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 2001 were as follows: 1) Application of biomaterials for gene delivery; 2) Analysis of Cyclin D1-induced skin carcinogenesis; 3) Role of HST-1 gene expression in spermatogenesis.
Atelocollagen-based Gene Transfer in Cells

    Atelocollagen, used clinically for wound healing, is a reliable safe carrier for gene delivery.⁽²⁰⁹⁾ To obtain phenotypic changes by gene expression of cDNA, we developed an efficient technique for high-throughput gene transfer and expression screening in mammalian cells in microarrays by pre-coating a microplate with an Atelocollagen complexed with cDNA to which cells are then seeded.⁽²¹⁰⁾ The complexes with a nano-particle form were efficiently transduced into cells without the use of any additional transfection reagent, and they allowed for long-term gene expression without apparent chromosomal integration. The complex spotted onto the well of a microplate was stable for a long period and allowed the cells to transduce and express reporter genes in a dose-dependent manner. We also showed that the present method using Atelocollagen-based gene transfer is applicable to gene medicines such as antisense ODNs and adenovirus vectors. These results suggest that Atelocollagen may be appropriate for general use in high-throughput screening of large sets of gene medicines for functional analyses in mammalian cells.
Enhanced Skin Carcinogenesis in Cyclin D1 Conditional Transgenic Mice

    Gene amplification and abnormal expression of Cyclin D1 have been described in several human cancers. To understand their biological significance in skin carcinogenesis, Cyclin D1-conditional transgenic mice with C57BL/6J background were established, in which skin-specific overexpression of Cyclin D1 transgene was observed. The mice were subjected to dimethyl benz[a]anthracene (DMBA) complete skin carcinogenesis studies. After 40 weeks of once weekly repeated administration of DMBA on the skin, all the mice with high Cyclin D1 expression had papillomas, while only 9.5% of the control mice without the transgene developed papillomas. Primary cultured keratinocytes with induced-Cyclin D1 transgene expression showed resistance to calcium-induced terminal differentiation and continued to replicate in vitro. These results clearly provide us with direct experimental evidence that overexpression of Cyclin D1 induces excessive dermal cell proliferation via the altered differentiation state of keratinocytes. The conditional transgenic mice described here provide us with excellent in vivo and in vitro model systems to understand the role of Cyclin D1 and deregulation of the cell cycle in carcinogenesis.
HST-1/FGF-4 acts as a survival factor for germ cells

    In our previous study, the HST-1/FGF-4 gene was expressed in Sertoli cells and germ cells in adult human and mouse testis, implying that it might play a role in spermatogenesis. For an understanding of its functional significance in spermatogenesis and therapeutic potency, transgenic mice with enhanced HST-1/FGF-4 gene expression in the testes were established, and it has been shown that HST-1/FGF-4 significantly increased the sperm count and prevented ADR-induced testicular toxicity. This evidence suggests that HST-1/FGF-4 may protect male germ cells from apoptosis. To investigate the potential role of HST-1/FGF-4 as an anti-apoptotic agent, the testes of adult mice that overexpressed HST-1/FGF-4 were exposed to mild hyperthermia, which induces germ cell specific apoptosis. The results indicate that HST-1/FGF-4 significantly reduced the apoptotic death of germ cells, decreased the weight of the testes, and reduced the sperm count. Thus, these results should shed light on the important roles of HST-1/FGF-4 during spermatogenesis. HST-1/FGF-4 may act as a survival factor for germ cells in adult mouse testes, and HST-1/FGF-4-based gene therapy may present a new paradigm for male infertility.




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 2001 were as follows: 1) Application of biomaterials for gene delivery; 2) Analysis of Cyclin D1-induced skin carcinogenesis; 3) Role of HST-1 gene expression in spermatogenesis. Atelocollagen-based Gene Transfer in Cells Atelocollagen, used clinically for wound healing, is a reliable safe carrier for gene delivery.⁽²⁰⁹⁾ To obtain phenotypic changes by gene expression of cDNA, we developed an efficient technique for high-throughput gene transfer and expression screening in mammalian cells in microarrays by pre-coating a microplate with an Atelocollagen complexed with cDNA to which cells are then seeded.⁽²¹⁰⁾ The complexes with a nano-particle form were efficiently transduced into cells without the use of any additional transfection reagent, and they allowed for long-term gene expression without apparent chromosomal integration. The complex spotted onto the well of a microplate was stable for a long period and allowed the cells to transduce and express reporter genes in a dose-dependent manner. We also showed that the present method using Atelocollagen-based gene transfer is applicable to gene medicines such as antisense ODNs and adenovirus vectors. These results suggest that Atelocollagen may be appropriate for general use in high-throughput screening of large sets of gene medicines for functional analyses in mammalian cells. Enhanced Skin Carcinogenesis in Cyclin D1 Conditional Transgenic Mice Gene amplification and abnormal expression of Cyclin D1 have been described in several human cancers. To understand their biological significance in skin carcinogenesis, Cyclin D1-conditional transgenic mice with C57BL/6J background were established, in which skin-specific overexpression of Cyclin D1 transgene was observed. The mice were subjected to dimethyl benz[a]anthracene (DMBA) complete skin carcinogenesis studies. After 40 weeks of once weekly repeated administration of DMBA on the skin, all the mice with high Cyclin D1 expression had papillomas, while only 9.5% of the control mice without the transgene developed papillomas. Primary cultured keratinocytes with induced-Cyclin D1 transgene expression showed resistance to calcium-induced terminal differentiation and continued to replicate in vitro. These results clearly provide us with direct experimental evidence that overexpression of Cyclin D1 induces excessive dermal cell proliferation via the altered differentiation state of keratinocytes. The conditional transgenic mice described here provide us with excellent in vivo and in vitro model systems to understand the role of Cyclin D1 and deregulation of the cell cycle in carcinogenesis. HST-1/FGF-4 acts as a survival factor for germ cells In our previous study, the HST-1/FGF-4 gene was expressed in Sertoli cells and germ cells in adult human and mouse testis, implying that it might play a role in spermatogenesis. For an understanding of its functional significance in spermatogenesis and therapeutic potency, transgenic mice with enhanced HST-1/FGF-4 gene expression in the testes were established, and it has been shown that HST-1/FGF-4 significantly increased the sperm count and prevented ADR-induced testicular toxicity. This evidence suggests that HST-1/FGF-4 may protect male germ cells from apoptosis. To investigate the potential role of HST-1/FGF-4 as an anti-apoptotic agent, the testes of adult mice that overexpressed HST-1/FGF-4 were exposed to mild hyperthermia, which induces germ cell specific apoptosis. The results indicate that HST-1/FGF-4 significantly reduced the apoptotic death of germ cells, decreased the weight of the testes, and reduced the sperm count. Thus, these results should shed light on the important roles of HST-1/FGF-4 during spermatogenesis. HST-1/FGF-4 may act as a survival factor for germ cells in adult mouse testes, and HST-1/FGF-4-based gene therapy may present a new paradigm for male infertility.