| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1995: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
In the present study, we attempted to immortalize human distal airway epithelial cells in vitro by transfecting viral oncogenes and to analyze the mechanisms of their immortalization.
Primary bronchiolar epithelial cells were obtained by incubating fragments of normal lung tissue from adult lung cancer patients (5th to 6th decade) in Ham's F12 supplemented with growth factors for one to 2 weeks. Cells outgrown on the dish were harvested and replated for the second culture in growth factor-supplemented MCDB 153, and then were transfected by lipofection with plasmids containing genomes of human papilloma virus (HPV) 16, HPV18, or SV 40. Culture of transfected cells was continued and, when the cells reached an -80% confluence, they were split at 1 : 5 for passage.
The results of three similar experiments showed that transfectants of SV40 were able to be passaged up to 9 times (120 days ; -20 population doubling), while transfectants of HPV16 and of HPV18 were able to be passaged only 2 or 3 times (2 to 6 population doubling). Immunocytochemical analysis of SV40-transfected cells revealed an expression of large T antigen and accumulation of p53 protein in the nuclei. Telomerase activity, measured by terminal repeat amplifying protocol assay, was not demonstrated in SV40-transfected cells examined at the 7th passage.
Our data indicated that the proliferative activity of human bronchiolar epithelial cells was markedly enhanced by the transfection of SV40 genome but not by HPV16 or HPV18 genome. This enhancing effect of SV40 may be related to the binding and inhibiting effects of large T antigen on the p53 protein. Immortalization of the cells, however, was unable to be induced in the present study, presumably because of a limited proliferative activity of the primary cells used here. Further studies are required with primary cells that possess more active proliferative potential, for example those from the lungs of younger people.
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