This project will test a hypothesis that carcinogenesis evolves through multiple stages by degradation of growth control pathways. Loss of cell cycle checkpoint controls gives initiated cells a growth advantage and induces genetic instability which accelerates malignant progression. Chemically initiated rat hepatocytes will be isolated based upon their selective ability to grow in vitro under conditions in which normal hepatocytes die. Such extended-lifespan variants correspond to the first stage of growth disregulation in models of multistage carcinogenesis. Extended-lifespan hepatocytes may progress during growth in vitro to immortal lines with indefinite lifespan. Immortal lines may evolve further to hepatocellular carcinoma. Normal rat hepatocytes in primary culture and initiated hepatocytes at these stages of neoplastic evolution will be tested for expression of checkpoints which regulate the transitions between cycle phases. When cellular DNA is damaged by radiation, growth normally is arrested at three checkpoints which control the rates of transition from G1 into S, rates of replicon initiation in S phase cells, and rates of transition from G2 into M. We will establish biological and biochemical features of checkpoint response to DNA damage in normal hepatocytes and determine the patterns of degradation of control at stages of neoplastic evolution. Gene amplification ability also will studied as a quantitative marker of the genetic instability that is associated with loss of checkpoint control. The liver tumor promoter, phenobarbital (PB), is required for expression of the extended lifespan phenotype by initiated hepatocytes in vitro and PB-dependent immortal hepatocyte lines have been isolated. We will determine whether PB promotes the extended and indefinite proliferative lifespans of initiated hepatocytes by inducing an autocrine loop and whether this promoter may suppress checkpoint function in initiated hepatocytes by inhibiting expression of the tumor suppressor gene, p53. We will determine the alterations in p53 structure and expression which may occur as hepatocytes progress to cancer. TGF-alpha is a hepatocyte growth factor which is frequently expressed in hepatocellular neoplasms but rarely expressed in pre-neoplastic foci. We will determine whether acquisition of an autocrine growth pathway can speed malignant conversion by hepatocyte lines. This project will connect a well-described animal model of chemical carcinogenesis to current theories on cell cycle control in cancer and multi-stage carcinogenesis.
NIH ExPorter
NIH ExPorter