| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2000: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
We established a functional analysis system for cancer-derived p53 gene in mammalian cultured-cells. Entire open reading frame of p53 cDNA was amplified from head and neck cancer cells by RT-PCR. The amplified p53 cDNA fragment was subcloned into pEGFP-C3 which is a mammalian expression vector containing the enhanced green fluorescent protein (GFP) gene under the transcriptional control of the cytomegalovirus immediate early promoter. We transfected a human osteosarcoma cell line, Saos2 with the expression vector containing tumor derived-p53 gene. Then, we examined the localization of the tumor-derived p53 proteins in the cells and the transcriptionai activity of the tumor-derived p53 proteins by luciferase reporter plasmids containing p21waf1, MDM2, or BAX promoter. A mutated-p53 (Glu17Lys, His193Leu) or a truncated-p53 (delta 121) did not activate the reporters at all, however, a mutated-p53 (Asn30Ser) showed the transcriptionai activity on all of the reporters as wild type p53 did. On the other hand, a mutated-p53 (Asp281His) activated the p21waf1 promoter strongly and the MDM2 promoter faintly, but did not activate the BAX promoter. Interestingly, this mutated-p53prevented Saos-2 cells from undergoing apoptosis after treatment with, adriamycine. This mutated-p53 probably induced cell cycle arrest but not apoptosis. Furthermore, another mutated-p53 (Glu17Lys, His193Leu) also prevented the cells from undergoing apoptosis after DNA damage. This mutated-p53 may gain undetermined new function, and this type of mutation can be called as an oncogenic mutation. These results suggest that some cancer cells may contain the oncogenic mutation of p53 gene, and the oncogenic mutation of p53 gene prevents cancer cells from undergoing apoptosis after DNA damage.
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