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Combination therapy with replicative oncolytic viruses is a recent topic in innovative cancer therapy, but few studies have examined the efficacy of oncolytic adenovirus plus replication-deficient adenovirus carrying a suicide gene. We aim to evaluate whether an E1A, E1B double-restricted oncolytic adenovirus, AxdAdB-3, can improve the efficacy for gallbladder cancers (GBCs) of the replication-deficient adenovirus-based herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) therapy directed by the carcinoembryonic antigen (CEA) promoter. Cytopathic effects of AxdAdB-3 plus AxCEAprTK (an adenovirus expressing HSVtk directed by CEA promoter) or AxCAHSVtk (an adenovirus expressing HSVtk directed by a nonspecific CAG promoter) with GCV administration were examined in several GBC lines and normal cells. Efficacy in vivo was tested in severe combined immunodeficiency disease mice with GBC xenografts. Addition of AxdAdB-3 (1 multiplicity of infection, MOI) significantly enhanced the cytopathic effects of AxCEAprTK (10 MOI)/GCV on GBC cells. The augmented effect was attributable to the replication of the AxCEAprTK and also to the enhanced CEA promoter activity, which was presumably transactivated by E1A. In normal cells, AxdAdB-3 (20 MOI) plus AxCEAprTK (200 MOI)/GCV was not cytopathic, whereas AxdAdB-3 (1 MOI) plus AxCAHSVtk (10 MOI)/GCV was significantly toxic. Low-dose AxdAdB-3 (2 x 10(7) PFU, plaque-forming unit) plus AxCEAprTK (2 x 10(8) PFU)/GCV significantly suppressed the growth of GBC xenografts as compared with either AxdAdB-3 (2 x 10(7) PFU)/GCV or AxCEAprTK (2 x 10(9) PFU)/GCV alone. E1A, E1B double-restricted replicating adenovirus at low dose significantly augmented the efficacy of CEA promoter-directed HSVtk/GCV therapy without obvious toxicity to normal cells, suggesting a potential use of this combination for treating GBC and other CEA-producing malignancies.

An adenovirus (Adv) retaining normal E1A but lacking the 55 kDa E1B protein replicates preferentially in TP53-deficient cancer cells including pancreatic cancer cell lines, resulting in the oncolysis of the tumor. When tumor cells are exposed to hypoxia, hypoxia-inducible factor-1alpha (HIF-1alpha) is stabilized and activated to promote the transcription of several genes such as vascular endothelial growth factor (VEGF), but in the presence of E1A hypoxia-induced VEGF m-RNA synthesis is inhibited by E1A binding to p300. In this study, we demonstrated that the cancer cells infected with a mutant Adv in which the p300 binding site in E1A was partially deleted induced a higher expression level of VEGF as compared to those of Adv with normal E1A. An immunoprecipitation study for E1A confirmed that mutant E1A had a reduced binding capacity for p300. Although the expressions of HIF-1alpha m-RNA were almost the same in both cancer cells infected with the mutant Adv and those with the wild Adv, the amount of HIF-1alpha protein in cancer cells infected with the wild E1A Adv was lower than in those infected with the mutant E1A type Adv. In vivo, in contrast to the angiogenesis treated with mutant E1A, wild-E1A inhibited tumor angiogenesis significantly. These results suggested that E1A suppressed the production of VEGF and inhibited tumor angiogenesis by binding with p300, resulting in the inhibition of the HIF-1alpha-mediated transcription of genes through binding to HRE. This study demonstrates, for the first time, the effect of an oncolytic replication-competent Adv in inhibiting tumor angiogenesis.

Radiation therapy is an established modality for the treatment of malignant gliomas. Several reports have shown the advantage of additional radiation in combination with gene therapy. In this study, we investigated the ability of radiation therapy to enhance 5-fluorocytosine (5-FC)/cytosine deaminase (CD) plus uracil phosphoribosyltransferase (UPRT) gene therapy in malignant gliomas. In vitro study suggested evidence of a significant cytotoxic interaction between radiation therapy and 5-FC/CD + UPRT gene therapy for glioma cells. In vivo experiments demonstrated that the combination of gene therapy and radiation possessed superior antitumor effect in comparison to single therapy. However, the adverse effects of radiation therapy in combination with the gene therapy were observed with respect to normal brain. This combination therapy may be feasible for the treatment of gliomas, although the radiation dose and area should be reduced in order to prevent side effects.

Advanced esophageal cancers are highly malignant and frequently resistant to 5-fluorouracil (5-FU). Escherichia coli uracil phosphoribosyltransferase (UP) is a pyrimidine salvage enzyme that alters 5-FU metabolism and sensitivity. A recombinant adenovirus encoding the UP gene (AxCA.UP) has been applied in gastric cancer gene therapy to sensitize cancer cells to lower concentrations of 5-FU. We have generated a recombinant adenovirus (AxCA.UT) encoding UP and herpes simplex virus thymidine kinase fusion protein (UT) to examine whether it would enhance the antitumor activity of AxCA.UP treatment. AxCA.UT treatment significantly enhanced the sensitivity of human esophageal cancer cells to and significantly enhanced the growth inhibition effects of UP gene therapy in vitro. Moreover, both 5-FU and ganciclovir showed bystander effects on growth inhibition. In an in vivo study, the therapeutic outcome of AxCA.UT treatment significantly enhanced the antitumor activity of AxCA.UP treatment. These observations suggest that AxCA.UT may be useful in esophageal cancer gene therapy.

Gliomas express a higher amount of Fas than normal brain tissue. It is of interest to know whether expression of the Fas receptor is unfavorable to the antiapoptotic pathways in gliomas. In this study, we introduced the Fas gene via an adenovirus vector (Adeno-Fas) into the A-172, U251, and U-373 MG glioma cell lines, each of which expresses Fas on the cell surface. Infection of Adeno-Fas induced apoptosis in each glioma cell line. In U251 cells and A-172 cells that express the same level of Fas as a result of infection with Adeno-Fas, a much higher percentage of U251 cells underwent apoptosis than did A-172 cells. This suggests that each glioma cell line has its own threshold of Fas expression, above which apoptosis is induced, and that the constitutive expression of Fas is below the level of this threshold. It was found that the constitutive expression of the anti-apoptotic gene Bcl-X(L) is higher in A-172 cells than in U251 cells. Adenovirus-mediated transduction of the Bcl-X(L) gene into U251 cells effectively suppressed Adeno-Fas-mediated apoptosis. These data indicate that the Bcl-X(L) gene is one of the important determinants of the threshold for Fas-mediated apoptosis. When U251 and U-373 MG cells were transduced with the Fas gene controlled by the myelin basic protein promoter, which had been shown to be active in gliomas but not in neural tissues, the cells underwent markedly enhanced apoptosis. Taken together, these results indicate that the overexpression of Fas alone induced apoptosis in each glioma cell line. The degree of Fas-mediated apoptosis was attenuated by the expression of an anti-apoptotic gene, Bcl-X(L). The adenovirus-mediated induction of Fas gene controlled by a tissue-specific promoter (e.g., myelin basic protein promoter) would be a promising therapeutic approach for malignant glioma.

Current clinical success rates of adenoviral vector (Adv)-based gene therapy of squamous cell carcinoma (SCC) of the head and neck remain unsatisfactory. A major problem with this approach is thought to be related to low Adv transduction efficiency due to weak expression of the adenovirus receptor, coxsackie-adenovirus receptor (CAR), in SCC. To improve the limited infectivity of Adv in oral SCC, we constructed mutated Adv incorporating the integrin-binding motif, RGD, in the HI loop of the fiber knob. The mutated Adv infected target cells through integrins commonly expressed in oral SCC. LacZ marker gene expression after infection with this mutated Adv (Adv-F/RGD) in oral SCC cell lines that showed reduced expression of CAR was approximately 5-10 times higher than that obtained with the parental Adv containing wild-type fiber knob (Adv-F/wt). In an in vitro study, transduction of oral cancer cell lines with Adv-F/RGD expressing human IL-2 (AxCAhIL2-F/RGD) resulted in greater production of cytokine than AxCAhIL2-F/wt infection. In an in vivo therapeutic xenograft model of oral SCC in nude mice, AxCAhIL2-F/RGD demonstrated antitumor effects superior to those of AxCAhIL2-F/wt. These data suggest that exploitation of genetically altered adenovirus vectors with integrin-binding motifs may offer significant improvements in oral SCC gene therapy.

The combined therapeutic effect of cytokine-producing cancer cell vaccines and local radiotherapy was studied in a mouse glioma 261 (GI261) brain tumor model. Brain tumor-bearing mice were treated with cytokine (IL -4, IL-6, IL-7, GM-CSF, TNF-alpha, LIF, LT) producing vaccines made by in vitro transduction of GI261 cells with the corresponding adenoviral vectors. Vaccines producing either IL-4 or GM-CSF cured 20-40% of mice. The antitumor effect strongly depended on the secreted cytokine level. Vaccination therapy induced specific activation of cytotoxic T lymphocytes measured by cell-mediated cytotoxicity assay. Brain tumors were heavily infiltrated by CD4+ lymphocytes after treatment with IL-4- or GM-CSF-secreting cells. GM-CSF vaccination induced moderate CD8+ infiltration, as well. Depleting either CD4+ or CD8+ lymphocyte subsets abolished the anticancer effect of GM-CSF-expressing cells. Strong synergism was observed by combining cytokine vaccination (GM-CSF, IL-4, IL-12) with local tumor irradiation: about 80-100% of the glioma-bearing mice was cured. The high efficiency of combined treatment was maintained even under suboptimal conditions when neither of the modalities cured any of the mice alone. This suggests that vaccination therapy might open a new potential in the clinical treatment of high-grade gliomas when applied as adjuvant to existing treatment modalities.

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.

The transduction of Bax protein, which is up-regulated in radiation- and chemotherapy-induced apoptosis, augments the cytotoxicity of radiotherapy and chemotherapy for cancers. The cytotoxicity of Bax overexpression is caused primarily by mitochondrial dysfunction, which is also involved in the apoptosis triggered by caspase-8. In this study, we transduced the Bax gene in combination with caspase-8 gene to evaluate whether or not this approach induces effective cytotoxicity in glioma cells. In terms of cancer gene therapy, it is critically important to induce cytotoxic genes in a cancer-specific manner. Therefore, we used the myelin basic protein promoter to drive cytotoxic genes. The expression level controlled by the myelin basic protein promoter was relatively low in gliomas. In U251 and U-373 MG glioma cells, adenovirus-mediated transduction of the Bax gene combined with caspase-8 gene induced enhanced apoptosis and cell death as determined by morphological analysis and assay for dead cells, hypodiploid cells, and DNA fragmentation (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling method). This therapeutic modality would be useful to induce a specific and enhanced cytotoxic effect for gliomas.

Various therapeutic approaches toward killing glioma cells by inducing apoptosis have been developed, but these approaches are often hampered by anti-apoptotic mechanisms. In this study, we attempted to develop a technique that overrides the resistance toward apoptosis in glioma cells. To date, p53- and Fas-mediated apoptotic pathways have been shown to be different. Therefore, we carried out a gene therapy that combines the pro-apoptotic effect of these two different pathways. The recombinant adenoviruses (Advs) for p53 and Fas ligand (FL) (Adv-p53 and Adv-FL, respectively) were constructed. Transfecting the p53 gene into glioma cell lines (A-172 and U251 glioma cells) led to overexpression of Bax, a protein that induces permeability transition; at the same time, this transfection brought about an overexpression of Fas. To intensify Fas-mediated apoptosis, we transferred the FL gene together with the p53 gene by Adv-mediated gene transduction into A-172 and U251 cells. Coinfecting Adv-p53 and Adv-FL into A-172 cells, which are relatively resistant to apoptosis by infection with Adv-p53 or Adv-FL alone (Adv-p53, multiplicity of infection (MOI) of 100: 8.5 +/- 0.7%; Adv-FL, MOI of 100: 3.0 +/- 0.1%) resulted in a drastic enhancement of the percentage of apoptotic cells (Adv-p53 and Adv-FL, each at an MOI of 30: 24.2 +/- 0.9%). Coinfection with Adv-p53 and Adv-FL in U251 cells resulted in a similar enhancement of the percentage of apoptotic cells (Adv-p53 and Adv-FL, each at an MOI of 30: 59.0 +/- 2.3%) compared with that seen in U251 cells transfected with Adv-p53 or Adv-FL alone (Adv-p53, MOI of 30: 3.1 +/- 0.3%; Adv-FL, MOI of 30: 18.1 +/- 0.3%). Regardless of whether a cell line is resistant or sensitive to FL- and p53-mediated apoptosis, coinfection with Adv-p53 and Adv-FL dramatically enhanced the degree of apoptosis of glioma cells. Our results indicate that the coinfection approach can be used as a modality for the gene therapy of gliomas, overriding the apoptosis-resistant mechanisms in glioma cells.

Uracil phosphoribosyltransferase (UPRT) of Escherichia coli origin can convert 5-fluorouracil (5-FU), a chemotherapeutic agent widely used for solid tumors, to an active intermediate, 5-fluorouridine-5'-monophosphate, as mammalian orotate phosphoribosyltransferase does. To examine whether the E. coli UPRT gene expressed in tumor cells can confer increased sensitivity to 5-FU, we retrovirally transduced Colon 26 cells, a murine colon carcinoma cell line, with the UPRT gene (Colon 26/UPRT cells) and tested the in vivo antitumoral effect of 5-FU in syngeneic immunocompetent mice. After 5-FU administration, tumors of Colon 26/UPRT cells regressed, whereas those of wild-type cells were unaffected. The mice that once eliminated Colon 26/UPRT tumors after 5-FU treatment rejected wild-type cells that were subsequently inoculated but not irrelevant syngeneic tumor cells. This suicide gene/prodrug system was less efficient in nude mice, suggesting that mature alphabeta T cells play a role in the antitumoral effect. The cytotoxicity mediated by the bystander effect was marginal in this system, contrary to the herpes simplex virus-thymidine kinase gene/ganciclovir system. Therefore, expression of the UPRT gene in tumor cells followed by 5-FU administration is a possible strategy for cancer gene therapy, but potentiation of the bystander effect is required for its therapeutic application.

Suicide gene therapy has been studied intensively for the treatment of cancer. A limited antitumoral effect was obtained by intratumoral injection of adenovirus harboring Escherichia coli cytosine deaminase gene (AdCD) in tumor-bearing mice followed by continuous administration of 5-fluorocytosine (5FC). To address the drawbacks of the limited potential for the induction of antitumoral immunity by CD suicide gene therapy, we hypothesized that antigen-presenting cells (APCs) might contribute to the efficient induction of an antitumoral immune response in tumor-bearing mice undergoing suicide gene therapy. We preinjected the mice with murine stem cell factor (SCF)-encoding adenovirus (AdSCF) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-encoding adenovirus (AdGM-CSF); after 7 days, the mice were inoculated with CT26 colon adenocarcinoma. AdCD was injected intratumorally into tumor-bearing mice followed by 5FC administration. The results showed that AdSCF/AdGM-CSF treatment could increase the number, surface molecule expression, and function of APCs efficiently. A more significant growth inhibition of established tumors and a prolongation of the survival period were observed in tumor-bearing mice after AdSCF/AdGM-CSF pretreatment in combination with AdCD/5FC therapy when compared with mice treated with AdSCF or AdGM-CSF in combination with AdCD/5FC, or AdCD/5FC alone (P.01). Cytotoxic T-lymphocyte activity was induced efficiently after the combined therapy, and mRNA of tumor necrosis factor-alpha, interleukin-4, interferon-gamma, and interleukin-2 was present in the tumor mass after combined therapy, suggesting that a more potent antitumoral response was induced by enhanced APCs. Our results demonstrated that AdSCF/AdGM-CSF pretreatment could activate APCs, and that these APCs could present the tumor antigens released from AdCD/5FC-killed tumor cells and activate the antitumoral response of the host, thus increasing the therapeutic efficiency of suicide gene therapy.