Project: Research project

Project Details


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.
Effective start/end date1/1/8812/31/88


  • National Cancer Institute
