It is known that long-term withdrawal of choline from the diet induces hepatocellular carcinomas in animal models in the absence of known carcinogens. We hypothesize that a choline deficient diet (CD) alters the balance of cell growth and cell death in hepatocytes and thus promotes the survival of clones of cells capable of malignant transformation. When grown in CD medium (5 μM or 0 μM choline) CWSV-1 rat hepatocytes immortalized with SV40 large T-antigen underwent p53-independent apoptosis (terminal dUTP end-labeling of fragmented DNA; laddering of DNA in agarose gel). CWSV-1 cells which were adapted to survive in 5 μM choline acquired resistance to CD-induced apoptosis and were able to form hepatocellular carcinomas in nude mice. These adapted CWSV-1 cells express higher amounts of both the 32 kDa membrane-bound and 6 kDa mature form of TGFα compared to cells made acutely CD. Control (70 μM choline) and adapted cells, but not acutely deficient hepatocytes, could be induced to undergo apoptosis by neutralization of secreted TGFα. Protein tyrosine phosphorylation is known to protect against apoptosis. We found decreased EGF receptor tyrosine phosphorylation in acutely choline deficient CWSV-1 cells. TGFβ1 is an important growth- regulator in the liver. CWSV-1 cells express TGFβ1 receptors and this peptide induced cell detachment and death in control and acutely deficient cells. Hepatocytes adapted to survive in low choline were also resistant to TGFβ1, although TGFβ1 receptors and protein could be detected in the cytoplasm of these cells. The non-essential nutrient choline is important in maintaining plasma membrane structure and function, and in intracellular signaling. Our results indicate that acute withdrawal of choline induces p53- independent programmed cell death in hepatocytes, whereas cells adapted to survive in low choline are resistant to this form of apoptosis, as well as to cell death induced by TGFβ1. Our results also suggest that CD may induce alterations (mutations?) in growth factor signaling pathways which may enhance cell survival and malignant transformation.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)