TACHIBANA Masaaki Tokyo Medical University, Medicine, Professor, 医学部, 教授 (70129526)
HATANO Tadashi Tokyo Medical University, Medicine, Professor, 医学部, 教授 (10101924)
YOSHIOKA Kunihiko Tokyo Medical University, Medicine, Assistant professor, 医学部, 講師 (60220589)
|Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2002: ¥2,200,000 (Direct Cost: ¥2,200,000)
Preparation of prostate cancer model with nude mice
For prostate cancer cell lines, LNCaP cells in which existence of the PSMA antigen has been confirmed, C4-2 cells, their subline, and PSMA-negative PC-3 cells were used. 1×10^6 LNCaP and C4-2 cells were subcutaneously injected, respectively. The C4-2 cells formed a tumor mass more easily (100%) than LNCaP cells (70%) showing appropriate as a model.
In vitro study using PSMA antibody conjugated Caspase 8 plasmid
At the beginning, the hTERT promoter driven Capase 8 plasmid was prepared. First, the cloned hTERT promoter (384bp) was integrated into the pGL3 basic vector. Next, the luciferase gene of the pGL3 vector was replaced by the Caspase 8 gene. Then, the PSMA antibody and the vector prepared above or pGL3 control vector (SV4O promoter driven luciferase gene) were biotynilated. At the same time, the PSMA antibody was biotynilated. The PSMA antibody conjugated luciferase plasmid (PSMA antibody and pGL3 control vector) and PSMA antibody C
aspase B DNA plasmid (PSMA antibody and hTERT promoter driven Caspase 8 plasmid) were prepared by conjugating the biotynilated vector and antibody with avidin, respectively. The luciferase assay was conducted by introducing the PSMA antibody conjugated luciferase plasmid into the LNCaP, C4-2 and PC-3 cells. The luciferase activities of the LNCaP and C4-2 cells were high, showing 10.3 and 28.4, respectively, when letting those of the PC-3 cells to be 1.0. In addition, the status of apoptosis in each cell line in administration of the PSMA antibody Caspase 8 DNA plasmid was confirmed by the FACS following the tunel staining. The results were 1.2% inPC3, 3.0% in LNCaP and 4.2% in C4-2, showing no significant difference.
In vivo study using PSMA antibody conjugated Caspase 8 plasmid
1, 10 and 100 μg of the PSMA antibody conjugated luciferase plasmid were administered to the homologous prostate cancer models(PC-3 and C4-2),respectively. The tumor was taken three days after administration and immunostained with the anti-luciferase antibody. The C4-2 tumor was partially stained in 10 and 100 μg administration, whereas the PC-3 tumor was not stained at all. Next, 100 μg of the PSMA antibody Caspase 8 DNA plasmid was administered daily when the tumor grew to the size of 10mm in diameter in the homologous prostate cancer models(PC-3 and C4-2),and its antiproliferative effects were examined. There was no significant difference though some antiproliferative effects were found, compared with the control group (the PSMA antibody conjugated luciferase plasmid administration group). Less