PATHOLOGY OF CHEMICAL CARCINOGENISIS

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This proposal will explore a number of interrelated commonalities produced in hepatocytes by chemical carcinogens, including altered nucleocytoplasmic RNA transport, nuclear enlargement, and alterations in nuclear lamina nucleosidetriphosphatase (NTPase), an enzyme thought to regulate RNA transport. Carcinogens selectively produce permanent elevations in activity of the 46-kD NTPase. Recent sequencing data demonstrate that the 46-kD NTPase represents 3 alpha-helical domains from the N- terminal region of lamins A/C. On the basis of differential photoaffinity labeling and phosphorylation, we hypothesize that the NTPase (which also shows weak homology to a number of nuclear oncogene proteins and an E. coli ATPase) may represent the kinase activity which phosphorylates intact nuclear lamins; phosphorylation of lamins is integrally related to cellular (nuclear) growth and mitosis. Rats will be fed a carcinogenic diet and sacrificed after various intervals. Hyperplastic foci (vs. background liver) and frank hepatocellular carcinomas will be examined. To provide a biological context for identification of changes pertinent to carcinogenesis, we will also examine livers undergoing the acute phase response, livers regenerating from chemical and surgical insult, and livers following a single exposure to hepatocarcinogens. These preparations will be analyzed for alterations in: 1) RNA processing and compartamentation, using a variety of cDNA probes and an in vitro transport assay which maintains appropriate restriction of nuclear RNA sequences; 2) nuclear size/ploidy and nuclear pore number/density, using flow cytometric and freeze-fracture techniques; 3) lamin A/C phosphorylation; and 4) NTPase activity, using enzymatic assays and cDNA probes. NTPase cDNA probes will be obtained from rat cDNA libraries using synthetic polynucleotides designed from amino acid sequencing data, or by cross-hybridizing rat cDNA libraries with human lamin A/C probes, and will be cloned and sequenced. These probes will also be used to examine whether carcinogens produce changes in the NTPase gene. Our basic hypothesis is that carcinogens modify expression of the nuclear lamina NTPase, leading to alterations in nuclear structure (via lamin phosphorylation) and RNA transport: These changes would produce a cascade of phenotypic alterations, leading (under suitable promotional influences) to eventual malignant transformation, thus providing a heuristic explanation for the altered phenotyic expression associated with carcinogenesis.