OBJECTIVETo develop a new kind of vector system called gene-viral vector, which combines the advantages of gene and virus therapies.

METHODSUsing recombinant technology, an anti-tumor gene was inserted into the genome of replicative virus specific for tumor cells. The cell killing effect, reporter gene expression of the green fluorescence protein, anti-tumor gene expression of mouse interleukin-12 (mIL-12) and replication of virus were observed by the methods of cell pathology, fluorescence microscopy, ELISA and electron microscopy, respectively.

RESULTSA new kind of gene-viral vector system of adenovirus, in which the E1b-55 kD gene was deleted but the E1a gene was preserved, was constructed. The vector system, like the replicative virus ONYX-015, replicated and proliferated in tumor cells but not in normal ones. Our vector had an advantage over ONYX-015 in that it carried different kinds of anti-tumor genes to enhance its therapeutic effect. The reporter gene expression of the green fluorescence protein in tumor cells was much better than the adenovirus vector employed in conventional gene the rapy, and the expression in our vector system was as low as or even less than that in the conventional adenovirus gene therapy system. Similar results were observed in experiments with this vector system carrying the anti-tumor gene mIL-12. Replication and proliferation of the virus carrying the mIL-12 gene in tumor cells were confirmed by electron microscopy.

CONCLUSIONSGene-viral vectors are new vectors with an anti-tumor gene inserted into the genome of replicative virus specific for tumor cells. Because of the specific replication and proliferation of the virus in tumor cells, expression of the anti-tumor gene is increased hundreds to thousands of times. This approach takes full advantages of gene therapy and virus therapy to enhance the effect on the tumor. It overcomes the disadvantages of conventional gene therapy, such as low transfer rate, low gene expression, lack of target tropism, and low anti-tumor activity. We believe that this is a promising means for future tumor treatment.

Adenoviridae ; genetics ; Adenovirus E1A Proteins ; genetics ; Adenovirus E1B Proteins ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Humans ; Interleukin-12 ; genetics ; Neoplasms ; therapy ; Recombination, Genetic ; Tumor Cells, Cultured ; Virus Replication

BACKGROUND: The association of simian virus 40 (SV40) with certain types of human cancers, including malignant lymphomas, has been a topic of interest for some time. Although the virus is distributed worldwide, its incidences vary according to the specific types of tumors, and the epidemiological areas. The aim of this study was to investigate the frequency of SV40 in malignant lymphomas among Korean patients. METHODS: One hundred seventy three cases of malignant lymphomas were evaluated by immunohistochemical staining for SV40 large T antigen (TAg), using an extremely sensitive, tyramide based, catalyzed signal amplification method. RESULTS: From 158 non-Hodgkin's lymphomas, including 115 diffuse large B-cell lymphomas, and 15 Hodgkin's lymphomas, none of the cases were positive for SV40 TAg. CONCLUSIONS: SV40 does not appear to be related to the pathogenesis of malignant lymphomas among Koreans.
Antigens, Polyomavirus Transforming ; Antigens, Viral, Tumor ; Hodgkin Disease ; Humans ; Incidence ; Korea ; Lymphoma ; Lymphoma, B-Cell ; Lymphoma, Non-Hodgkin ; Simian virus 40 ; Viruses

BACKGROUNDOur previous studies have demonstrated potent oncolysis efficacy of the E1A, E1B double-restricted replication-competent oncolytic adenovirus AxdAdB-3 for treatment of bladder cancer. Here, we reported the feasibility and efficacy of AxdAdB-3 alone, or in combination with gemcitabine for treating renal cell carcinoma.

METHODSCytopathic effects of AxdAdB-3 were evaluated in human renal cell carcinoma cell lines TOS-1, TOS-2, TOS-3, TOS-3LN, SMKT-R3, SMKT-R4 and ACHN, and in normal human renal proximal tubule epithelial cells (RPTEC). AxdAdB-3 induced down-regulation of the cell cycle was determined by flow cytometry. Combination therapies of AxdAdB-3 with gemcitabine were evaluated in vitro and in vivo on subcutaneous TOS-3LN tumors in a severe combined immunodeficiency disease (SCID) mouse model.

RESULTSAxdAdB-3 was potently cytopathic against the tested most renal cell carcinoma cell lines including TOS-2, TOS-3, TOS-3LN, SMKT-R3 and SMKT-R4, while normal human RPTEC were not destroyed. AxdAdB-3 effectively induced cell cycle S-phase entry. Combined therapy of AxdAdB-3 with gemcitabine demonstrated stronger antitumor effects in vitro and in vivo compared with either AxdAdB-3 or gemcitabine alone.

CONCLUSIONAxdAdB-3 alone, or in combination with gemcitabine may be a promising strategy against renal cell carcinoma.

Adenoviridae ; genetics ; metabolism ; physiology ; Adenovirus E1A Proteins ; genetics ; Adenovirus E1B Proteins ; genetics ; Animals ; Antimetabolites, Antineoplastic ; pharmacology ; therapeutic use ; Carcinoma, Renal Cell ; drug therapy ; therapy ; Cell Cycle ; drug effects ; genetics ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Coxsackie and Adenovirus Receptor-Like Membrane Protein ; Deoxycytidine ; analogs & derivatives ; pharmacology ; therapeutic use ; Flow Cytometry ; Humans ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred BALB C ; Mice, SCID ; Oncolytic Virotherapy ; Receptors, Virus ; genetics ; metabolism ; Xenograft Model Antitumor Assays

No abstract available.
Antigens, Viral, Tumor* ; Simian virus 40*

The coeneal endothelium is essential for the maintenance of normal corneal hydration, thickness, and transparency. However, corneal endothelial cells are incapable of significant proliferation in vivo. As we age, the density of corneal endothelial (CEN) cells gradually decreases. The goal of our study is to explore the possibility of enhancing the proliferation of corneal endothelial cells by introduction of SV 40 large T antigen, a transforming protein. To this end, introduction of protein into CEN cells was assessed by liposome assisted beta-galactosidase transfection in vivo, ex vivo, and in vivo. In all cases, cells treated with liposome-protein complex have shown dramatic blue stain in beta-galactosidase activity staining. This result convinced us that we could artificially introduce a foreign protein into a cell. To ascertain where SV 40 large T antigen is localized in the cell, purified SV 40 large T antigen was transfected into the cells using liposome and its presence was determined immunohistochemically. We show that the liposome delivered SV 40 large is localized in the nucleus and mitotic figures which may suggest its functional activity.
Antigens, Viral, Tumor* ; beta-Galactosidase ; Endothelial Cells* ; Endothelium ; Liposomes ; Transfection*

PURPOSE: To determine whether the delivery of the SV40 large T-antigen is a feasible method for transiently inducing proliferation of corneal endothelial cells, we delivered liposome-protein complex into bovine corneal endothelial cells(BCEC). METHOD: SV40 large T-antigen protein was introduced into BCEC and positive cells were identified by immunohistochemistry. Quiescent BCECs were double-labeled using BrdU as a measure of de novo DNA synthesis and the Ki-67 was detected by standard immunohistochemical methods. RESULT: The treatment of quiescent BCECs with large T antigen caused an increase in BrdU incorporation and Ki-67 expression. It was tested by time-course study. CONCLUSION: This finding suggests that liposome-mediated delivery of transforming proteins could be a method to transiently induce corneal endothelial cell proliferation.
Antigens, Viral, Tumor* ; Bromodeoxyuridine ; Cell Proliferation ; DNA ; Endothelial Cells* ; Immunohistochemistry

BACKGROUND: Conditionally immortalized hepatocytes (CIH) can be cultured almost indefinitely at permissive temperatures (33 degrees C), but they undergo apoptosis at nonpermissive temperatures (37~39 degrees C) by the release of p53 through inactivation of T antigen, which is called T antigen dependency. This study was aimed at examining if T antigen-independent clones can develop from CIH. METHODS: CIH established with a temperature-sensitive T antigen (WA1) were cultured continuously at 39 degrees C. Three clones (W39B, W39C, and W39J) survived at this temperature and was subject to following analyses: the morphology, growth, apoptosis, the expression of T antigen and p53, telomerase, and the T antigen gene sequence. RESULTS: WA1 proliferated at 33 degrees C with the population doubling time of 30.8 +/- 1.7 hours, but they underwent cell death at 39 degrees C. However, T antigen-independent clones (W39B, W39C, and W39C) proliferated at 39 degrees C without undergoing apoptosis, suggesting they lost the temperature-sensitive characteristics. WA1 expressed the T antigen at 33 degrees C, but not at 39 degrees C, and this temperature-sensitive pattern was maintained in T antigen-independent clones. In p53 expression, however, T antigen-independent clones revealed a different pattern. p53 was detected even at 39 degrees C where it normally would not be detected. Telomerase was activated in all the analyzed cell lines. A temperature-sensitive point mutation at nucleotide position 3505 of the WA1 was retained in all T antigen-independent clones. CONCLUSION: CIH can lost temperature-sensitive characteristics and acquire an ability to proliferate at nonpermissive temperatures. These changes might be related to the change of p53 rather than the change of T antigen itself in these cell lines.
Antigens, Viral, Tumor* ; Apoptosis ; Cell Death ; Cell Line ; Clone Cells ; Hepatocytes* ; Point Mutation ; Telomerase

OBJECTIVE: The ubiquitous human polyomavirus, JC virus(JCV) is the etiologic agent of the fatal demyelinating central nervous system(CNS) disease, progressive multifocal leukoencephalopathy(PML). Recent studies have reported the detection of the JCV in samples derived from several type of human neural tumors and suggested the possible association of JCV with CNS tumors. Here we report for the first time, the presence of JCV in Korean glioblastoma multiforme(GM) patients. METHODS: Two Korean GM patients were assayed for JCV. To detect JCV, we performed immunohistochemical analysis using anti-JCV and anti-glial fibrillary acidic protein(GFAP) serum and polymerase chain reaction(PCR) using primers. RESULTS: JCV antigen was detected in cytoplasm abundantly in cells of this tumor case. Also, GFAP immunoreactivity was predominantly observed in cytoplasm of the cells that were morphologically bizarred appearing astrocytes in GM. In addition, both of the large T antigen gene and the VP1 gene were detected and this result correspond with previous result of immunohistochemistry. CONCLUSION: Although it is not certain that GM is associated with the JCV, we are attempted to elucidate the possible implication of JCV in the tumorigenesis of certain human malignant gliomas.
Antigens, Viral, Tumor ; Astrocytes ; Brain* ; Carcinogenesis ; Cytoplasm ; Glioblastoma* ; Glioma ; Humans ; Immunohistochemistry ; JC Virus*

OBJECTIVE: It is still unclear how the abnormal hTERT expression is involved in the process of ovarian carcinogenesis. A recent report demonstrated that the introduction of c-erbB-2 could efficiently induce tumorigenicity of cells with the transfection of SV40 large T antigen and hTERT. It is designed to find correlation between overexpression of hTERT and c-erbB-2 in ovarian carcinogenesis. METHODS: Using immunohistochemistry, we tested whether overexpression of hTERT and c-erbB-2 were associated in ovarian cancer. Immunohistochemical staining of hTERT and c-erbB-2 was done in 63 cases of ovarian cancer. Overexpression of hTERT and c-erbB-2 were correlated to clinicopathological variables. RESULTS: Overexpression of hTERT was found in 7 (11.1%) of cases, whereas overexpression of c-erbB2 was founded in 3 (4.8%) of cases. It was found that overexpression of hTERT and c-erbB-2 were significantly correlated (p=0.03). Neither overexpression of hTERT nor that of c-erbB-2 was associated with any of clinicopathological variables, such as stage, grade, and histology. CONCLUSION: Although the significant correlation between hTERT and c-erbB-2 was found, the low frequency of overexpression of hTERT and c-erbB-2 suggests that cooperation of hTERT and c-erbB-2 may be minor mechanism of ovarian carcinogenesis.
Antigens, Viral, Tumor ; Carcinogenesis ; Immunohistochemistry ; Ovarian Neoplasms ; Receptor, erbB-2 ; Transfection

OBJECTIVE: It is still unclear how the abnormal hTERT expression is involved in the process of ovarian carcinogenesis. A recent report demonstrated that the introduction of c-erbB-2 could efficiently induce tumorigenicity of cells with the transfection of SV40 large T antigen and hTERT. It is designed to find correlation between overexpression of hTERT and c-erbB-2 in ovarian carcinogenesis. METHODS: Using immunohistochemistry, we tested whether overexpression of hTERT and c-erbB-2 were associated in ovarian cancer. Immunohistochemical staining of hTERT and c-erbB-2 was done in 63 cases of ovarian cancer. Overexpression of hTERT and c-erbB-2 were correlated to clinicopathological variables. RESULTS: Overexpression of hTERT was found in 7 (11.1%) of cases, whereas overexpression of c-erbB2 was founded in 3 (4.8%) of cases. It was found that overexpression of hTERT and c-erbB-2 were significantly correlated (p=0.03). Neither overexpression of hTERT nor that of c-erbB-2 was associated with any of clinicopathological variables, such as stage, grade, and histology. CONCLUSION: Although the significant correlation between hTERT and c-erbB-2 was found, the low frequency of overexpression of hTERT and c-erbB-2 suggests that cooperation of hTERT and c-erbB-2 may be minor mechanism of ovarian carcinogenesis.
Antigens, Viral, Tumor ; Carcinogenesis ; Immunohistochemistry ; Ovarian Neoplasms ; Receptor, erbB-2 ; Transfection