A Mr 14,000 polypeptide (p14), identified as liver fatty acid binding protein, in normal liver cytosol was shown previously to be the principal target of the carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene), early during hepatic carcinogenesis in rats. Immunohistochemical analyses using rabbit antiserum against pure p14/liver fatty acid binding protein revealed marked increases in the levels of the protein in cytoplasm specifically during mitosis in normal and regenerating hepatocytes, and throughout the cell cycle in hyperplastic and malignant hepatocytes brought about by carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene) or 3′-methyl-4-dimethylaminoazobenzene. Present also in normal hepatocytes was a nuclear antigen that was not detected in the hyperplastic hepatocytes, benign hepatocytic adenomas, and hepatocellular carcinomas produced by these carcinogens. The nuclear antigen was discerned to be a Mr 17,000 polypeptide (p17) in extracts of normal liver nuclei and nucleosomes.
In the present study, the p17 was purified by high-performance liquid chromatography and identified as being the three variants of histone H3, based on common molecular size, amino acid composition, electrophoretic migration in Triton-acetic acid-urea gels, and Western blot and histochemical reactions using affinity-purified antibodies. The histone H3 of all tested organs reacted specifically with the antiserum in Western blots following sodium dodecyl sulfate gel electrophoresis. In contrast, in a survey of 23 normal rat organs, nuclei of virtually only hepatocytes were reactive immunohistochemically. In view of the exceptional immunohistochemical reactivity of nuclei of normal hepatocytes, attributable to accessible histone H3, and the lack of such reaction in carcinogen-altered hepatocytes, the collected evidence indicates that normal hepatocytes contain uniquely available histone H3 sites that become cryptic during the chemical carcinogenesis.
Supported in part by NIH Grant CA-05945, institutional NIH Grants CA-06927 and RR-05539, and an appropriation from the Commonwealth of Pennsylvania.