Takemi Sakamoto

Expression of a retrovirally transduced gene under control of an internal housekeeping gene promoter does not persist due to methylation and is restored partially by 5-azacytidine treatment

Although expression of transgenes under the control of a retroviral long terminal repeat (LTR) promoter has been shown not to persist due to methylation, it has been observed that internal promoter may be active even if expression from the LTR promoter is silent. We constructed a retroviral vector carrying the herpes simplex virus thymidine kinase (HSVtk) gene under the control of the albumin gene promoter and transduced the HSVtk gene into hepatocellular carcinoma cells. Three of 14 mice, however, could not eradicate HSVtk-transduced grafts completely despite ganciclovir (GCV) treatment. These GCV-refractory cell lines exhibited resistance to GCV after recultivation. Subsequent Southern blot analysis revealed that the HSVtk gene was not deleted but extensively or completely methylated in GCV-refractory lines. Treatment with 5-azacytidine, a demethylating agent, partially restored the sensitivity of GCV-refractory lines to GCV. These results indicate that expression of retrovirally transduced gene may not persist in vivo due to methylation even when the gene is directed by an internal housekeeping gene promoter. These observations may also have important implications for future clinical applications of retrovirus-mediated gene therapy.

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 hosts immunocompetence may be a critical factor for achieving successful gene therapy against cancer. Int. J. Cancer 81:592–597, 1999.

Complete cure of established murine hepatocellular carcinoma is achievable by repeated injections of retroviruses carrying the herpes simplex virus thymidine kinase gene

Although xenotransplantation of retrovirus-producing cells into a tumor has been shown to be effective for the treatment of cancer, injections of recombinant retroviruses are much more feasible for clinical applications. We established a clone producing retroviruses carrying the herpes simplex virus thymidine kinase (HSVtk) gene with titers of up to 4 × 107 colony-forming units/ml, and examined the effectiveness of in vivo gene therapy against cancer. Syngeneic mice were inoculated subcutaneously with murine hepatocellular carcinoma (HCC) cells, BNL1ME A.7R.1, and the treatment was initiated after tumors were established. When mice were given an intratumoral injection of HSVtk-carrying retroviruses or their producing cells followed by ganciclovir (GCV) treatment, significantly pro- longed survival periods were observed. When mice were treated with repeated intratumoral injections of HSVtk-carrying retrovirus-producing cells, significant antitumor responses and some cures were induced by GCV treatment. Furthermore, repeated intratumoral injections of HSVtk-carrying retroviruses and GCV treatment resulted in complete regression of established HCC tumors in all animals used in the experiment. Mice that completely eradicated tumors exhibited protective immunity against wild-type HCC tumors. These results suggest that repeated injections of HSVtk-carrying retroviruses followed by GCV treatment is a potent modality for the treatment of solid tumors.

Tissue-specific expression of HSV-tk gene can induce efficient antitumor effect and protective immunity to wild-type hepatocellular carcinoma.

The efficacy of expression of the herpes simplex virus thymidine kinase (HSV‐tk) gene under the transcriptional control of the liver‐specific albumin gene promoter, followed by ganciclovir treatment, was investigated both in vitro and in vivo. Murine and rat hepatocellular carcinoma (HCC) cells infected with retroviruses carrying the HSV‐tk gene under the control of the murine albumin gene promoter were selectively killed by ganciclovir treatment in vitro, whereas non‐HCC cells, such as murine mammary tumor cells and fibroblast cells, which were infected with the same retroviruses, were not. Susceptibility of the retroviral‐infected HCC cells to ganciclovir was more than 100‐fold higher than that of the retroviral‐infected non‐HCC cells. When mice bearing a bulky HCC mass consisting of the retroviral‐infected HCC cells were treated with systemic ganciclovir administration, complete regression of the tumors was observed without any signs of overt toxicity. Profound antitumor effects on preestablished murine HCCs were observed when wild‐type HCC cells were implanted into animals with a small percentage of the retroviral‐infected counterparts. When only 5% of the cells were infected with retroviruses carrying the HSV‐tk gene, significant inhibition of tumor development was observed with systemic ganciclovir treatment. Importantly, animals that were treated with implantation of mixtures of the retroviral‐infected and parental HCC cells, followed by ganciclovir administration, did not exhibit tumor formation and resisted subsequent rechallenge with wild‐type HCC cells. Our results indicate the feasibility of combination therapy with the HSV‐tk gene and ganciclovir for the treatment of HCC. Int. J. Cancer 71:470‐475, 1997.

Gene therapy for the treatment of hepatoma by retroviral-mediated gene transfer of the herpes simplex virus thymidine kinase

Abstract We previously demonstrated that a foreign gene transferred by means of a retroviral vector can be expressed selectively in hepatoma cells when a liver-specific promoter was used to direct its expression. We now describe an approach for the treatment of hepatoma by the introduction of herpes simplex virus thymidine kinase (HSV-TK) gene. Expression of HSV TK gene in hepatoma cells was evaluated as an elimination system for a potential use in therapies. A murine retroviral vector was constructed in which the HSV-TK gene was expressed under control of the murine albumin enhancer and promoter elements. Replication-defective vector viral particles were obtained by transfer of the vector DNA into the ecotropic packaging cell line psi2 and were used to infect murine hepatoma cells. The introduction of the HSV-TK gene into hepatoma cells by infection of the recombinant retrovirus did not affect their proliferation at all. The sensitivity of those infected cells to the toxic effects of the nucleoside analog ganciclovir was found to be significantly increased by transfer of the HSV-TK gene. The difference in sensitivity between infected and uninfected cells to ganciclovir concentrations should give the utility for a clinical application indicating the feasibility of gene therapy toward hepatoma by the retroviral-mediated HSV-TK gene transfer.

Construction of retroviral vectors to induce strong hepatoma cell-specific expression of cytokine genes

Continuing advances in molecular biology have provided tools for a promising approach to the treatment of cancer. Among the various strategies of gene therapy for cancer, many are aimed at killing tumour cells indirectly by the induction or reinforcement of a host immune response by gene transduction of various cytokines, major histocompatibility complex or immune accessory molecules. In the present study, we selected the tumour necrosis factor‐α, interleukin‐2 and interleukin‐3 genes as potential cytokine genes to induce antitumour effects. We constructed retroviral vectors carrying these cytokine genes under the control of the murine albumin enhancer and promoter and retrovirally transduced these genes into hepatoma and non‐hepatoma cell lines. Strong expression of the cytokine genes was induced in transduced hepatoma cells, while no evident expression was detected in transduced non‐hepatoma cells. These results demonstrate the hepatoma‐specific expression of cytokine genes and imply the feasibility of in vivo gene transfer into hepatomas without affecting any other tissues. Furthermore, these cytokine genes were expressed much more intensively when they were derived from the albumin enhancer and promoter than when derived from the simian virus 40 early region promoter. These results indicate that transcriptional regulatory sequences specific for the target tissues could be preferable to viral promoters for the gene therapy of cancer.

Interferon treatment of chronic hepatitis C in patients with hemophilia or von Willebrand's disease in Japan

Seven patients with chronic hepatitis C, six hemophiliacs and a patient with von Willebrands disease, were treated with interferon-alpha (IFN-alpha). Either 9 MU of recombinant IFN-alpha 2a or 3 MU of lymphoblastoid alpha-IFN was administered daily for 2 weeks and then three times a week for 22 weeks. Liver histology, hepatitis C virus (HCV) genotypes, and HCV-RNA levels in sera were investigated in all of the patients before IFN therapy was instituted. Liver histology was classified by the European classification. HCV genotyping conformed to the so-called Okamotos classification. HCV-RNA levels in sera were quantitated by competitive polymerase chain reaction, using mutant RNA. Liver histology, HCV genotype, and serum HCV-RNA level (copies/ml) in each patient were: patient 1, chronic persistent hepatitis, type II, 3×103 respectively; patient 2, chronic active hepatitis (CAH) 2a, type III, 6×104; patient 3, CAH2a, type IV, 2×105; patient 4, CAH2b, type I, 2×107; patient 5, CAH2b, type II, 8×104; patient 6, CAH2b, type III, 7×106; and patient 7, CAH2b, type IV, 1×107. Sustained elimination of HCV was achieved in patient 3 and temporary elimination was achieved in patients 1 and 2. The other patients showed persistent HCV-RNA positivity in sera both during and after IFN treatment. Poor responsiveness to IFN was observed in patients with relatively progressive liver histology and high levels of HCV viremia.

Bacterial cytosine deaminase suicide gene transduction renders hepatocellular carcinoma sensitive to the prodrug 5-fluorocytosine

Abstract One of the most promising approaches to the treatment of cancer is to render tumor cells susceptible to normally non-toxic chemotherapeutic agents utilizing so-called ‘suicide genes’. The herpes simplex virus thymidine kinase gene is a prototypic suicide gene and clinical trials of gene therapy using it have already been approved for the treatment of glioma and ovarian cancer. We previously demonstrated that transduction of the herpes simplex virus thymidine kinase gene into hepatocellular carcinoma (HCC) cells resulted in the selective killing of transduced cells by ganciclovir treatment. There is, however, another potential suicide gene. Bacterial cytosine deaminase (CD) converts the innocuous compound 5-fluorocytosine (5-FC) to the highly toxic compound 5-fluorouracil (5-FU). Attractively, both compounds have been clinically available so that much is known of their pharmacokinetics. We transferred the bacterial CD gene with a retroviral vector into HCC cells and determined the sensitivity to 5-FC. Transduced HCC cells were susceptible to 5-FC toxicity in a dose-dependent manner and exhibited 0% survival at a 5-FC concentration of 100 μg/ml, while untransduced cells exhibited nearly 100% survival at the same concentration. Sensitivity of the former cells to 5-FC was approximately 100-fold higher than the latter cells, indicating the feasibility of this strategy for the treatment of HCC. Furthermore, it was demonstrated that bacterial CD genetransduced HCC cells eliminated untransduced cells in the presence of 5-FC whose concentration was essentially non-toxic to untransduced cells. This phenomenon, the so-called ‘bystander effect’, provides therapeutic advantages for gene therapy for cancers, even if it is impossible to introduce a foreign gene into all target cells of a cancer. These results demonstrate that retroviral-mediated bacterial CD gene transfer has potential for the treatment of HCC.

Autoimmunity during alpha-interferon therapy for chronic hepatitis C

SummaryOne hundred twenty-five patients with chronic hepatitis C were treated with natural IFNα or recombinant IFNα-2a, daily doses of 3 MU or 9MU, respectively. IFNs were given 6 times a week for the first 2 weeks followed by thrice weekly administration for 12 weeks or more. ANA, TMA, AMA and anti-DNA antibody newly developed in 8, 1, 2 and 1 patient, respectively. Furthermore, we encountered some cases in which underlying autoimmune disorders were throught to be exacerbated by IFNs. It is important to pay sufficient attention to the development of autoimmune diseases in IFN therapy for chronic hepatitis C.

A case of early gastric malignant lymphoma diagnosed and completely resected by strip biopsy

A case of early gastric malignant lymphoma definitively diagnosed by strip biopsy is reported. The subsequent operation revealed that the strip biopsy had resulted in radical resection. A 55-year-old woman visited our hospital for detailed examination of a small gastric lesion. Histologic findings of the specimens obtained by conventional forceps biopsy indicated reactive lymphoid hyperplasia, although the possibility of malignant lymphoma was not completely ruled out. Strip biopsy was, therefore, performed to establish a definitive diagnosis. Histopathological examinations of the strip biopsy specimen revealed definitive findings of malignant lymphoma, which was B-cell phenotype immunocytochemically. The margin of the resected specimen was free of invasion by malignant lymphoma and no lymph node involvement was suggested by endoscopic ultrasonography, computed tomography, and gallium scintigram. Subtotal gastrectomy was subsequently performed to rule out the possibility of remaining malignant lymphoma cells. It was proven that the strip biopsy removed the lesion completely and no perigastric lymph nodes were involved. While is still controversial as to whether strip biopsy should be adopted for the radical resection of early gastric lymphoma, this procedure can definitely provide excellent specimens for the accurate diagnosis of gastric malignant lymphoma and probably for group III lesions in the stomach.