SECTION FOR STUDIES ON METASTASIS

15.SECTION FOR STUDIES ON METASTASIS

　The focus of the Section for Studies on Metastasis lies on the development of novel animal models, methods,and strategies to study tumorigenesis and metastasis. The specific activities in 1999 were as follows:1) Development of a novel gene transfer method through the atelocollagen minipellet;2) Evaluation of cationic liposomes suitable for gene and oligonucleotide transfer into animals;3)Adenovirus- mediated transfer of the HST- 1/FGF- 4 gene to protect mice from lethal irradiation.
Controlled Release of Plasmid DNA in vivo

　A novel gene transfer method which allows prolonged- release and expression of plasmid DNA in vivo in normal adult animals has been established. ⁽²²²⁾ In this system, atelocollagen, a biocompatible polymer, acts as the carrier. The Minipellet, a biodegradable cylindrical solid type dosage formulation and made of atelocollagen, carrying plasmid DNA was administered by intramuscular (im)injection into animals. Once introduced into the animals, the Minipellet gradually releases plasmid DNA in vivo. A single im injection of as little as 50 m g of plasmid DNA- Minipellet produced physiologically significant levels of recombinant protein in the systemic circulation of animals and resulted in biological effects that were stable for more than 60 days post- administration. Furthermore, the Minipellet can be removed surgically. These results suggest that the Minipellet carrying plasmid DNA may enhance the clinical potency of plasmid- based gene transfer, facilitating a more effective and long- term use of naked plasmid vectors for gene therapy. Application of our system for the augmentation of the bioavailability of low molecular weight materials such as antisense oligonucleotides or adenovirus vectors has also been studied.
Cationic Liposome-based Gene Delivery in vivo

　Cationic liposome- mediated in vivo gene transfer represents a promising approach for somatic gene therapy. To assess the most suitable liposome for gene delivery into a wide range of organs and fetuses in mice, we have explored several types of cationic liposomes conjugated with plasmid DNA carrying the b - galactosidase gene through intravenous injection into pregnant animals. Through the analysis of several types of recently synthesized cationic liposome/lipid formulations, DMRIE- C reagent, a liposome formulation of the cationic lipid DMRIE (1,2- dimyristyloxypropyl- 3- dimethyl- hydroxy ethyl ammonium bromide)and cholesterol in membrane- filtered water met our requirements.⁽²²³⁾ Furthermore, our results highlight that cationic lipids are essential for antisense oligonucleotide delivery in vitro and in vivo.⁽²²⁴⁾
Toward Targeted Killing of Tumor-specific Antigen-producing Cancer Cells

　Cancer- specific antigens are promising for the specific delivery of certain drugs or genes to cancer cells in cancer therapy, as well as in fetal gene therapy. Toward targeted killing of tumor- specific antigen- producing cells, we used retroviral vectors co- displaying an anti- carcinoembryonic antigen (CEA)single chain variable fragment (scFv)- envelope chimeric protein and an unmodified envelope protein to deliver a gene for herpes simplex virus thymidine kinase. Ganciclovir treatment of human cancer cells after the infection of the HSV- tk virus with the chimeric envelope showed an efficient specific killing of the target cells. ⁽²²⁵⁾ These results may have significant implications for the use of scFv directed against tumor- specific antigens for targeting cancer cells.
Therapeutic Potential of Adex1HST-1 Against Lethal Irradiation

　The therapeutic potential of Adex1HST- 1, an adenovirus vector carrying the HST- 1/ FGF- 4 gene (fibroblast growth factor- 4), on severely injured C57BL/6 mice after exposure to lethal irradiation was studied.⁽²²⁶⁾ The group of mice that received Adex1HST- 1 three days prior to lethal irradiation (9 Gy)with ⁶⁰Co gamma- rays had a dramatic and significant increase in survival to 90% compared with control animals (0%)that received wild type adenoviruses (p<0.0001).adex1HST- 1 administration immediately after irradiation could also prolong survival significantly. Histological sections of bone marrow and spleen indicated that the surviving irradiated- animals expressing Adex1HST- 1 showed a significant recovery of hematological cells. In addition, histopathological analysis revealed that the extent to which Adex1HST- 1 protects against irradiation in this prevention model may also be involved in gastro- intestinal injury. This dual potential of HST- 1 may lend strong support to the concept of the therapeutic effectiveness of adenoviruses carrying HST- 1 to lessen the side effects of current adjuvant cancer therapy regimens.
NG2 as the Target Molecule in the Tumor Vasculature

　NG2 is a large, integral membrane, chondroitin sulfate proteoglycan that was first identified as a cell surface molecule expressed by immature neural cells. Subsequently NG2 was found to be expressed by a wide variety of immature cells as well as several types of tumors with high malignancy. To elucidate the possible effects of NG2 in tumorigenesis and neovasculature, knockout mice and transgenic mice were established. Vascular smooth muscle cells from NG2- knockout mice showed an altered response to platelet- derived growth factor AA. ⁽²²⁷⁾ These animal models carrying NG2 gene modifications will be useful to address the question of whether NG2 can be a target molecule in the tumor vasculature.

15.SECTION FOR STUDIES ON METASTASIS

Controlled Release of Plasmid DNA in vivo

Cationic Liposome-based Gene Delivery in vivo

Toward Targeted Killing of Tumor-specific Antigen-producing Cancer Cells

Therapeutic Potential of Adex1HST-1 Against Lethal Irradiation

NG2 as the Target Molecule in the Tumor Vasculature




15.SECTION FOR STUDIES ON METASTASIS



	The focus of the Section for Studies on Metastasis lies on the development of novel animal models, methods,and strategies to study tumorigenesis and metastasis. The specific activities in 1999 were as follows:1) Development of a novel gene transfer method through the atelocollagen minipellet;2) Evaluation of cationic liposomes suitable for gene and oligonucleotide transfer into animals;3)Adenovirus- mediated transfer of the HST- 1/FGF- 4 gene to protect mice from lethal irradiation. Controlled Release of Plasmid DNA in vivo 　A novel gene transfer method which allows prolonged- release and expression of plasmid DNA in vivo in normal adult animals has been established. ⁽²²²⁾ In this system, atelocollagen, a biocompatible polymer, acts as the carrier. The Minipellet, a biodegradable cylindrical solid type dosage formulation and made of atelocollagen, carrying plasmid DNA was administered by intramuscular (im)injection into animals. Once introduced into the animals, the Minipellet gradually releases plasmid DNA in vivo. A single im injection of as little as 50 m g of plasmid DNA- Minipellet produced physiologically significant levels of recombinant protein in the systemic circulation of animals and resulted in biological effects that were stable for more than 60 days post- administration. Furthermore, the Minipellet can be removed surgically. These results suggest that the Minipellet carrying plasmid DNA may enhance the clinical potency of plasmid- based gene transfer, facilitating a more effective and long- term use of naked plasmid vectors for gene therapy. Application of our system for the augmentation of the bioavailability of low molecular weight materials such as antisense oligonucleotides or adenovirus vectors has also been studied. Cationic Liposome-based Gene Delivery in vivo 　Cationic liposome- mediated in vivo gene transfer represents a promising approach for somatic gene therapy. To assess the most suitable liposome for gene delivery into a wide range of organs and fetuses in mice, we have explored several types of cationic liposomes conjugated with plasmid DNA carrying the b - galactosidase gene through intravenous injection into pregnant animals. Through the analysis of several types of recently synthesized cationic liposome/lipid formulations, DMRIE- C reagent, a liposome formulation of the cationic lipid DMRIE (1,2- dimyristyloxypropyl- 3- dimethyl- hydroxy ethyl ammonium bromide)and cholesterol in membrane- filtered water met our requirements.⁽²²³⁾ Furthermore, our results highlight that cationic lipids are essential for antisense oligonucleotide delivery in vitro and in vivo.⁽²²⁴⁾ Toward Targeted Killing of Tumor-specific Antigen-producing Cancer Cells 　Cancer- specific antigens are promising for the specific delivery of certain drugs or genes to cancer cells in cancer therapy, as well as in fetal gene therapy. Toward targeted killing of tumor- specific antigen- producing cells, we used retroviral vectors co- displaying an anti- carcinoembryonic antigen (CEA)single chain variable fragment (scFv)- envelope chimeric protein and an unmodified envelope protein to deliver a gene for herpes simplex virus thymidine kinase. Ganciclovir treatment of human cancer cells after the infection of the HSV- tk virus with the chimeric envelope showed an efficient specific killing of the target cells. ⁽²²⁵⁾ These results may have significant implications for the use of scFv directed against tumor- specific antigens for targeting cancer cells. Therapeutic Potential of Adex1HST-1 Against Lethal Irradiation 　The therapeutic potential of Adex1HST- 1, an adenovirus vector carrying the HST- 1/ FGF- 4 gene (fibroblast growth factor- 4), on severely injured C57BL/6 mice after exposure to lethal irradiation was studied.⁽²²⁶⁾ The group of mice that received Adex1HST- 1 three days prior to lethal irradiation (9 Gy)with ⁶⁰Co gamma- rays had a dramatic and significant increase in survival to 90% compared with control animals (0%)that received wild type adenoviruses (p<0.0001).adex1HST- 1 administration immediately after irradiation could also prolong survival significantly. Histological sections of bone marrow and spleen indicated that the surviving irradiated- animals expressing Adex1HST- 1 showed a significant recovery of hematological cells. In addition, histopathological analysis revealed that the extent to which Adex1HST- 1 protects against irradiation in this prevention model may also be involved in gastro- intestinal injury. This dual potential of HST- 1 may lend strong support to the concept of the therapeutic effectiveness of adenoviruses carrying HST- 1 to lessen the side effects of current adjuvant cancer therapy regimens. NG2 as the Target Molecule in the Tumor Vasculature 　NG2 is a large, integral membrane, chondroitin sulfate proteoglycan that was first identified as a cell surface molecule expressed by immature neural cells. Subsequently NG2 was found to be expressed by a wide variety of immature cells as well as several types of tumors with high malignancy. To elucidate the possible effects of NG2 in tumorigenesis and neovasculature, knockout mice and transgenic mice were established. Vascular smooth muscle cells from NG2- knockout mice showed an altered response to platelet- derived growth factor AA. ⁽²²⁷⁾ These animal models carrying NG2 gene modifications will be useful to address the question of whether NG2 can be a target molecule in the tumor vasculature.