Zusammenfassung
Phenol-lösliche Chromatinprotein wurden aus Zellkernen normaler und leukämischer menschlicher Leukozyten isoliert. Die Proteine, deren Bedeutung im Rahmen der Kontrolle des Transkriptionsvorganges diskutiert wird, wurden durch Analyse ihrer gelelektrophoretischen Verteilungsmuster und ihrer Markierung mit14C-Leuzin oder32P-Orthophosphat verglichen. Außerdem wurde der Einfluß interkalierender Agentien wie Adriamycin untersucht.
Die Fraktion phenol-löslicher Nicht-Histon-Proteine des Chromatins menschlicher Leukozyten enthält mindestens 20–35 individuelle Proteine, welche mit zunehmender Laufgeschwindigkeit auf 10%-Polyacrylamidgelen numeriert wurden. Zwischen normalen Lymphozyten und normalen Granulozyten ergaben sich charakteristische Unterschiede mit jeweils einer spezifischen Proteinbande (Nr. 26a bei Lymphozyten; Nr. 25 bei Granulozyten) für beide Zelltypen. Interessanterweise war das Protein Nr. 25 in Myelozyten (CML) nicht vorhanden, jedoch in reifen Granulozyten desselben Patienten (CML) nachweisbar. Die radioaktiven Markierungen der Proteine mit32P (Phosphorilierung) bzw.14C-Leuzin (Umsatz) nahmen ganz allgemein mit zunehmender Zelldifferenzierung ab. Leukämische Lymphozyten unterschieden sich von normalen Lymphozyten durch erhöhte Protein-Konzentrationen im hochmolekularen Bereich. Beim Vergleich von Zellen aus AML, ALL und der CML-Blastenkrise ergaben sich bei weitgehend ähnlichen Mustern keine typischen Differenzen. Ebenso waren gemeinsame leukämiespezifische Abweichungen gegenüber Normalzellen nicht nachzuweisen. Lymphosarkomzellen zeigten allerdings quantitative und qualitative Abweichungen vom gewöhnlichen Verteilungsmuster der Zellkernproteine.
Nach Einwirkung von Adriamycin in vitro kam es bei den verschiedensten Zellinien dosisabhängig zu einer selektiven Abnahme eines bestimmten Proteins (Nr. 30) dessen mögliche Funktion als „marker“ für den Interkalationslocus von Adriamycin an der DNA diskutiert wird.
Summary
Phenol-soluble chromatin proteins which may be involved in gene control mechanisms have been isolated from citric acid nuclei of normal and leukaemic human leukocytes derived from freshly obtained venous blood. They were compared by one-dimensional gel electrophoresis and by comparative labelling with14C-leucine or32P-orthophosphate. In addition, the influence of intercalating agents such as adriamycin and daunomycin was studied.
Between 20–35 individual proteins were found in the phenol-soluble nonhistone protein fraction of human leukocytes. They were numbered with increasing mobility on 10% polyacrylamide gels. Distinct differences were found between normal lymphocytes and normal granulocytes, with one specific protein (no. 26 a in lymphocytes; no. 25 in granulocytes) for both cell types. Interestingly, protein no. 25 was not present in CML myelocytes but in CML granulocytes.32P-and leucine labels were generally found decreased with increasing cell differentiation. Leukaemic lymphocytes differed from normal lymphocytes by increased protein concentrations in the high molecular weight region.
In comparisons of AML and ALL cells, including blast cells of CML blast crisis, no constant differences nor any markers common to leukaemic cells as compared to normal cells were detectable. However, lymphosarcoma cells showed quantitiative and qualitative aberrations from the usual leukaemia pattern. Afterin vitro incubations of cells with adriamycin a selective decrease of an individual protein (no. 30) was noted. This protein may serve as a marker for the intercalation site.
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References
Arnold, E.A., Buksas, M.M., Young, K.E.: A comparative study of some properties of chromatin from two “minimal deviation” hepatomas. Cancer Res.33, 1169–1176 (1973)
Barrett, T., Maryanka, D., Hamlyn, P.H., Gould, H.J.: Nonhistone proteins control gene expression in reconstituted chromatin. Proc. nat. Acad. Sci. USA71, 5057–5061 (1974)
Biessmann, H., Rajewsky, M.F.: Nuclear protein patterns in developing and adult brain and in ethylnitrosourea-induced neuroectodermal tumours of the rat. J. Neurochem.24, 387–393 (1975)
Boyum, A.: Separation of leucocytes from blood and bone marrow. Scand. J. clin. Lab. Invest.21, Suppl. No. 97 (1968)
Busch, H., Ballal, N.R., Olson, M.O.J., Yeoman, L.C.: Chromatin and its nonhistone proteins. In: Methods in Cancer Research (H. Busch, ed.), Vol. 11, pp. 43–121. New York: Academic Press 1975
Chae, C.B., Smith, M.C., Morris, H.P.: Chromosomal nonhistone proteins of rat hepatomas and normal rat liver. Biochem. biophys. Res. Commun.60, 1468–1474 (1974)
Chiu, J.F., Tsai, Y.H., Sakuma, K., Hnilica, L.S.: Regulation ofin vitro mRNA transcription by a fraction of chromosomal proteins. J. biol. Chem.250, 9431–9433 (1975)
Desai, L.S.: Mechanism of gene interaction and histone deacetylation in human leukemic cells. J. Cell Biol.63, 82a, Abstr. no. 163 (1974)
Desai, L.S., Wulff, U.C., Foley, G.E.: Properties of chromosomal proteins of human leukemic cells. Biochimie57, 315–324 (1975)
Di Marco, A., Arcamone, F.: DNA complexing antibiotics — daunomycin, adriamycin and their derivatives. Arzneim.-Forsch.25, 368–375 (1975)
Dounce, A.L.: The isolation and composition of cell nuclei and nucleoli. In: The Nucleic Acids. (E. Chargaff and J.N. Davidson, eds.), Vol. 2, pp. 93–153. New York: Academic Press 1955
Johnson, E.M., Karn, J., Allfrey, V.G.: Early nuclear events in the induction of lymphocyte proliferation by mitogens. Effects of concanavalin A on the phosphorylation and distribution of nonhistone chromatin proteins. J. biol. Chem.249, 4990–4999 (1974)
Kostraba, N.C., Wang, T.Y.: Differential activation of transcription of chromatin by non-histone fractions. Biochim. biophys. Acta (Amst.)262, 169–180 (1972)
Levy, R., Levy, S., Rosenberg, S.A., Simpson, R.T.: Selective stimulation of nonhistone chromatin protein synthesis in lymphoid cells by phytohemagglutinin. Biochemistry12, 224–228 (1973)
MacGillivray, A.J., Carroll, D., Paul, J.: The heterogeneity of the non-histone chromatin proteins from mouse tissues. FEBS Lett.13, 204–208 (1971)
MacGillivray, A.J., Cameron, A., Krauze, R.J., Rickwood, D., Paul, J.: The non-histone proteins of chromatin. Their isolation and composition in a number of tissues. Biochim. biophys. Acta (Amst.)277, 384–402 (1972)
Marushige, K., Bonner, J.: Template properties of liver chromatin. J. molec. Biol.15, 160–174 (1966)
Marushige, K., Brutlag, D., Bonner, J.: Properties of chromosomal nonhistone protein of rat liver. Biochemistry7, 3149–3155 (1968)
Masera, P., Pileri, A., Brachet, J., Hulin, N.: Lymphocyte actinomycin binding capacity in chronic lymphocytic leukaemia. Experientia (Basel)28, 1484 (1972)
McClure, M.E., Hnilica, L.S.: Nuclear proteins in genetic restriction. III. The cell cycle. Sub-cell. Biochem.1, 311–332 (1972)
Noodén, L.D., van den Broek, H.W.J., Sevall, J.S.: Stabilization of histones from rat liver. FEBS Lett.29, 326–328 (1973)
Paul, J., Gilmour, R.S.: Organ-specific restriction of transcription in mammalian chromatin. J. molec. Biol.34, 305–316 (1968)
Pigram, W.J., Fuller, W., Hamilton, L.D.: Sterochemistry of intercalation: interaction of daunomycin with DNA. Nature, New Biol.235, 17–19 (1972)
Pileri, A., Masera, P., Hulin, N.: Brachet, J.: Actinomycin binding capacity in human leukaemic lymphoid cells. Acta haemat. (Basel)48, 89–97 (1972)
Platz, R.D., Kish, V.M., Kleinsmith, L.J.: Tissue specificity of non-histone chromatin phosphoproteins. FEBS Lett.12, 38–40 (1970)
Sawada, H., Gilmore, V.H., Saunders, G.F.: Transcription from chromatins of human lymphocytic leukemia cells and normal lymphocytes. Cancer Res.33, 428–434 (1973)
Seeber, S., Schmidt, C.G.: Isolation of rapidly labelled nuclear RNA of high specific activity from human leukaemic cells. Klin. Wschr.51, 677–679 (1973)
Seeber, S., Brucksch, K.P., Käding, J., Schmidt, C.G., Busch, H.: Oligonucleotides of ribosomal 28 S RNA in human leukemic cells and normal lymphocytes. Cancer Res.34, 1281–1288 (1974a)
Seeber, S., Käding, J., Brucksch, K.P., Schmidt, C.G.: Defective rRNA synthesis in human leukaemic blast cells? Nature (Lond.)248, 673–675 (1974b)
Seeber, S., Schmidt, C.G., Busch, H.: Isolation, separation and fractionation of human leukemic and normal leukocytes. Comparative studies on preribosomal and ribosomal RNA and on non-histone chromatin proteins. In: Methods in Cancer Research (H. Busch, ed.), Vol. 14, pp. 131–190. New York: Academic Press 1978
Spelsberg, T.C., Hnilica, L.S., Ansevin, A.T.: Proteins of chromatin in template restriction. III. The macromolecules in specific restriction of the chromatin DNA. Biochim. biophys. Acta (Amst.)228, 550–562 (1971)
Stein, G.S., Borun, T.W.: The synthesis of acidic chromosomal proteins during the cell cycle of HeLa S-3 cells. I. The accelerated accumulation of acidic residual nuclear protein before the initiation of DNA replication. J. Cell Biol.52, 292–307 (1972)
Stein, G.S., Park, W., Thrall, C., Mans, R., Stein, J.: Regulation of cell cycle stage-specific transcription of histone genes from chromatin by non-histone chromosomal proteins. Nature (Lond.)257, 764–767 (1975)
Taylor, C.W., Yeoman, L.C., Daskal, I., Busch, H.: Two-dimensional electrophoresis of proteins of citric acid nuclei prepared with aid of a Tissumizer®. Exp. Cell Res.82, 215–226 (1973)
Teng, C.S., Hamilton, T.H.: Role of chromatin in estrogen action in the uterus. II. Hormone-induced synthesis of nonhistone acidic proteins which restore histone-inhibited DNA-dependent RNA synthesis. Proc. nat. Acad. Sci. USA63, 465–472 (1969)
Teng, C.S., Teng, C.T., Allfrey, V.G.: Studies of nuclear acidic proteins. Evidence for their phosphorylation, tissue specificity, selective binding to deoxyribonucleic acid, and stimulatory effects on transcription. J. biol. Chem.246, 3597–3609 (1971)
Viñuela, E., Algranati, I.D., Ochoa, S.: Synthesis of virus-specific proteins in Escherichia coli infected with the RNA bacteriophage MS2. Europ. J. Biochem.1, 3–11 (1967)
Wang, T.Y.: Restoration of histone-inhibited DNA-dependent RNA synthesis by acidic chromatin proteins. Exp. Cell Res.53, 288–291 (1968)
Weisenthal, L.M., Ruddon, R.W.: Characterization of human leukemia and Burkitt lymphoma cells by their acidic nuclear protein profiles. Cancer Res.32, 1009–1017 (1972)
Weisenthal, L.M., Ruddon, R.W.: Catabolism of nuclear proteins in control and phytohemagglutinin-stimulated human lymphocytes, leukemic leukocytes, and Burkitt lymphoma cells. Cancer Res.33, 2923–2935 (1973)
Wu, F.C., Elgin, S.C.R., Hood, L.E.: Nonhistone chromosomal proteins of rat tissues. A comparative study by gel electrophoresis. Biochemistry12, 2792–2797 (1973)
Yeoman, L.C., Taylor, C.W., Jordan, J.J., Busch, H.: Two-dimensional polyacrylamide gel electrophoresis of chromatin proteins of normal rat liver and Novikoff hepatoma ascites cells. Biochem. biophys. Res. Commun.53, 1067–1076 (1973)
Yeoman, L.C., Taylor, C.W., Jordan, J.J., Busch, H.: Differences in chromatin proteins of growing and non-growing tissues. Exp. Cell Res.91, 207–215 (1975)
Yeoman, L.C., Seeber, S., Taylor, C.W., Fernbach, D.J., Falletta, J.M., Jordan, J.J., Busch, H.: Differences in chromatin proteins of resting and growing human lymphocytes. Exp. Cell Res.100, 47–55 (1976)
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These studies were supported by grant Se 161/6 from Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg
Supported by Humboldt-Stiftung
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Seeber, S., Meshkov, T., Brucksch, K.P. et al. Comparative studies on phenol-soluble nonhistone chromatin proteins in normal and leukaemic human leukocytes. Klin Wochenschr 57, 257–265 (1979). https://doi.org/10.1007/BF01476506
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DOI: https://doi.org/10.1007/BF01476506