Section for Studies on Metastasis

15. Section for Studies on Metastasis

　The research in the Section for Studies on Metastasis has been focused on the development of novel models, methods, and strategies to study tumorigenesis and metastasis. The specific activities in 1998 were: 1) Molecular cloning and expression of a cDNA encoding a rat leukemia inhibitory factor (LIF) to generate pluripotent rat embryonic stem (ES) cells; 2) generation of conditional transgenic mice carrying HST-1(FGF-4) gene by cre/loxP system; 3) development of a novel gene transfer method through intramuscular injection of atelocollagen minipellet; and 4) inhibition of in vitro growth of human germ cell tumor by antisense oligonucleotide (AS ODNs) and an antagonist peptide for HST-1.
Culture of Rat ES Cells

　The creation of targeted mutations in the animals has been a valuable source of animal models of human disease. Mouse LIF has little activity for the maintainance of the developmental potential of rat ES cells which can contribute to the generation of germ-line chimeras, resulting in no knockout rats being available to date. In order to facilitate studies on rat ES cells, full-length rat LIF cDNA was isolated and the total nucleotide sequence was determined.⁽¹⁷⁸⁾ A rat cell line which was transduced by our rat LIF cDNA could maintain the derivatives of rat blastocysts with an undifferentiated phenotype. Therefore, the availability of our rat LIF will open up the possibility of the use of rat ES cells in studies of normal physiology and generation of malignant tumor models. Work is in progress to establish rat ES cells to generate germ-line chimera using our cloned rat LIF.
Targeted HST-1 Gene Activation in vivo

　In studies on the biological function of the HST-1 gene in vivo and in vitro,⁽¹⁷⁹⁾ an efficient and accurate method for controlled in vivo transgene modulation by site-directed recombination was used for HST-1 gene analysis. Five transgenic mouse founder lines were produced carrying the universal promoter and the human HST-1 gene sequence, separated by a neomycin stop sequence that contains elements preventing expression of the transgene and Cre recombinase recognition sites. Progeny from two of these lines were systemically injected with adenoviruses carrying Cre recombinase. Subsequent analysis by the use of ELISA method confirmed that Cre-mediated recombination elicited systemic HST-1 gene expression in mice and resulted in thrombocytosis. Induction of HST-1 in a targeted region of transgenic mice by inoculating the Cre-expressing adenoviruses positionally into brain and testis is now underway.
Controlled Release of Plasmid DNA in vivo

　Direct gene transfer was achieved with several viral vectors, naked plasmid DNA or cationic liposomes. A non-viral vector-system such as plasmid DNA-based gene transfer is an attractive model for treatment of acquired metabolic disorders. However, several potential problems need to be resolved to improve the transfection efficiency, high-level and long-lasting in vivo gene expression prior to clinical application. We attempted to develop a novel gene transfer method using atelocollagen as a carrier. The atelocollagen minipellet is biodegradable, and once introduced into animals releases conjugated plasmid DNA gradually in vivo. Furthermore, most importantly the controlled release of plasmid DNA facilitates a longer lasting biological effect than intramuscular injection of plasmid DNA alone. Thus, these results suggest that an atelocollagen minipellet may enhance clinical potency of plasmid-based gene transfer, facilitating a more effective and long-term use of naked plasmid vectors for gene therapy.
Antisense Strategy against Human Testicular Tumor

　To evaluate possible antisense therapy to inhibit human testicular tumor, the growth inhibitory effects of AS ODNs against human HST-1-producing tumor cells (NEC-8) were studied. Results demonstrate that at least one HST-1 AS ODN designed against exon 3 markedly inhibited growth of NEC-8 cells in a dose-dependent manner. This AS ODN also had no inhibitory effect on cells which have no HST-1 gene expression. Synthetic peptides corresponding to sequences against the high-affinity receptor binding sites and two heparansulfate binding sites behave as antagonists and showed a striking inhibition of the proliferation of NEC-8 cells in vitro. These findings suggest that reduction of HST-1 protein in human testicular tumor cells, and possibly also in a human germ cell tumors, may be a novel and rational gene therapy to improve treatment outcome.

15. Section for Studies on Metastasis

Culture of Rat ES Cells

Targeted HST-1 Gene Activation in vivo

Controlled Release of Plasmid DNA in vivo

Antisense Strategy against Human Testicular Tumor




	15. Section for Studies on Metastasis



		The research in the Section for Studies on Metastasis has been focused on the development of novel models, methods, and strategies to study tumorigenesis and metastasis. The specific activities in 1998 were: 1) Molecular cloning and expression of a cDNA encoding a rat leukemia inhibitory factor (LIF) to generate pluripotent rat embryonic stem (ES) cells; 2) generation of conditional transgenic mice carrying HST-1(FGF-4) gene by cre/loxP system; 3) development of a novel gene transfer method through intramuscular injection of atelocollagen minipellet; and 4) inhibition of in vitro growth of human germ cell tumor by antisense oligonucleotide (AS ODNs) and an antagonist peptide for HST-1. Culture of Rat ES Cells 　The creation of targeted mutations in the animals has been a valuable source of animal models of human disease. Mouse LIF has little activity for the maintainance of the developmental potential of rat ES cells which can contribute to the generation of germ-line chimeras, resulting in no knockout rats being available to date. In order to facilitate studies on rat ES cells, full-length rat LIF cDNA was isolated and the total nucleotide sequence was determined.⁽¹⁷⁸⁾ A rat cell line which was transduced by our rat LIF cDNA could maintain the derivatives of rat blastocysts with an undifferentiated phenotype. Therefore, the availability of our rat LIF will open up the possibility of the use of rat ES cells in studies of normal physiology and generation of malignant tumor models. Work is in progress to establish rat ES cells to generate germ-line chimera using our cloned rat LIF. Targeted HST-1 Gene Activation in vivo 　In studies on the biological function of the HST-1 gene in vivo and in vitro,⁽¹⁷⁹⁾ an efficient and accurate method for controlled in vivo transgene modulation by site-directed recombination was used for HST-1 gene analysis. Five transgenic mouse founder lines were produced carrying the universal promoter and the human HST-1 gene sequence, separated by a neomycin stop sequence that contains elements preventing expression of the transgene and Cre recombinase recognition sites. Progeny from two of these lines were systemically injected with adenoviruses carrying Cre recombinase. Subsequent analysis by the use of ELISA method confirmed that Cre-mediated recombination elicited systemic HST-1 gene expression in mice and resulted in thrombocytosis. Induction of HST-1 in a targeted region of transgenic mice by inoculating the Cre-expressing adenoviruses positionally into brain and testis is now underway. Controlled Release of Plasmid DNA in vivo 　Direct gene transfer was achieved with several viral vectors, naked plasmid DNA or cationic liposomes. A non-viral vector-system such as plasmid DNA-based gene transfer is an attractive model for treatment of acquired metabolic disorders. However, several potential problems need to be resolved to improve the transfection efficiency, high-level and long-lasting in vivo gene expression prior to clinical application. We attempted to develop a novel gene transfer method using atelocollagen as a carrier. The atelocollagen minipellet is biodegradable, and once introduced into animals releases conjugated plasmid DNA gradually in vivo. Furthermore, most importantly the controlled release of plasmid DNA facilitates a longer lasting biological effect than intramuscular injection of plasmid DNA alone. Thus, these results suggest that an atelocollagen minipellet may enhance clinical potency of plasmid-based gene transfer, facilitating a more effective and long-term use of naked plasmid vectors for gene therapy. Antisense Strategy against Human Testicular Tumor 　To evaluate possible antisense therapy to inhibit human testicular tumor, the growth inhibitory effects of AS ODNs against human HST-1-producing tumor cells (NEC-8) were studied. Results demonstrate that at least one HST-1 AS ODN designed against exon 3 markedly inhibited growth of NEC-8 cells in a dose-dependent manner. This AS ODN also had no inhibitory effect on cells which have no HST-1 gene expression. Synthetic peptides corresponding to sequences against the high-affinity receptor binding sites and two heparansulfate binding sites behave as antagonists and showed a striking inhibition of the proliferation of NEC-8 cells in vitro. These findings suggest that reduction of HST-1 protein in human testicular tumor cells, and possibly also in a human germ cell tumors, may be a novel and rational gene therapy to improve treatment outcome.