Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Apr;81(7):2092–2096. doi: 10.1073/pnas.81.7.2092

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

S A Vinores, J J Churey, J M Haller, S J Schnabel, R P Custer, S Sorof
PMCID: PMC345443  PMID: 6585789

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, short-term 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 peroxidase-antiperoxidase 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.

Full text

PDF
2092

Images in this article

2093Image
on p.2093
2094Image
on p.2094
2094Image
on p.2094
2095Image
on p.2095
2095Image
on p.2095
2095Image
on p.2095

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blackburn G. R., Andrews J. P., Custer R. P., Sorof S. Early events during liver carcinogenesis involving two carcinogen:protein complexes. Cancer Res. 1981 Oct;41(10):4039–4049. [PubMed] [Google Scholar]
  2. Blackburn G. R., Andrews J. P., Rao K. V., Sorof S. An early event associated with liver carcinogenesis involving loss of a polypeptide that binds carcinogen. Cancer Res. 1980 Dec;40(12):4688–4693. [PubMed] [Google Scholar]
  3. Blackburn G. R., Schnabel S. J., Danley J. M., Hogue-Angeletti R. A., Sorof S. Principal polypeptide target of carcinogen at the beginning of liver carcinogenesis by three carcinogens. Cancer Res. 1982 Nov;42(11):4664–4672. [PubMed] [Google Scholar]
  4. Burgoyne L. A., Wagar M. A., Atkinson M. R. Initiation of DNA synthesis in rat thymus: correlation of calcium-dependent initiation in thymocytes and in isolated thymus nuclei. Biochem Biophys Res Commun. 1970 Jun 5;39(5):918–922. doi: 10.1016/0006-291x(70)90411-0. [DOI] [PubMed] [Google Scholar]
  5. Gronow M., Thackrah T. Changes in the composition of rat liver chromatin fractions during nitrosamine carcirogenesis. Eur J Cancer. 1974 Jan;10(1):21–25. doi: 10.1016/0014-2964(74)90033-4. [DOI] [PubMed] [Google Scholar]
  6. Hewish D. R., Burgoyne L. A. Chromatin sub-structure. The digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonuclease. Biochem Biophys Res Commun. 1973 May 15;52(2):504–510. doi: 10.1016/0006-291x(73)90740-7. [DOI] [PubMed] [Google Scholar]
  7. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  8. Lennox R. W., Oshima R. G., Cohen L. H. The H1 histones and their interphase phosphorylated states in differentiated and undifferentiated cell lines derived from murine teratocarcinomas. J Biol Chem. 1982 May 10;257(9):5183–5189. [PubMed] [Google Scholar]
  9. MacLeod M. C., Pelling J. C., Slaga T. J., Noghrei-Nikbakht P. A., Mansfield B. K., Selkirk J. K. Specificity of interaction between carcinogenic polynuclear aromatic hydrocarbons and nuclear proteins: widespread occurrence of a restricted pattern of histone binding in intact cells. Prog Nucleic Acid Res Mol Biol. 1983;29:111–115. doi: 10.1016/s0079-6603(08)60437-7. [DOI] [PubMed] [Google Scholar]
  10. Miller E. C., Miller J. A. Mechanisms of chemical carcinogenesis. Cancer. 1981 Mar 1;47(5 Suppl):1055–1064. doi: 10.1002/1097-0142(19810301)47:5+<1055::aid-cncr2820471302>3.0.co;2-3. [DOI] [PubMed] [Google Scholar]
  11. Miller E. C., Miller J. A. Searches for ultimate chemical carcinogens and their reactions with cellular macromolecules. Cancer. 1981 May 15;47(10):2327–2345. doi: 10.1002/1097-0142(19810515)47:10<2327::aid-cncr2820471003>3.0.co;2-z. [DOI] [PubMed] [Google Scholar]
  12. Noll M., Thomas J. O., Kornberg R. D. Preparation of native chromatin and damage caused by shearing. Science. 1975 Mar 28;187(4182):1203–1206. doi: 10.1126/science.187.4182.1203. [DOI] [PubMed] [Google Scholar]
  13. Panyim S., Chalkley R. High resolution acrylamide gel electrophoresis of histones. Arch Biochem Biophys. 1969 Mar;130(1):337–346. doi: 10.1016/0003-9861(69)90042-3. [DOI] [PubMed] [Google Scholar]
  14. Sarrif A. M., Bertram J. S., Kamarck M., Heidelberger C. The isolation and characterization of polycyclic hydrocarbon-binding proteins from mouse liver and skin cytosols. Cancer Res. 1975 Mar;35(3):816–824. [PubMed] [Google Scholar]
  15. Schmidt W. N., Gronert B. J., Page D. L., Briggs R. C., Hnilica L. S. Antigenic changes in nonhistone proteins during azo dye hepatocarcinogenesis. Cancer Res. 1982 Aug;42(8):3164–3174. [PubMed] [Google Scholar]
  16. Sorof S., Sani B. P., Kish V. M., Meloche H. P. Isolation and properties of the principal liver protein conjugate of a hepatic carcinogen. Biochemistry. 1974 Jun 4;13(12):2612–2620. doi: 10.1021/bi00709a022. [DOI] [PubMed] [Google Scholar]
  17. Sorof S., Young E. M., McBride R. Z., Coffey C. B., Luongo L. Increased selectivity of interaction between fluorenylamine carcinogens and liver proteins during hepatocarcinogenesis. Mol Pharmacol. 1969 Nov;5(6):625–639. [PubMed] [Google Scholar]
  18. Stein G. S., Stein J. L., Thomson J. A. Chromosomal proteins in transformed and neoplastic cells: a review. Cancer Res. 1978 May;38(5):1181–1201. [PubMed] [Google Scholar]
  19. Takami H., Busch F. N., Morris H. P., Busch H. Comparison of salt-extractable nuclear proteins of regenerating liver, fetal liver, and Morris hepatomas 9618A and 3924A. Cancer Res. 1979 Jun;39(6 Pt 1):2096–2105. [PubMed] [Google Scholar]
  20. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  21. Yankner B. A., Shooter E. M. Nerve growth factor in the nucleus: interaction with receptors on the nuclear membrane. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1269–1273. doi: 10.1073/pnas.76.3.1269. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES