 5-FU. Hoechst33258 staining of the nuclei and methylthiazolyl tetrazolium(MTT) assay were applied to detect apoptosis and cell survivability, respectively. In addition, the anti-tumor effects were examined in vivo by injecting cells with different vectors into nude mice. Our results revealed a notable killing effect of combined treatment with 5-FC and radiation on CNE-2 cells transfected with vectors in vitro. This effect was especially notable on pEC-TS transferred cells, which showed 57% of cells were killed. In vivo, an obvious suppression of tumor was displayed in pEC-TS group, which was significantly different from other groups (p < 0.05). Consequently, this expression cassette may have a great therapeutic potential for the treatment of NPC.