Mechanisms of cellular immortalization and, carcinogenesis

通过引入人端粒酶逆转录酶（hTERT） cDNA，人正常成纤维细胞表达端粒酶活性，端粒长度延长，增殖寿命延长，但无一例外不能永生。我们将hTERT cDNA转染到转化了SV40 t抗原基因的人成纤维细胞中。转染hTERT后，t抗原转化的细胞死亡，但无一例外地永生。因此，hTERT和t抗原的存在足以使人成纤维细胞永生化。t抗原产物是一种多功能蛋白。温度敏感型t抗原突变体在非允许温度下失去其功能，如-病毒复制、SV40早期基因抑制、建立转化、维持转化表型、与p53蛋白结合等。永生的人类成纤维细胞。然而，即使在非允许温度下，hTERT和t抗原的作用也没有阻止细胞的增殖。关于t -抗原的永生化功能有两种可能的解释，一种是t -抗原在非允许温度下存活的功能非常有限，另一种是t -抗原只需要建立而不需要维持永生化表型。2)建立了一种高灵敏度、高定量的端粒酶活性测定新方法。我们用新方法测定了人不同肝脏组织的端粒酶活性，发现可以区分慢性肝炎、肝硬化等非肿瘤性肝脏病变和高分化肝细胞癌的低活性。我们还发现，大约一半的癌前肝病变的端粒酶活性比非肿瘤病变的端粒酶活性非常弱，但水平显著高于非肿瘤病变。我们建议，为了患者的安全，这些癌前病变应作为早期癌症进行治疗。少

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 … More 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. Less