The oncolytic effect of recombinant vesicular stomatitis virus is enhanced by expression of the fusion cytosine deaminase/uracil phosphoribosyltransferase suicide gene

Vesicular stomatitis virus (VSV) has recently been demonstrated to exhibit significant oncolytic capabilities against a wide variety of tumor models in vitro and in vivo. To potentially enhance the oncolytic effect, we generated a novel recombinant VSV (rVSV) that expressed the fusion suicide gene Escherichia coli cytosine deaminase (CD)/uracil phosphoribosyltransferase (UPRT). rVSV encoding the CD/UPRT fusion gene (VSV-C:U) exhibited normal growth properties and generated high levels of biologically active CD/UPRT that could catalyze the modification of 5-fluorocytosine into chemotherapeutic 5-fluorouracil (5-FU), which exhibited considerable bystander effect. Intratumoral inoculation of VSV-C:U in the presence of the systemically administered prodrug 5-fluorocytosine produced statistically significant reductions in the malignant growth of syngeneic lymphoma (A20) or mammary carcinoma (TSA) in BALB/c mice compared with rVSV treatments or with control 5-FU alone. Aside from detecting prolonged therapeutic levels of 5-FU in VSV-C:U-treated animals harboring TSA tumors and enhancing bystander killing of tumor cells, we demonstrated marked activation of IFN-gamma-secreting cytotoxic T cells by enzyme-linked immunospot analysis that may have also facilitated tumor killing. In conclusion, the insertion of the fusion CD/UPRT suicide gene potentiates the oncolytic efficiency of VSV by generating a strong bystander effect and by contributing to the activation of the immune system against the tumor without detrimentally altering the kinetics of virus-mediated oncolysis and may be useful in the treatment of malignant disease.     

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.     

KDR promoter can transcriptionally target cytosine deaminase suicide gene to cancer cells of nonendothelial origin

The KDR/flk-1 gene promoter is considered to be endothelial cell-specific. We show its activity in two cancer cell lines of non-endothelial origin: in murine L1 sarcoma and OVP-10 human ovarian carcinoma cell lines. KDR promoter-driven cytosine deaminase gene can be efficiently expressed in these cells leading to sensitization to 5-fluorocytosine, as demonstrated both in vitro and in vivo. Our results indicated that KDR promoter activity is not endothelial cell-exclusive and that this promoter can also be used to obtain specific expression of therapeutic genes in certain cancer cells.     

Usefulness of repeated direct intratumoral gene transfer using hemagglutinating virus of Japan-liposome method for cytosine deaminase suicide gene therapy

To investigate the feasibility of repeated gene transfection in suicide gene therapy against human solid tumors by a combination of 5- fluorocytosine (5-FC) and its converting enzyme, cytosine deaminase (CD), we repeatedly transfected the yeast CD gene into the human pancreatic cancer cell line BXPC3 using the hemagglutinating virus of Japan-liposome in a new gene transfer method. The in vivo growth of the s.c. transplanted BXPC3 tumor in nude mice given CD-gene transfection was significantly suppressed by i.p. injection of 5-FC when compared with tumors treated with the control vector. Furthermore, the tumor transfected with the CD gene during a 7-day interval was suppressed much more than that of a single transfection. These results suggest that repeated transfection of the suicide gene together with the combination of 5-FC and the yeast CD gene using the hemagglutinating virus of Japan-liposome gene transfer method may be useful for the treatment of human solid tumors, including pancreatic cancer.     

Induction of protective immunity against syngeneic rat cancer cells by expression of the cytosine deaminase suicide gene

The use of the cytosine deaminase (CD)/5-fluorocytosine suicide system as a cancer gene therapy approach enables selective killing of CD-modified cells as well as the ablation of non-modified tumor cells due to a bystander effect that has been suggested to involve the immune system in vivo. Using a stable CD transfectant of the tumorigenic rat adenocarcinoma cell line AS (AS/CD), an antitumoral response against the CD expressing cell line as well as the parental cell line could be induced by stepwise vaccinations in syngeneic animals. AS/CD tumor regression occurred independently of 5-fluorocytosine treatment and was sufficient to protect 37% of the animals against subsequent challenge with tumorigenic doses of the parental AS cell line. Immune rats contained lymphocytes able to specifically lyse CD modified as well as unmodified AS tumor cells in vitro, most likely contributing to the in vivo antitumoral reaction. Thus, the CD suicide system seems to be suitable not only for a local tumor gene therapy but also for the application as therapy of metastatic tumors and minimal residual disease.     

Efficacy of gene therapy-delivered cytosine deaminase is determined by enzymatic activity but not expression

The potential utility of tumour-selective 5-fluorouracil treatment using attenuated Salmonella serovar typhimurium recombinant for cytosine deaminase (TAPET-CD) has been documented in experimental settings. The present data demonstrate that in vivo (19)F-magnetic resonance spectroscopy measurements allow the outcome prediction of this prokaryotic-based therapy, demonstrating the necessity of non-invasive real-time imaging techniques for treatment monitoring.     

胞嘧啶脱氨酶/氟胞嘧啶体系对人乳腺癌基因的实验治疗作用

目的：研究胞嘧啶脱氨酶（ＣＤ）／氟胞嘧啶（５－ＦＣ）体系对人乳腺癌的实验治疗作用。方法：应用ＭＴＴ法测定人乳腺细胞对５－ＦＣ的敏感性。结果：５－ＦＣ对导入ＣＤ基因的人乳腺癌细胞有明显的细胞毒作用,而对未导入ＣＤ基因的人乳腺癌细胞的毒性较低,５－ＦＣ对导入ＣＤ基因的人乳腺癌细胞的ＩＣ５０分别为４１８μｇ／ｍｌ和１４２９μｇ／ｍｌ。结论：ＣＤ／４－ＦＣ体系对体外转基因的人乳腺癌细胞具有抗肿瘤作用。     

Cytosine deaminase suicide gene therapy for peritoneal carcinomatosis

Gene therapy is a novel therapeutic approach that might soon improve the prognosis of some cancers. We investigated the feasibility of cytosine deaminase (CD) suicide gene therapy in a model of peritoneal carcinomatosis. DHD/K12 colorectal adenocarcinoma cells transfected in vitro with the CD gene were highly sensitive to 5-fluorocytosine (5-FC), and a bystander effect could also be observed. Treating CD+ cells with 5-FC resulted in apoptosis as detected by terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling. In vitro, several human cell lines derived from ovarian or colorectal carcinomas, as well as the rat glioblastoma 9 L cell line, responded to CD/5-FC and showed a very strong bystander effect. 5-FC treatment of peritoneal carcinomatosis generated in syngeneic BDIX rats by CD-expressing DHD/K12 cells led to a complete and prolonged response and to prolonged survival. Our study thus demonstrated the efficacy of CD suicide gene therapy for the treatment of peritoneal carcinomatosis.     

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.     

Mechanisms of thymidine kinase/ganciclovir and cytosine deaminase/ 5-fluorocytosine suicide gene therapy-induced cell death in glioma cells

Suicide gene transfer using thymidine kinase (TK) and ganciclovir (GCV) treatment or the cytosine deaminase (CD)/5-fluorocytosine (5-FC) system represents the most widely used approach for gene therapy of cancer. However, molecular pathways and resistance mechanisms remain controversial for GCV-mediated cytotoxicity, and are virtually unknown for the CD/5-FC system. Here, we elucidated some of the cellular pathways in glioma cell lines that were transduced to express the TK or CD gene. In wild-type p53-expressing U87 cells, exposure to GCV and 5-FC resulted in a weak p53 response, although apoptosis was efficiently induced. Cell death triggered by GCV and 5-FC was independent of death receptors, but accompanied by mitochondrial alterations. Whereas expression of Bax remained unaffected, in particular, GCV and also 5-FC caused a decline in the level of Bcl-2. Similar findings were obtained in 9L and T98G glioma cells that express mutant p53, and also underwent mitochondrial apoptosis in both the TK/GCV and CD/5-FC system. Upon treatment of 9L cells with 5-FC, Bcl-xL expression slowly declined, whereas exposure to GCV resulted in the rapid proapoptotic phosphorylation of Bcl-xL. These data suggest that TK/GCV- and CD/5-FC-induced apoptosis does neither require p53 nor death receptors, but converges at a mitochondrial pathway triggered by different mechanisms of modulation of Bcl-2 proteins.     

Increased sensitivity of glioma cells to 5-fluorocytosine following photo-chemical internalization enhanced nonviral transfection of the cytosine deaminase suicide gene

Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80 % of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments.PCI was performed with the photosensitizer AlPcS_2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS_2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect.     

Selection of drug-resistant transduced cells with cytosine nucleoside analogs using the human cytidine deaminase gene

Hematopoietic toxicity produced by most anticancer drugs limits their potential for curative therapy. We have shown previously that the human cytidine deaminase (CD) gene can confer drug resistance in murine bone marrow cells (BMCs) to the nucleoside analog, cytosine arabinoside (ARA-C). In the present study, as the first objective we showed that the CD gene can also render drug resistance in BMCs to related analogs, 2',2'-difluorodeoxycytidine (dFdC) and 5-azadeoxycytidine (5-AZA-CdR). As a second objective, we investigated the potential of ex vivo selection with cytosine nucleoside analogs of CD-transduced BMC. The goal of this approach was to enrich the fraction of CD-transduced BMCs so as to increase the transgene expression and level of drug resistance before transplantation. This strategy may have the potential to circumvent the problem in clinical gene therapy of low level of gene transfer and adequate long-term gene expression. Using a bicistronic retroviral vector containing the CD and the green fluorescent protein (CDiGFP), we transduced murine L1210 leukemic cells. All three analogs, ARA-C, dFdC, and 5-AZA-CdR were demonstrated in vitro to enrich (>95%) the population of leukemic cells expressing the GFP transgene. However, with CD-transduced primary murine BMCs cultivated at high cell density we observed that in vitro selection with ARA-C was not possible due to release of CD into the culture medium at amounts that were sufficient to inactivate the analog. The CD-containing medium produced a chemoprotective effect on mock BMCs as shown by lack of significant growth inhibition in the presence of ARA-C. However, at low cell density in a cell mixture containing CD-transduced cells, the mock BMCs showed marked drug sensitivity to ARA-C as determined by clonogenic assay. Selection with ARA-C was shown to significantly increase the CD enzyme activity in transduced BMC. These results suggest that CD gene has the potential to be a good selectable marker and a possible tool for chemoprotection in cancer gene therapy.     

逆转录病毒介导的CD/5-FC对胰腺癌细胞的杀伤作用

目的 :探讨大肠埃希菌胞嘧啶脱氨基酶 (CD) /5 -氟胞嘧啶 ( 5 FC)系统对胰腺癌细胞的体外生长抑制作用。方法 :将含CD基因的重组逆转录病毒载体导入胰腺癌细胞形成转化细胞系 ,对转化细胞进行体外药物敏感实验 ,包括 :1)转化细胞在前体药物 5 FC作用下的细胞生长抑制率 ;2 )MTT法检测旁观者效应。结果 :体外实验 5 FC的有效浓度 >2mmol/L时 ,就表现出杀伤作用 ,当 5 FC的有效浓度 >6mmol/L时 ,几乎未见细胞生长 ,PA3 17/CD与TD2混育比例在 90 %时 ,杀伤作用最大 ,相同药物浓度下 ,混育 96h杀伤作用明显高于 2 4h。结论 :CD/5 FC系统对胰腺癌细胞系细胞具有实验性基因治疗作用     

Direct and bystander killing of sarcomas by novel cytosine deaminase fusion gene

Soft tissue and bone sarcomas of the extremities can be difficult to eradicate, and standard treatment may require limb amputation. New therapies to decrease tumor size could improve the effectiveness of treatment and decrease the frequency of limb amputation. Cytosine deaminase (CD)-based gene therapy has been shown to be effective in decreasing growth of solid tumors when animals with CD-expressing tumor cells are treated with 5 fluorocytosine (5FC), an inert prodrug that is converted to 5-fluorouracil (5FU) by CD. In this investigation, we used a novel CD-containing fusion gene to determine whether CD-based gene therapy affected soft tissue or bone sarcomas. The novel fusion gene (NGFR-CD) encodes for a protein with extracellular and transmembrane domains of human nerve growth factor receptor (NGFR) and cytoplasmic CD. Murine 2472 (2) sarcoma cells were transduced with fusion genes containing either the bacterial (NGFR-(b)CD) or yeast (NGFR-(y)CD) CD gene. 5FC treatment killed NGFR-(b)CD- and NGFR-(y)CD-transduced sarcoma cells in vitro through direct and bystander effects (P < 0.01). In contrast, 5FC treatment of mice with s.c. 2NGFR-(b)CD or 2NGFR-(y)CD tumors affected only 2NGFR-(y)CD tumors. 5FC had no effect on growth of NGFR-(b)CD tumors but caused significant decrease in the size of 2NGFR-(y)CD tumors (51 +/- 60 versus 938 +/- 767 mm(3), treated versus control, P < 0.01). Evaluation of bystander killing in vivo revealed significant tumor killing, with a 5-fold reduction in s.c. tumor volume evident in saline versus 5FC-treated mice when tumors were comprised of 90% 2472 cells and 10% 2NGFR-(y)CD selected for fluorescence-activated cell sorting (P < 0.01). Bone sarcomas were eliminated in 9 of 10 5FC-treated mice, compared with 11.8 +/- 6.0 mm(2) in saline-treated mice (P < 0.002). In addition, 5FC treatment of bone sarcomas caused a significant reduction in cancer-induced bone destruction (P < 0.002) and resulted in a reduction in the number of osteoclasts. Finally, 5FC treatment had no effect on animal weight or survival, whereas doses of 5FU providing equivalent tumor reduction as 5FC resulted in treatment-associated deaths and significant weight loss (P < 0.001).     

Adenovirus-mediated tissue-specific cytosine deaminase gene therapy for human hepatocellular carcinoma with different AFP expression levels

To evaluate whether in vitro and in vivo transferring of Escherichia coli cytosine deaminase gene to a solid tumor will confer the sensitivity to the prodrug 5-fluorocytosine (5FC) on these cells, we constructed two replication-defective adenovirus vector in which the cytosine deaminase gene was driven by CAG promoter (Adex1CACD) and AFP gene 5'-flanking region (Adex1AFPCD), respectively. By transferring these two vectors to SMMC7721AFP(-) and HepG2 human hepatocellular carcinoma (HCC) cells in vitro, we found that Adex1CACD vector could effectively suppress SMMC7721AFP(-) and HepG2 cells growing in the presence of 5FC even if the infected cell is less to 20%, while Adex1AFPCD vector only conferred HepG2 cells sensitivity to 5FC. When Adex1CACD was directly injected into established subcutaneous SMMC7721AFP(-) tumors in nude mice receiving 5FC, the tumor growth was inhibited significantly, which was consistent with those in vitro results. Furthermore, the Adex1AFPCD plus 5FC suppressed SMMC7721AFP(+) tumor growth in vivo, but not SMMC7721AFP(-) tumor. The results suggested that the CAG promoter-controlled CD gene could effectively mediate the growth inhibition in different kinds of HCC combined with administration of 5FC, and the AFP promoter-controlled CD gene could only suppress the HCCs expressing high levels of AFP. Therefore, adenovirus-mediated tumor-specific gene transfer may be a potential strategy for local control of tumor growth.     

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.