SECTION FOR STUDIES ON METASTASIS

15.SECTION FOR STUDIES ON METASTASIS

    The Section for Studies on Metastasis focuses on the development of novel animal models, methods, and strategies to study tumorigenesis and metastasis including the scope of cancer gene therapy. Specific activities in 2000 were as follows: 1) Application of a novel gene transfer method through biomaterial for DNA vaccination; 2) Identification of HST-1 gene expression in adult animals and analysis of its testicular functions; 3) Generation of Cyclin D1 transgenic mice.
A method to augment gene delivery for DNA vaccination through atelocollagen

    Muscle and skin represent major targets for gene therapy applications and are now also the main targets for DNA vaccination. Non-viral gene transfer into these tissues by direct injection of plasmid DNA is simple, but provides relatively low level and short-term expression of the transferred genes. A novel gene transfer method using atelocollagen dramatically sustained release of plasmid DNA in vivo, thereby keeping constant the levels of secreted proteins, and may induce immune responses to vaccine DNA with far less DNA than necessary for non-augmented vaccination. It demonstrates that our gene delivery with minimal invasive skin injection causes similar increases in gene expression with intramuscular injection of plasmid DNA encoding the envelope protein of the hepatitis B virus (HBV). Relatively high level expression of the transferred gene was detectable in the circulation and increased immune responses to a secreted surface antigen were achieved within 2 weeks after atelocollagen-mediated DNA injection into the skin. Five to 6 weeks after injection, an IgM to IgG class switch occurred. These results suggest that our atelocollagen-mediated DNA vaccination-induced humoral responses in mice mimic those observed in humans with natural HBV infection.
Detection of spatial localization of Hst-1/Fgf-4 gene expression in brain and testes from adult mice

    HST-1, a member of the fibroblast growth factor (FGF) family (FGF-4), has not been detected or reported in adult tissues analyzed to date. To investigate whether there is a possible role of HST-1/FGF-4 in the adult stage, a highly sensitive RT-PCR analysis in adult murine tissues was carried out. The results revealed Hst-1/Fgf-4 gene expression in the nervous system, intestines, and testes of normal adult mice.⁽¹⁹²⁾ In situ hybridization was used to localize Hst-1/Fgf-4 gene expression in the cerebellum and testes of 10-week-old mice. Cell type-specific gene expression was detected: Purkinje cells in the cerebellum and Sertoli cells in the testes. These findings suggest that the Hst-1/Fgf-4 gene also plays an important role in adult tissues, and may offer insights into the biological significance of HST-1/FGF-4 in cerebellar and testicular functions.
Functional analysis of HST-1/FGF-4 in spermatogenesis

    To understand the functional significance of HST-1/FGF-4 in the testes, the HST-1/FGF-4 gene was specifically expressed in the testes of adult transgenic animals carrying the human HST-1/FGF-4 gene using a Cre/lox conditional gene expression system. It was found that overexpression of HST-1/FGF-4 in the testes resulted in an increased sperm count. To verify the function of HST-1/FGF-4 as a regulatory factor in spermatogenesis, a Sertoli cell culture system in which the HST-1/FGF-4 gene switches on/off was used. Expression profiling based on cDNA chip analysis indicated that induced expression of HST-1/FGF-4 in Sertoli cells markedly enhanced the induction of genes related to spermatogenesis. The mechanisms by which HST-1/ FGF-4 supports spermatogenesis will be further analyzed using a Sertoli-germ cell co-culture system.
Generation of conditional Cyclin D1 transgenic mice

    Cyclin D1 is a gene critically involved in the regulation of progression through the G1 phase of the cell cycle, which thereby contributes to cell proliferation. Abnormal expression of Cyclin D1 has been described in several human cancers. To understand the biological significance of Cyclin D1 expression in carcinogenesis, a Cyclin D1-conditional transgenic C57BL/6J mouse was established. Induced expression of Cyclin D1 was achieved by site-directed Crerecombinase. Using this system, skin-specific expression of Cyclin D1 is allowed, and the mice were assessed for DMBA complete skin carcinogenesis. After 30 weeks, all of the mice with Cyclin D1 expression had papillomas, while only 9.5% of wild type mice without Cyclin D1 expression had papillomas. This result clearly shows that Cyclin D1 plays an important role in mouse skin carcinogenesis. Thus, conditional transgenic mice provide excellent in vivo and in vitro model systems for elucidating the role of Cyclin D1 and deregulation of the cell cycle in carcinogenesis.




15.SECTION FOR STUDIES ON METASTASIS



	The Section for Studies on Metastasis focuses on the development of novel animal models, methods, and strategies to study tumorigenesis and metastasis including the scope of cancer gene therapy. Specific activities in 2000 were as follows: 1) Application of a novel gene transfer method through biomaterial for DNA vaccination; 2) Identification of HST-1 gene expression in adult animals and analysis of its testicular functions; 3) Generation of Cyclin D1 transgenic mice. A method to augment gene delivery for DNA vaccination through atelocollagen Muscle and skin represent major targets for gene therapy applications and are now also the main targets for DNA vaccination. Non-viral gene transfer into these tissues by direct injection of plasmid DNA is simple, but provides relatively low level and short-term expression of the transferred genes. A novel gene transfer method using atelocollagen dramatically sustained release of plasmid DNA in vivo, thereby keeping constant the levels of secreted proteins, and may induce immune responses to vaccine DNA with far less DNA than necessary for non-augmented vaccination. It demonstrates that our gene delivery with minimal invasive skin injection causes similar increases in gene expression with intramuscular injection of plasmid DNA encoding the envelope protein of the hepatitis B virus (HBV). Relatively high level expression of the transferred gene was detectable in the circulation and increased immune responses to a secreted surface antigen were achieved within 2 weeks after atelocollagen-mediated DNA injection into the skin. Five to 6 weeks after injection, an IgM to IgG class switch occurred. These results suggest that our atelocollagen-mediated DNA vaccination-induced humoral responses in mice mimic those observed in humans with natural HBV infection. Detection of spatial localization of Hst-1/Fgf-4 gene expression in brain and testes from adult mice HST-1, a member of the fibroblast growth factor (FGF) family (FGF-4), has not been detected or reported in adult tissues analyzed to date. To investigate whether there is a possible role of HST-1/FGF-4 in the adult stage, a highly sensitive RT-PCR analysis in adult murine tissues was carried out. The results revealed Hst-1/Fgf-4 gene expression in the nervous system, intestines, and testes of normal adult mice.⁽¹⁹²⁾ In situ hybridization was used to localize Hst-1/Fgf-4 gene expression in the cerebellum and testes of 10-week-old mice. Cell type-specific gene expression was detected: Purkinje cells in the cerebellum and Sertoli cells in the testes. These findings suggest that the Hst-1/Fgf-4 gene also plays an important role in adult tissues, and may offer insights into the biological significance of HST-1/FGF-4 in cerebellar and testicular functions. Functional analysis of HST-1/FGF-4 in spermatogenesis To understand the functional significance of HST-1/FGF-4 in the testes, the HST-1/FGF-4 gene was specifically expressed in the testes of adult transgenic animals carrying the human HST-1/FGF-4 gene using a Cre/lox conditional gene expression system. It was found that overexpression of HST-1/FGF-4 in the testes resulted in an increased sperm count. To verify the function of HST-1/FGF-4 as a regulatory factor in spermatogenesis, a Sertoli cell culture system in which the HST-1/FGF-4 gene switches on/off was used. Expression profiling based on cDNA chip analysis indicated that induced expression of HST-1/FGF-4 in Sertoli cells markedly enhanced the induction of genes related to spermatogenesis. The mechanisms by which HST-1/ FGF-4 supports spermatogenesis will be further analyzed using a Sertoli-germ cell co-culture system. Generation of conditional Cyclin D1 transgenic mice Cyclin D1 is a gene critically involved in the regulation of progression through the G1 phase of the cell cycle, which thereby contributes to cell proliferation. Abnormal expression of Cyclin D1 has been described in several human cancers. To understand the biological significance of Cyclin D1 expression in carcinogenesis, a Cyclin D1-conditional transgenic C57BL/6J mouse was established. Induced expression of Cyclin D1 was achieved by site-directed Crerecombinase. Using this system, skin-specific expression of Cyclin D1 is allowed, and the mice were assessed for DMBA complete skin carcinogenesis. After 30 weeks, all of the mice with Cyclin D1 expression had papillomas, while only 9.5% of wild type mice without Cyclin D1 expression had papillomas. This result clearly shows that Cyclin D1 plays an important role in mouse skin carcinogenesis. Thus, conditional transgenic mice provide excellent in vivo and in vitro model systems for elucidating the role of Cyclin D1 and deregulation of the cell cycle in carcinogenesis.

15.SECTION FOR STUDIES ON METASTASIS

A method to augment gene delivery for DNA vaccination through atelocollagen

Detection of spatial localization of Hst-1/Fgf-4 gene expression in brain and testes from adult mice

Functional analysis of HST-1/FGF-4 in spermatogenesis

Generation of conditional Cyclin D1 transgenic mice