|Budget Amount *help
¥12,300,000 (Direct Cost : ¥12,300,000)
Fiscal Year 1999 : ¥3,800,000 (Direct Cost : ¥3,800,000)
Fiscal Year 1998 : ¥8,500,000 (Direct Cost : ¥8,500,000)
By introduction of human telomerase reverse transcriptase (hTERT) cDNA, human normal fibroblasts expressed telomerase activity, elongated telomere size, extended proliferative life sapn, but were not immortalized without exception. We transfected hTERT cDNA into human fibroblasts that were transformed with SV40 T-antigen gene. T-antigen transformed cells were mortal but were immortalized, without exception, after the transfection with hTERT. Therefore, the presence of both hTERT and T-antigen were sufficient for immortalization of human fibroblasts. T-antigen product is known to be a multifunctional protein. Temperature sensitive mutant of T-antigen loses its functions, at the non-permissive temperature, such as- virus replication, repression of SV40 early genes, establishment transformation, maintenance of transformed phenotype, and binding with p53 protein. Immortalized human fibroblasts .by hTERT and T-antigen, however, did not stop proliferation even at the non-permissive temperatu
re where activity of T-antigen was lost. There, are two possible explanations on required function of T-antigen for immortalization, one is that a function of T-antigen required for immortalization is very limited that survives at the non-permissive temperature and the other is that T-antigen is required only for establishment but not for the maintenance of immortalized phenotype. 2) We established a new method to measure telomerase activity that was highly sensitive and highly quantitative. By using new method we assayed telomerase activity in various human liver tissues and found that we could discriminate low activities in non-tumor liver lesions such as chronic hepatitis and liver cirrhosis from in well-differentiated hepatocellular carcinomas. We also found that about a half of pre-cancerous hepatic lesions had very weak but significant level of telomerase activity higher than non-tumor lesions and suggested that these pre-cancerous lesions should be treated as early carcinomas for the safety of patients.