Late Target Protein of the Carcinogen N-2-Fluorenylacetamide in Rat Liver

Abstract

In previous studies, administration of a radioactive tracer dose of the liver carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene; FAA), to normal or carcinogen-fed rats led to the presence of one or two principal labeled carcinogen:protein complexes in liver cytosol. The early target M_r 14,000 protein of the carcinogen in normal rats was identified as being liver fatty acid-binding protein and was associated in hepatocytes with normal mitosis and the cell proliferation brought about by either of the two liver carcinogens, FAA or 3′-methyl-4-dimethylaminoazobenzene. Continued ingestion of any of the three hepatocarcinogens, FAA, 3′-methyl-4-dimethylaminoazobenzene, or ethionine, resulted in the progressive loss of the early radioactive complex and the concurrent gain in liver cytosol of the late carcinogen:protein complex (M_r ∼150,000) formed by the tracer dose of FAA.

Present attempts to extract FAA derivatives from the late carcinogen:protein complex with organic solvents indicated that virtually all of the carcinogen was apparently covalently bound to the resultant denatured protein. It is unknown whether the covalent interaction occurred in vivo or as an accompaniment of the protein denaturation associated with the solvent extractions. In support of a possible noncovalent interaction, treatment of the unextracted complex with sodium dodecyl sulfate, urea, and β-mercaptoethanol followed by electrophoresis readily dissociated the majority of the bound carcinogen. The late carcinogen:protein complex was shown to contain a 55 kDa subunit (p55), which was purified to homogeneity according to molecular size. The subunit is a relatively basic polypeptide with a pI of 8.4 to 8.6. In Western blots using rabbit immunoglobulins against the p55, the late target protein was found to be present at low concentrations in liver cytosols of normal rats, and was induced to relatively high levels by ingestion of the carcinogen for 3 to 5 weeks. The induction of high levels of the late target protein explains in part the progressive elevation in content of the late carcinogen:protein complex in rat liver during carcinogenesis by FAA. The isolated p55 was susceptible to a spontaneous stepwise breakdown, resulting in a ladder of decreasing molecular sizes with an average unit difference of 5.8 kDa per step over six size intervals. The p55 subunit was detected in nonhepatic organs of normal rats, but unlike levels in liver, the levels there were not affected by ingestion of the carcinogen. The purification of the p55 subunit and preparation of its immunoglobulins provide the basis of future studies on the functional roles of the late target protein and its relationships to the early target protein in hepatocytes.