| Project/Area Number |
12670824
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Dermatology
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| Research Institution | Ehime University |
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Principal Investigator |
MURAKAMI Sinji Ehime University, University Hospital, Assistant Professor, 医学部・附属病院, 講師 (50175626)
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| Co-Investigator(Kenkyū-buntansha) |
HANAKAWA Yasushi Ehime University, Faculty of Medicine, Instructor, 医学部, 助手 (90284398)
SHIRAKATA Yuji Ehime University, Faculty of Medicine, Instructor, 医学部, 助手 (50226320)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | melanoma / ASK1 / adenovirus vector / apoptosiss / gene therapy / nude mouse / メラノーマ |
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| Research Abstract |
In order to assess whether adenovirus vector is useful for melanoma, we constructed adenovirus vector carrying apoptosis signal regulating kinase 1. We introduced full length of ASK1 cDNA into plasmid, transfected to E. coli and then amplified. Next, we purified the plasmid and introduced into cosmid cassette. We co-transfected both cosmid cassette and DNA-TPC into 293 cells and further cultured. After several days, we obtained adenovirus vector carrying ASK1 DNA. Then, adenovirus vector was amplified in 293 cells, and vector was purified with cesium chloride gradient centrifugation method followed by dialysis, and stored at -70 C. Twelve melanoma cell line were cultured in 10% FCS/DME or RPMI1640 and divided into 2 groups according to the cell growth assessed by growth assay. We infected adenovirus vector into melanoma cell line and observed by phase contrast microscopy. The infected melanoma cells turned into apoptosis at a density of MOI=20. These results indicate that ASK! induces apoptosis of melanoma cells. Next we investigated the efficacy of adenovirus vector for gene therapy of melanoma in vivo condition. One million of melanoma cells were injected into the subcutaneous of dorsal skin of nude mouse. After 4 weeks, the tumor grew about 15 mm in diameter. Adenovirus vector was injected directly into the tumor, and then we measured the size of tumor. The size of ASK1 injected melanoma decreased whereas that of control virus injected melanoma increase. Histopathological analysis showed a lot of apoptotic cells in the ASK1 injected tumor. Taking these results together, we conclude that ASK1 has the potential to reduce the size of melanoma and should be useful tool for melanoma gene therapy.
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