Normal liver chromatin contains a firmly bound and larger protein related to the principal cytosolic target polypeptide of a hepatic carcinogen.

Abstract

A 14,000-dalton polypeptide was previously reported to be the principal protein target of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) in liver cytosol at the start of hepatocarcinogenesis in rats. The 14,000-dalton polypeptide was purified to homogeneity according to gel electrophoreses in both NaDodSO4-containing medium and acetic acid/urea and also by immunogenicity. An immunologically related form of the cytosolic target polypeptide has now been found to be present in the nuclei of normal rat liver as a 17,500-dalton polypeptide that is firmly and ionically bound to chromatin. Serial salt extractions of isolated liver nuclei or chromatin at 0.15 and 0.35 ionic strengths fail to dissolve the bound polypeptide, according to electrophoretic transfer immunoblot analyses. Most of the 17,500-dalton polypeptide is extracted at 0.65 ionic strength, the remainder at 1.2, and none at 2.0, nor thereafter in 8 M urea. In addition, shortterm digestion of purified liver nuclei with micrococcal nuclease solubilizes the 17,500-dalton polypeptide. All three protocols also solubilize low levels of intermediate 17,500-to 14,000-dalton species, the latter size being the same as that of the cytosolic protein target of the carcinogen. The presence of protease inhibitors during the isolations and extractions of the nuclei and chromatin reduces the amounts of these smaller polypeptides. In normal rat liver only nuclei and cytoplasm of hepatocytes contain reactive antigen according to peroxidaseantiperoxidase immunohistochemistry, staining most intensely perilobularly, less in the lobular midzone, and least centrilobularly. The nuclei of the perilobular hepatocytes constitute the strongest staining compartment within all of normal liver. Of 22 nonhepatic tissues of normal rats, 16 contain relatively few cells with immunoreactive cytoplasm. Nonhepatic nuclear antigen is present only in villar crest cells of duodenum (which are normally exposed to liver bile), also having cytoplasmic antigen as well. Five kinds of evidence appear to connect the chromatin-bound 17,500-dalton polypeptide of normal liver nuclei to the cytosolic 14,000-dalton polypeptide that is the principal target of the carcinogen early during hepatocarcinogenesis in rats. The present findings indicate a direct connection between a chromosomal protein and the immediate principal cytosolic protein target of a carcinogen.

Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes.

“…In hepatocytes of normal and regenerating livers, the cytoplasmic level of immunodetected p14 is markedly elevated only during mitosis as compared to that in adjacent interphase hepatocytes (4)(5)(6). In contrast, the concentration of discernible p14 is greatly increased throughout the cell cycle in proliferating hepatocytes of hyperplastic foci and hepatocellular carcinomas in livers of rats fed either of the carcinogens FAA or 3'-methyl-4-dimethylaminoazobenzene (4,7).…”

“…Previously isolated pl4 from normal rat liver was homogeneous in molecular size and charge (3,5). Rabbit antiserum raised against p14 was immunologically specific in reaction with normal rat liver cytosol (3,7).…”

“…In immunoaffinity purifications of p14 for use in the present study, rabbit anti-p14 immunoglobulins were coupled to Affi-Gel 10 (Bio-Rad). Nonspecifically bound 2S proteins were removed by successively washing the column with (i) 25 mM Tris-HCl (pH 7.5) containing 0.25% Triton X-100 and 0.5 M NaCl, (it) 25 mM Tris HCl, pH 7.5/0.15 M NaCl, (iii) 25 gag tgc gaa ctg gag acc atg act ggg gaa aag gtc aag gca gtg gtt aag atg gag ggt GAG TGC GAA CTG GAG ACC ATG ACT GGG GAA AAG GTC AAG GCA GTG GTT AAG ATO GAG GGT compared with matched immunoblots (3,5). Proteins were similarly analyzed after electrophoresis in 12% polyacrylamide slab gels containing 6 mM Triton X-100, 5% acetic acid, and 7.5 M urea (Triton/acetic acid/urea gels) (20).…”

“…In most immunoblots, p14 and L-FABP were subjected to NaDodSO4 gel electrophoresis and were electrotransferred onto nitrocellulose filter paper (0.1 ,um) (3,5). In other experiments, proteins in Triton/acetic acid/urea gels were transferred similarly, except for the addition of 0.1% NaDodSO4.…”

“…A donated cDNA expression library in bacteriophage Xgtll was previously generated from liver poly(A)+ RNA of normal adult Fischer rats (22). Recombinant phage clones were screened with p14 antiserum (3,5,23,24). Clones containing p14 cDNA were subcloned in plasmid vector pUC9 and then in bacteriophage M13mpl8 (24).…”