This grant has been continuously funded for more than nineteen years to study REGULATION OF DIFFERENTIATION IN HEPATOCYTES IN VITRO. The applicant has established two in vitro culture systems. (1) A long term culture system for primary hepatocytes in which the cells are maintained in a chemically-defined medium supplemented with dimethylsulfoxide (DMSO), and (2) A series of immortalized hepatocyte cell lines, transformed cell lines and tumor cell lines that retain the ability to express liver-specific functions, some at liver-like levels. During the current funding period, she has utilized both systems to make several important findings. She has demonstrated that hepatocytes in long-term DMSO culture: (1) Maintain the potential not only to undergo DNA synthesis but also to proliferate, (2) Can differentiate into bile duct-like cells, and (3) Can express specific oncogenes and growth factors in a similar pattern to what is observed in regenerating liver, when induced to synthesize DNA. Using immortalized hepatocyte cell lines and transformed and tumor cell lines derived from them, the applicant has made several contributions with regard to the effect of transformation on expression of differentiated functions, regulation of albumin and alpha 1 integrin expression, factors that suppress the transformed phenotype in transformed hepatocytes and differential responses of premalignant and malignant cells to TGF-beta. She has also made several important technical advances in gene delivery that will enable her to carry out both gain and loss of function studies in both culture systems. In the current application, the applicant proposes to pursue specific findings made during the past funding period and focus her studies on regulation of alpha 1 integrin expression in hepatocytes, the relationship between alpha 1 integrin expression and growth control in hepatocytes and TGF-beta 1 induced suppression of the transformed phenotype. She hypothesizes that transformation in hepatocytes is accompanied by loss of alpha 1 integrin expression which may be mediated by increased expression of ras, and that suppression of the transformed phenotype in hepatocytes induced by TGF-beta may be mediated, at least in part, by restoration of alpha 1 integrin expression.
The specific aims are: (1) To examine regulation of expression of alpha 1 integrin in hepatocytes and hepatocyte cell lines; (2) To determine whether alpha 1 integrin expression plays an active role in growth control; (3) To elucidate key players in the signal transduction pathway used by TGF-beta to suppress the transformed phenotype; (4) To determine whether the type II/type I TGF-beta receptor ratio is related to the differential responses to TFG-beta observed in hepatocytes and hepatocyte cell lines.
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