We are investigating the mechanisms by which a choline-deficient (CD) diet, which induces reactive oxygen species (ROS), results in hepatocarcinogenesis in vivo in rats. Our work with CWSV-1 rat hepatocytes links ROS with changes in mitochondrial membrane potential as a possible prelude to carcinogenesis. To enhance our ability to analyze this carcinogenesis process at different stages, we have developed an organotypic rat liver culture model in which viable tissue has been maintained in vitro as very thin slices (< 1mm thick) for longer than one year. Viability has been determined with dual vital staining (trypan blue and neutral red), as well as with histology of fresh and cultured tissue. Immunohistochemistry is being used to determine the presence of tumor markers including GST-P (glutathione S-transferase - placental form), CD44, c-Met (hepatocyte growth factor receptor), and PCNA, in long-term liver slices cultured in defined/serum-free media with choline-sufficient (HC = high choline) versus choline-deficient (CD = zero choline) media. Two experiments are being analyzed in which the organotypic cultures have been maintained for 6 months. The results show that choline deficiency (CD medium) results in a marked increased expression of GST-P, a widely used marker for the pre-cancerous state, between 12 and 16 weeks of culture. The presence of 1 mM PBN in the medium markedly inhibits this expression of GST-P, to near control (HC) levels. In contrast, CD44 (a stem and/or progenitor cell marker) is high in HC cultures beginning with day 1, is lower in CD cultures, and is virtually absent from CD cultures containing 1 mM PBN. c-Met and PCNA, which typically come on later in the in vivo model, are not seen in tissue sections of these cultures up to 16 weeks of CD treatment. Choline deficiency is known to result global changes in DNA methylation. Previous results from our group have shown that CD leads to hypomethylation of CG islands in the promoter regions of specific genes, which in turn correlates with increased gene expression. Specifically, CD leads to hypomethylation of the promoter of GST-P and its increased expression, in rat liver tissue in vivo, ultimately progressing to hepatocellular carcinoma (HCC). PBN in the diet inhibits the formation of HCC lesions. Thus, our organotypic culture model reproduces in vivo results, especially with respect to induction of GSTP expression by CD and inhibition of this induction by PBN. Supported by NIH grant # R01 CA82506.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]