Masaji Kikukawa

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.

Effective and safe gene therapy for colorectal carcinoma using the cytosine deaminase gene directed by the carcinoembryonic antigen promoter

We have recently isolated carcinoembryonic antigen (CEA) promoter regions consisting of 419 bp and 204 bp from CEA-producing human colorectal carcinoma (CRC). We constructed CEA419/CD and CEA204/CD retroviruses carrying the bacterial cytosine deaminase (CD) gene directed by the CEA promoter regions. pCD2 retroviruses carrying the CD gene directed by the retrovirus long terminal repeat promoter were also used. CEA419/CD or CEA204/CD retrovirus-infected CRC cells were found to be susceptible to 5-fluorocytosine (5-FC), while non-CRC cells infected with the same retroviruses were not. CD-transduced CRC xenografts in nude mice were sensitive to 5-FC treatment, resulting in arrest of tumor growth. When mice with intraperitoneally disseminated CRCs were given intraperitoneal injections of CEA419/CD retrovirus-producing cells followed by 5-FC treatment, significantly prolonged survival rates were observed compared with animals injected with pCD2 retrovirus-producing cells followed by 5-FC treatment. Importantly, bone marrow suppression was not observed in animals injected with CEA419/CD retrovirus-producing cells and 5-FC, while profound bone marrow suppression was observed in those injected with pCD2 retrovirus-producing cells and 5-FC. These results indicate that effective and safe in vivo gene therapy for advanced CRC may be feasible by transferring the CD gene controlled by the CEA promoter followed by 5-FC treatment.

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.

Hepatocellular carcinoma in an orthotopic mouse model metastasizes intrahepatically in cirrhotic but not in normal liver

Prognosis of hepatocellular carcinoma (HCC) still remains poor mainly because of intrahepatic metastasis. In the majority of cases, HCC is found in conjunction with liver cirrhosis. It is, therefore, of great importance to investigate the invasive and metastatic behavior of HCC in cirrhotic liver. To examine this, a liver cirrhosis model was produced by injecting thioacetamide i.p. into mice. Murine HCC cells were labeled with the fluorescent carbocyanine dye, DiI, and implanted directly under the capsule of cirrhotic and normal livers of syngeneic mice. DiI‐labeled HCC cells in the liver were observed under fluorescent and confocal microscopy. Histological analysis of cirrhotic and normal livers revealed that implanted HCC cells migrated to and invaded the adjacent periportal regions, but not the adjacent centrolobular areas. This characteristic behavior of HCC was more evident in cirrhotic liver than in normal liver. Furthermore, intrahepatic metastasis to unimplanted hepatic lobes was observed in cirrhotic liver as early as 7 days after implantation, while it was not detected in normal liver even 4 weeks later. Thus, an orthotopic animal model for HCC with cirrhosis described here may be suitable for investigating the invasive and metastatic behavior of HCC. Importantly, labeling tumor cells with a fluorescent dye before orthotopic implantation may be a convenient and useful method to investigate the invasive and metastatic behavior of various types of cancer. Int. J. Cancer 80:471–476, 1999.

Pancreatic pleural effusion with a pancreaticopleural fistula diagnosed by magnetic resonance cholangiopancreatography and cured by somatostatin analogue treatment.

AbstractA 69-year-old man with chronic alcoholic pancreatitis developed a left-sided massive pleural effusion. Magnetic resonance cholangiopancreatography clearly demonstrated the pancreatic cyst and the fistula connecting the cyst with the left pleural cavity, resulting in the diagnosis of pancreatic pleural effusion with a pancreaticopleural fistula. Conservative somatostatin analogue treatment completely eradicated the pancreatic pleural effusion and closed the pancreaticopleural fistula.

COMPARISON OF CARCINOEMBRYONIC ANTIGEN PROMOTER REGIONS ISOLATED FROM HUMAN COLORECTAL CARCINOMA AND NORMAL ADJACENT MUCOSA TO INDUCE STRONG TUMOR-SELECTIVE GENE EXPRESSION

To establish in vivo gene therapy against cancer, it is requisite to induce strong, cancer cell‐selective expression of a therapeutic gene. Comparison of the promoter activity of 5′ flanking regions of the carcinoembryonic antigen (CEA) gene isolated from various origins is therefore of considerable interest. The 5′ flanking region of the CEA gene between −135 and +69 bp upstream from the transcriptional start site, which is recognized as the core promoter region, was isolated from CEA‐producing human colorectal carcinoma (CRC), normal adjacent mucosa, CEA‐producing cell lines and CEA‐non‐producing cell lines. No mutations were observed by single‐strand conformation polymorphism in the CEA promoter regions. Subsequent sequence analysis revealed that there were no mutations in the CEA promoter regions isolated from CEA‐producing CRC and normal adjacent mucosa. Furthermore, nuclear extracts prepared from CEA‐producing human CRC cells could equally bind to both the CEA promoter fragments isolated from CEA‐producing CRC and normal mucosa. Both CEA promoter regions could direct 5‐ to 20‐fold higher expression of a luciferase reporter gene in CEA‐producing cells than in CEA‐non‐producing cells. Therefore, we suggest that the use of either CEA promoter region isolated from CRC or normal mucosa is equally effective to induce strong, CEA‐producing cancer‐selective expression of a therapeutic gene. Int. J. Cancer 78:242–247, 1998.© 1998 Wiley‐Liss, Inc.

Transient cyclophosphamide treatment before intraportal readministration of an adenoviral vector can induce re-expression of the original gene construct in rat liver

Although adenovirus is an attractive vehicle for transferring therapeutic genes in vivo, animal studies have indicated that the clinical usefulness of adenoviruses may be limited by their immunogenicity. Although immunosuppressive strategies around the time of initial exposure of adenoviruses have been shown to prevent the formation of neutralizing antibodies and permit the successful readministration of adenoviruses in animals, the practicality of the approaches remains questionable. Because the majority of prospective gene therapy patients have already been infected with wild-type adenoviruses, initial treatment with adenoviruses in humans may correspond to readministration of adenoviruses into animals. It is shown here that although intraportal infusion of adenoviruses carrying a reporter lacZ gene resulted in transient high levels of transgene expression in the rat liver, intraportal readministration of adenoviruses failed to induce detectable levels of transgene expression. Conversely, when animals were treated transiently with cyclophosphamide before the intraportal readministration of adenoviruses, development of neutralizing antibodies and antigen-specific T cell proliferation in response to adenoviral readministration was significantly suppressed and successful re-expression of the transgene was achievable. These results may have important implications for efficacy considerations when adenoviral vectors are employed in clinical settings.

Interaction of Kupffer cells to splenic macrophages and hepatocytes in endotoxin clearance: Effect of alcohol

An additional administration of high dose ethanol to chronic alcohol‐fed rats led to a decrease in endotoxin clearance and an increase in endotoxin accumulation in the spleen accompanied by an elevation of tumour necrosis factor (TNF) levels in the portal vein. Endotoxin uptake and TNF production by Kupffer cells (KC) and splenic macrophages in the chronic ethanol load rats were significantly greater than those in the control rats. When these cells were precultured in the medium containing 10 to 100mmol/L ethanol, the endotoxin uptake and TNF production of KC were decreased. However, this did not affect the endotoxin uptake and TNF production of splenic macrophages.