In this proposal, the mechanism for Collagen type I timer synthesIs by liver cells and the role of this molecule in hepatocellular carcinoma will be examined. Although collagen type I Chains are usually coordinately expressed, tumors, liver cirrhosis and transformed liver cells synthesize collagen type I timers, consisting of three alpha1(I) chains. Furthermore, cirrhosis appears to be a primary contributor to hepatocarcinoma. Because liver cells require a specific matrix for maintenance of their normal phenotype, the appearance of collagen type I timer could alter cell activity and cause abnormal cell proliferation and/or invasion.
The first aim i s to determine the mechanism for transcriptional inactivation of alpha2(I). A unique model system consisting of two liver cell lines will be utilized to examine collagen alpha2(I) gene expression. A carcinogen, 2-N-(acetoxyacetyl)-aminofluorine (AAF), has been used to transform rat liver epithelial-like cells, K16 . The resulting tumorigenic cell line, W8, produces type timer and does not transcribe alpha2(I) mRNA. The promoter-5' region of the alpha2(I) gene in W8 cells is methylated causing transcriptional inactivation. Methylation of the promoter/first exon at specific sites is hypothesized to alter binding and/or activity of proteins that regulate transcription. Experiments are proposed to examine how DNA-methylation inhibits alpha2(I) gene transcription at the basal transcription initiation region and at upstream regulatory sites. Methylation sites in the alpha2(I) promoter of W8 cells will be located by genomic sequencing. The binding of nuclear proteins to methylated regions of the alpha2(I) promoter will be investigated using DNA mobility gel shifts, Southwestern analysis and DNA footprinting. The function of specific DNA-methylation sites on alpha2(I) transcription will be tested by transfection and in vitro transcription assays.
The second aim i s to investigate the role of collagen type I timer in tumorigenicity. Recently, W8 cells have been transfected with alpha2(I) cDNA eukaryotic expression vectors. The new stable cell lines secrete normal collagen and form fewer colonies in soft agar, a hallmark of tumorigenicity. This data provides important evidence that decreased alpha2(I) gene expression contributes to the transformed phenotype. A logical extension of these studies is to test tumorigenicity by injecting transfected cell lines secreting varying ratios of type (I) timer and normal collagen with and without type (I) timer matrix into animals.
The third aim i s to examine normal, cirrhotic, and neoplastic liver tissues for the presence of collagen type (I) timers and DNA-methylation of the alpha2(I) gene. The presence of type (I) timer in cirrhosis could provide a matrix that promotes proliferation of cells contributing to the emergence of tumors. Preliminary immunohistochemistry of neoplastic tissue in cirrhotic liver demonstrates the presence of type I timer. In addition, type (I) timer formation in vivo is hypothesized to be due to DNA- methylation of the alpha2(I) gene. Therefore, the methylation status of the alpha2(I) gene in tissue containing type (I) timer will be examined.
