| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
To search new oncogenes in thyroid cancer, we performed cloning using new technique, which called smart method. The merit of this method is that very small amount of RNA (1μg) is good enough to make complete length cDNA. We constructed retroviral vector including cDNAs and transfeced to NIH3T3 cells. After 2 weeks of incubation, four colony piled up on the culture plate. When these transformed cells were subcutaneously injected to the nude mice, tumors developed within 2-3 weeks. We extracted DNA from the tumors and confirmed integration of human DNA by specific primers. Sequencing analysis revealed that one gene was truncated Raf-1 gene and other three were truncated BRAF gene. When these genes were transfected to the cells, we observed that the cells were transformed.
Next, we analyzed BRAF gene mutation using six human thyroid cancer cell lines and 186 thyroid tumor tissues. As the results, BRAF point mutation, V599E, was observed in four of six cell lines and 47 of 186 thyroid tumors. (25.3% ; 0/13 follicular adenoma, 0/7 follicular carcinoma, 45/160 papillary carcinomas and 2/6 undifferentiated carcinomas). Activation of MEK-MAP kinase pathway was observed in cell lines harboring BRAF mutation. BRAF mutation-associated enhanced cell growth was suppressed by MEK inhibitor, U0126. Examination of 135 patients with thyroid cancer showed that BRAF mutation correlated significantly with tumor size (P=0.015) and clinical stage (P=0.046). Our results indicate that activating mutation of BRAF gene could be a potentially useful marker of prognosis of patients with advanced thyroid cancers.
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