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.     

Apoptosis Induction With 5-Fluorocytosine/Cytosine Deaminase gene Therapy for Human Malignant Glioma Cells Mediated by Adenovirus

Previously, we evaluated the therapeutic efficacy of the adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for malignant gliomas. However, the molecular pathways that mediate the 5-FC/CD gene therapy-induced cell death remains to be elucidated. In this study, we examined the induction of apoptosis and the role of caspases in 5-FC/CD gene therapy using human malignant glioma cells [Gli36delta5 (mutated p53) and U87MG (wild p53)]. The treatment with 5-FC/CD gene-therapy-induced apoptosis both in Gli36delta5 cells and in U87MG cells according to flow cytometric analysis. Immunoblot analysis revealed that caspases 3 and 9 were processed in response to 5-FC/CD in a concentration- and time-dependent manner, but caspase 8 was not. Each caspase 3 and 9 inhibitor significantly reduced apoptosis triggered by 5-FC/CD, but the caspase 8 inhibitor did not affect apoptosis induction. 5-FC/CD significantly promoted the release of cytochorme c from mitochondria in a concentration-dependent manner. These results indicate that 5-FC/CD gene therapy induces apoptosis in human malignant glioma cells and that the apoptotic cell death is mediated by the activation of mitochondrial caspase cascades involving caspases 3 and 9. This is the first report concerning the apoptotic mechanism of 5-FC/CD gene therapy, and these findings could be used to increase the efficacy of suicide gene therapy systems for the treatment of malignant glioma.     

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.     

肿瘤双自杀基因治疗系统pTRKH2-PsT/CD,pTRKH2-PsT/UPRT在厌氧婴儿双歧杆菌中的构建

Objective: We recombine the suicide gene CD, UPRT into plasmid pTRKH2 and clone the recombinant dual suicide gene therapy system into tumor-hypoxia-targeting vector Bifidobacterium infantis and characterize its function. Methods: CD gene, UPRT gene and lactic acid bacteria expression plasmid pTRKH2 were digested by restriction endonuclease BamH I and Sal I, and constructed recombinant plasmids pTRKH2/CD and pTRKH2/UPRT in E. coll. The recombinant plasmids were then transfected into Bifidobacterium Infantis by electroporation. Identification of pTRKH2/CD and pTRKH2/UPRT was processed by dual restriction endonuclease digesting and sequencing. RT-PCR and SDS-PAGE were used to examine the expression of CD and UPRT genes at RNA and protein levels. The killing effects on Melanoma B16-F10 cells by pTRKH2/CD and pTRKH2/UPRT suicide gene therapy system with 5-FC were examined by MTT assay. Results: The CD gene and UPRT gene was successfully recombined into lactic acid bacteria expression plasmid pTRKH2. After dual endonuclease digestion of plasmid purified from the positively transfected E. co/i, two fragments of 6.9 Kb and 1.3 Kb were found for CD gene and two fragments of 6.9 Kb and 620 bp were found for UPRT gene. The sequencing of CD gene and UPRT gene proved consistent sequences with Genebank published data. A fragment of 1.3 Kb for CD gene and fragment of 620 bp for UPRT gene was found in recombinant Bifidobac- terium by RT-PCR. A 52 KDa protein for CD gene was identified in whole-cell protein of recombinant Bifidobacterium and a 26 KDa protein for UPRT gene was identified in supernatant fluid of recombinant Bifidobacterium. The survival rate of tumor cells treated by extracts from culture of recombinant Bifidobacterium with 5-FC showed a strong killing effects of pTRKH2/CD and pTRKH2/UPRT dual suicide gene therapy system on Melanoma B16-F10 cells. Conclusion: CD gene and UPRT gene are suc- cessfully inserted into pTRKH2 and transfected into tumor-hypoxia-targeting vector Bifldobacterium Infantis. This dual suicide gene therapy system shows a high efficiency for tumor cells killing.     

肿瘤厌氧靶向双自杀基因治疗系统pTRKH2-PsT/CD、pTRKH2-PsT/UPRT的构建

目的 利用婴儿双歧杆菌对实体瘤低氧区的靶向效应,构建肿瘤厌氧靶向双自杀基因治疗系统pTRKH2-PsT /CD和pTRKH2-PsT /UPRT。方法 用PCR的方法从质粒pGEX/CD和pGEX/UPRT中扩增出CD基因和UPRT基因,双酶切CD基因、UPRT基因和质粒pTRKH2-PsT,分别连接后重组于大肠杆菌中。之后用电转化的方法将重组质粒转入婴儿双歧杆菌中。用RT-PCR检测该系统mRNA水平的表达,SDS-PAGE检测该系统在蛋白质水平的表达。在黑色素瘤B16-F10细胞上检测该系统的体外肿瘤细胞杀伤效果。结果 成功地将CD基因和UPRT基因转入了质粒pTRKH2-PsT,CD基因和UPRT基因的测序结果表明序列与Genebank公布的序列一致。RT-PCR检测到CD和UPRT mRNA水平的明显表达。在含有CD基因的婴儿双歧杆菌细胞全蛋白中发现了CD蛋白质的表达,在含有UPRT基因的婴儿双歧杆菌上清液中发现了UPRT蛋白质的表达。黑色素瘤细胞的低存活率证明了pTRKH2-PsT/CD、pTRKH2-PsT/UPRT自杀基因治疗系统对黑色素瘤的显著杀伤作用。结论 肿瘤厌氧靶向双自杀基因治疗系统pTRKH2-PsT/CD、pTRKH2-PsT/UPRT构建成功并显示出杀伤肿瘤细胞的作用。     

Cytosine deaminase/5-fluorocytosine exposure induces bystander and radiosensitization effects in hypoxic glioblastoma cells in vitro

PURPOSE: 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. METHODS AND MATERIALS: 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. RESULTS: 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. CONCLUSION: Hypoxia-inducible CD/5-FC therapy in combination with radiation therapy shows both a pronounced BE and a radiosensitizing effect under hypoxic conditions.     

Chemovirotherapy for head and neck squamous cell carcinoma with EGFR-targeted and CD/UPRT-armed oncolytic measles virus

First-line treatment of recurrent and/or refractory head and neck squamous cell carcinoma (HNSCC) is based on platinum, 5-fluorouracil (5-FU) and the monoclonal antiEGFR antibody cetuximab. However, in most cases this chemoimmunotherapy does not cure the disease, and more than 50% of HNSCC patients are dying because of local recurrence of the tumors. In the majority of cases, HNSCC overexpress the epidermal growth factor receptor (EGFR), and its presence is associated with a poor outcome. In this study, we engineered an EGFR-targeted oncolytic measles virus (MV), armed with the bifunctional enzyme cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT). CD/UPRT converts 5-fluorocytosine (5-FC) into the chemotherapeutic 5-FU, a mainstay of HNSCC chemotherapy. This virus efficiently replicates in and lyses primary HNSCC cells in vitro. Arming with CD/UPRT mediates efficient prodrug activation with high bystander killing of non-infected tumor cells. In mice bearing primary HNSCC xenografts, intratumoral administration of MV-antiEGFR resulted in statistically significant tumor growth delay and prolongation of survival. Importantly, combination with 5-FC is superior to virus-only treatment leading to significant tumor growth inhibition. Thus, chemovirotherapy with EGFR-targeted and CD/UPRT-armed MV is highly efficacious in preclinical settings with direct translational implications for a planned Phase I clinical trial of MV for locoregional treatment of HNSCC.     

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.     

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.     

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.     

Therapeutic effect of suicide gene-transferred mesenchymal stem cells in a rat model of glioma

We evaluated a new therapeutic strategy for malignant glioma, which combines intratumoral inoculation of mesenchymal stem cells (MSCs) expressing cytosine deaminase gene with 5-fluorocytosine (5-FC) administration. For in vitro and in vivo experiments, MSCs were transfected with adenovirus carrying either enhanced green fluorescent protein gene (AdexCAEGFP) or cytosine deaminase gene (AdexCACD), to establish MSC-expressing EGFP (MSC-EGFP) or CD (MSC-CD). Co-culture of 9L glioma cells with MSC-CD in a medium containing 5-FC resulted in a remarkable reduction in 9L cell viability. The migratory ability of MSC-EGFP toward 9L cells was demonstrated by double-chamber assay. For the in vivo study, rats harboring 9L brain tumors were inoculated with MSC-EGFP or MSC-CD. Immunohistochemistry of rat brain tumors inoculated with MSC-EGFP showed intratumoral distribution of MSC-EGFP. Survival analysis of rats bearing 9L gliomas treated with intratumoral MSC-CD and intraperitoneal 5-FC resulted in significant prolongation of survival compared with control animals. In conclusion, molecular therapy combining suicide gene therapy and MSCs as a targeting vehicle represents a potential new therapeutic approach for malignant glioma, both with respect to the antitumor potential of this system and its neuroprotective effect on normal brain tissue.     

In vivo efficacy and toxicity of 5-fluorocytosine/cytosine deaminase gene therapy for malignant gliomas mediated by adenovirus

We evaluated the therapeutic efficacy and neurotoxicity of adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for experimental malignant brain tumors. The 5-FC sensitivity in 9 L cells infected by an adenovirus vector expressing CD (AdexCACD) was increased 1700-fold compared with control cells. Rats bearing 9 L brain tumors were treated with an intratumoral injection of AdexCACD followed by intraperitoneal administration of 5-FC. The rats demonstrated remarkable inhibition of tumor growth by magnetic resonance imaging, and 7 of 10 rats survived for >90 days. To evaluate the potential side-effects of the 5-FC/CD gene therapy, rats were treated with an intracerebral injection of AdexCACD into the right basal ganglia and with 5-FC. The magnetic resonance imaging showed a highly enhanced area on the gadollinium-enhanced T1-weighted image at 18 days postinjection. Pathologically, this corresponded to an area of necrosis with surrounding apoptotic cells. In addition, there was demyelination and gliosis with enlargement of the lateral ventricles. These results suggest that the 5-FC/CD gene therapy may provide an anticancer effect for malignant brain tumors in humans, but also show that there are neurotoxic effects on normal brain tissue.     

CD与bcl-Xs基因联合转染卵巢癌细胞株对5-氟胞嘧啶作用的影响

目的　了解自杀基因胞嘧啶脱氨酶基因 (cytosinedeaminase ,CD)及其前药 5 氟胞嘧啶 (5 fluorucytosine,5 FC)和bcl Xs基因转移联合作用对卵巢癌细胞株生长的影响。方法　以复制缺陷型腺病毒为载体将CD基因和bcl Xs基因体外转染大鼠卵巢癌细胞株NUTU 19细胞 ,加入含 5 FC的培养基。MTT法检测培养细胞吸光度值 ,计算细胞存活率。结果　单一bcl Xs基因转移及单一CD /5 FC系统作用对NUTU 19细胞的生长抑制作用均随病毒滴度的增加而增大 ;将两者联合作用于NU TU 19细胞 ,其生长抑制率比两者单独使用时的生长抑制率之和更高 ,表现为协同效应 (P <0 .0 0 0 1)。结论　CD /5 FC系统与bcl Xs基因转移联合使用对NUTU 19细胞的生长抑制具有协同作用。     

Expression of the bifunctional suicide gene CDUPRT increases radiosensitization and bystander effect of 5-FC in prostate cancer cells

Purpose

To test the hypothesis that, with 5-fluorocytosine (5-FC) treatment, the co-expression of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) can lead to greater radiosensitization and bystander effect than CD-expression alone.

Methods and materials

R3327-AT cell lines stably expressing CD or CDUPRT were generated. The 5-FC and 5-FU cytotoxicity, and the radiosensitivity with/without 5-FC treatment, of these cells were evaluated under both aerobic and hypoxic conditions. The bystander effect was assessed by apoptosis staining and clonogenic survival. The pharmacokinetics of 5-FU and 5-FC metabolism was monitored in mice bearing CD- or CDUPRT-expressing tumors using ¹⁹F MR spectroscopy (MRS).

Results

CDUPRT-expressing cells were more sensitive to 5-FC and 5-FU than CD-expressing cells. CDUPRT-expression further enhanced the radiosensitizing effect of 5-FC, relative to that achieved by CD-expression alone. A 25-fold lower dose of 5-FC resulted in the same magnitude of radiosensitization in CDUPRT-expressing cells, relative to that in CD-expressing cells. The 5-FC cytotoxicity in co-cultures of parental cells mixed with 10-20% CDUPRT cells was similar to that in 100% CDUPRT cells. ¹⁹F MRS measurements showed that expression of CDUPRT leads to enhanced accumulation of fluorine nucleotide (FNuc), relative to that associated with CD-expression alone.

Conclusion

Our study suggests that CDUPRT/5-FC strategy may be more effective than CD/5-FC, especially when used in combination with radiation.     

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.     

CD自杀基因联合淋巴细胞趋化因子基因疗法的肿瘤治疗作用及免疫机理研究

本研究以腺病毒作为载体,将大肠杆菌胞嘧啶脱氨酶(CD)基因与小鼠淋巴细胞趋化因子(Ltn)基因体内联合转染,观察了其抗肿瘤效应并分析了免疫机理.小鼠皮下接种结肠腺癌CT26细胞后3天,肿瘤局部注射表达Ltn的重组腺病毒AdLtn和表达CD的重组腺病毒AdCD,然后连续10天给予5一氟胞嘧啶(5-FC)300mg/kg进行治疗,结果表明,联合治疗组荷瘤小鼠皮下肿瘤结节的生长受到明显抑制,小鼠存活期明显长于单用AdLtn治疗组或单用AdCD/5-FC治疗组.经联合治疗后小鼠脾细胞的NK活性和对(37结肠腺癌细胞的CTL杀伤活性明显增强.瘤体细胞FACS分析结果表明,经联合基因治疗后,肿瘤组织CD4~ 、CD8~ 细胞浸润增加,结肠腺癌细胞表达H-2Kd和B7-1分子明显增加.提示经CD自杀基因和Ltn基因联合治疗后,肿瘤细胞免疫原性增加.本研究结果表明联合应用自杀基因和Ltn基因治疗可以提高机体对肿瘤细胞免疫的应答,增加机体的抗肿瘤作用,是肿瘤基因治疗中一条新的途径.     

Carcinoembryonic antigen-specific suicide gene therapy of cytosine deaminase/5-fluorocytosine enhanced by the cre/loxP system in the orthotopic gastric carcinoma model

Tumor-specific gene delivery is crucial to achieving successful effects in suicide gene therapy. Carcinoembryonic antigen (CEA) promoter has been widely used for this purpose, but the expression level of tumor-specific promoters such as CEA promoter is generally low. In the previous study, we used the Cre/loxP system and showed that LacZ expression by the CEA promoter was remarkably enhanced and maintained its specificity using the Cre/loxP regulation system. In this study, the Cre/loxP system was first applied to augmentation of selective expression of the cytosine deaminase (CD) gene as a suicide gene therapy in CEA-producing cells. The double infection with AxCEANCre expressing Cre recombinase under the control of the CEA promoter and AxCALNLCD expressing the CD gene under the control of the CAG promoter by the Cre switching system rendered CEA-producing tumor cells 13-fold more sensitive to 5-fluorocytosine (5-FC) compared with the single infection with AxCEACD expressing CD gene driven by the CEA promoter. The therapeutic efficacy of the enhanced CD/5-FC suicide gene therapy was evaluated in orthotopic implantation models of human gastric carcinoma. Adenovirus vectors (1 x 10(9) plaque-forming units) were administered i.p. into mice three times, and then 5-FC was administered i.p. for the next 10 days. Tumor volume and weight in mice treated with AxCEANCre and AxCALNLCD/5-FC were significantly reduced as compared with those in mice treated not only with Mock (AxCALacZ) but also with AxCEACD/5-FC (P < 0.0001). This beneficial effect on tumor burden was also reflected in the overall survival. The survival periods of the mice treated with AxCEANCre and AxCALNLCD/5-FC were longer than those of mice treated with Mock or AxCEACD/5-FC (P < 0.01). These results suggested that application of the Cre/loxP system could provide a new approach for enhanced selective suicide gene therapy of CD/5-FC for the treatment of advanced gastric carcinoma.     

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.     

Cytosine deaminase/5-fluorocytosine gene therapy can induce efficient anti-tumor effects and protective immunity in immunocompetent mice but not in athymic nude mice

Murine hepatocellular carcinoma cells were retrovirally transduced with the bacterial cytosine deaminase (CD) gene. CD-transduced cells exhibited more than 120-fold higher sensitivity to 5-fluorocytosine (5-FC) compared with parental cells. When syngeneic immunocompetent mice were inoculated s.c. with parental hepatocellular carcinoma cells containing as little as 5% CD-transduced cells, significant inhibition of tumor formation was induced by 5-FC treatment. Furthermore, established solid tumors in immunocompetent mice containing only 5% CD-transduced cells were infiltrated markedly with CD4- and CD8+ T lymphocytes and macrophages by 5-FC treatment, such that significant reduction or even complete regression of tumors was observed. These tumor-free mice resisted subsequent rechallenge with wild-type tumor. Conversely, when athymic nude mice were inoculated with a cell mixture containing CD-transduced cells and parental cells at a ratio of 40:60, all developed tumors despite 5-FC treatment. Our results indicate that gene therapy using the CD/5-FC system can induce efficient anti-tumor effects and protective immunity in immunocompetent mice but not in athymic immunodeficient mice, suggesting that the host's immunocompetence may be a critical factor for achieving successful gene therapy against cancer.     

Enhancement of Anti-tumor Activity of Newcastle Disease Virus by the Synergistic Effect of Cytosine Deaminase

This study was conducted to investigate enhancement of anti-tumor effects of the lentogenic Newcastledisease virus Clone30 strain (NDV rClone30) expressing cytosine deaminase (CD) gene against tumor cells andin murine groin tumor-bearing models. Cytotoxic effects of the rClone30-CD/5-FC on the HepG2 cell line wereexamined by an MTT method. Anti-tumor activity of rClone30-CD/5-FC was examined in H22 tumor-bearingmice. Compared to the rClone30-CD virus treatment alone, NDV rClone30-CD/5-FC at 0.1 and 1 MOIs exertedsignificant cytotoxic effects (P<0.05) on HepG2 cells. For treatment of H22 tumor-bearing mice, recombinantNDV was injected together with 5-FC given by either intra-tumor injection or tail vein injection. When 5-FCwas administered by intra-tumor injection, survival for the rClone30-CD/5-FC-treated mice was 4/6 for 80 daysperiod vs 1/6 , 0/6 and 0/6 for the mice treated with rClone30-CD, 5-FC and saline alone, respectively. When5-FC was given by tail vein injection, survival for the rClone30-CD/5-FC-treated mice was 3/6 vs 2/6 , 0/6 and0/6 for the mice treated with rClone30-CD, 5-FC or saline alone, respectively. In this study, NDV was used forthe first time to deliver the suicide gene for cancer therapy. Incorporation of the CD gene in the lentogenic NDVgenome together with 5-FC significantly enhances cell death of HepG2 tumor cells in vitro, decreases tumorvolume and increases survival of H22 tumor-bearing mice in vivo.