We established two in vitro culture systems that have enabled us to study regulation of expression of differentiated functions in hepatocytes in vitro and to determine how transformation alters the expression of differentiated functions in hepatocytes in vitro. The two systems are (1) a long term culture system for primary hepatocytes in which the cells are maintained in a chemically-defined medium supplemented with dimethylsulfoxide and (2) a series of SV40-immortalized hepatocyte cell lines that retain the ability to express liver-specific functions at liver-like levels. During the current funding period, we have extended the system to include primary hepatocytes that synthesize DNA, spontaneously transformed SV40 hepatocyte cell lines, ras-transformed SV40 immortalized hepatocyte cell lines, tumor cell lines and two systems for studying suppression of the transformed phenotype. We have made several important findings. first, we have transformed SV40-immortalized hepatocytes with activated c-Ha ras and shown that ras-transformation caused marked changes in the morphology and growth properties of the immortal cells but did not decrease transcription of albumin or expression of several other liver-specific genes. However, the albumin enhancer was unable to function in ras-transformed hepatocytes. Differentiation at the level of albumin transcription was maintained in ras-transformed cells, but the underlying molecular mechanism driving albumin transcription was altered by transformation. Second, we determined that TGFbeta1 suppresses the transformed phenotype in ras- transformed hepatocytes by regulating alpha1beta1 integrin expression.
The specific aims of this proposal, which are logical extensions of the work ongoing in our laboratory, are (1) to continue to characterize the methods used by the hepatocytes and hepatocyte cell lines in our system to transcribe albumin; (2) to further examine suppression of the transformed phenotype by TGFbeta1 and alpha1 integrin; (3) to continue to define what viral and cellular DNA sequences can immortalize, transform, or suppress transformation of hepatocytes; to continue to determine what changes in oncogene and growth factor expression accompany transformation of hepatocytes; and (4) to determine the effect of site of inoculation (in vivo cellular environment) on growth, tumorigenicity, morphology and gene expression of transformed hepatocyte cell lines and tumor cell lines. We will continue to use in vitro hepatocyte systems to study differences in gene expression that occur during the process of transformation and to unravel the cellular and molecular mechanisms underlying these changes.
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