Thioacetamide-stimulated expression of non-histone protein.

The search for cancer specific nuclear proteins, stimulated by the supposition that transition from the normal to the neoplastic state resulting from disturbances in the control mechanisms of gene expression, indicated that non-histone protein of MW 48 kD is much more abundant in animal tumour cells than in normal liver (Krajewska et al., 1990). A non-histone component of MW 48 kD was assessed for changes during chemically induced carcinogenesis. Rats were treated with the hepatocarcinogen thioacetamide (TAA) and the expression of the polypeptide studied, in total nuclear protein and nonhistone protein fractions, was tested by Western blot technique in the presence of antibodies developed against a component of MW 48 kD from Kirkman-Robbins hepatoma. It was demonstrated that TAA-induced hepatocarcinogenesis was accompanied by the expression of non-histone protein of MW 48 kD at a significantly elevated level. A clear and distinct change in the expression of the component studied in the spleen of TAA-treated rats was also observed. These results support the suggestion that over-expression of non-histone protein of MW 48 kD could contribute to neoplastic transformation.

Nuclear distribution pattern of tumour-associated nonhistone protein of mol. wt 48,000.

1. As a further step toward characterizing nonhistone protein of mol. wt 48,000 which was found to be much more abundant in animal tumour cells than in normal ones [Krajewska W.M., Lipínska A., Marszatek M., Kilianska Z., Wojtkowiak Z. and Klyszejko-Stefanowicz L. Cell. Biochem. Funct. 8, 79-89 (1990)] its intranuclear localization in hamster liver and Kirkman-Robbins hepatoma was studied. The protein was identified by immunoblotting technique in the presence of antibodies against polypeptide of mol. wt about 48,000 from Kirkman-Robbins hepatoma. 2. Distribution of antigen with mol. wt of 48,000 in nuclear fractions representing different levels of nuclear material organization, i.e. in nucleoli, nuclease-sensitive and nuclease-resistant fractions, and extensive nuclease digestion products separated by size on Bio-Gel A-50m; implied the structural role of this component. 3. Fractionation of endogenously digested nuclei into low salt extract, high salt extract and nuclear matrix revealed that in normal liver the antigen studied is associated with nuclear matrix while in hepatoma this component appeared in high salt extract. 4. These results suggest that polypeptide with mol. wt of 48,000 is a shuttling protein which may be involved in reorganization of nuclear matrix during neoplastic transformation.

Nuclear proteins of hamster hepatoma after administration of antitumour agents.

One- and two-dimensional gel electrophoretic analysis of nuclear proteins of Kirkman-Robbins hepatoma was used to study the effects of the tumour growth inhibitors methotrexate (MTX) and acyclonucleoside (DHPQtB) on protein composition. MTX and DHPQtB inhibited Kirkman-Robbins hepatoma growth by 89.2 +/- 3.5% and 16.3 +/- 6.1% respectively. The biosynthesis and/or metabolism of some polypeptide spots was affected by these antitumour agents, especially among components with molecular wt/isoelectric points of 52,000-64,000/4.9-5.5, 69,000-78,000/5.0-5.9 and 88,000-100,000/5.1-5.9.

Hepatoma-associated nuclear matrix nonhistone antigens.

Polyclonal antibodies generated against a group of high molecular weight nonhistone proteins from Morris hepatoma 7777 were used in immunological studies of hepatoma-associated nonhistone proteins in rat and hamster. We revealed the presence of cross-reactive antigens in rat Morris hepatomas 7777 and 8994, and in hamster Kirkman-Robbins hepatoma, but not in normal rat or hamster livers. These specific nonhistone proteins were found to be preferentially localized in the nuclear matrix of rat Morris hepatoma 7777 as well as hamster Kirkman-Robbins hepatoma.

Nuclear antigen with a molecular weight of 48,000 associated with malignant transformation.

1. Analysis of immunoblots of one- and two-dimensional electropherograms of non-histone proteins from Kirkman-Robbins hepatoma, Morris hepatoma 7777, regenerating liver and normal liver demonstrated the presence of elevated level of nuclear antigen with mol. wt of 48,000 and pI of 5.4 in tumour cells. 2. Small amounts only were detected in proliferating and quiescent cells suggesting that different expression of this component may reflect biochemical events related to malignant transformation rather than to general cell activation.

Studies on low molecular weight nuclear protein of tumour and normal cells.

1. Preliminary results of comparative electrophoretical and immunological analyses of the components of two classes of non-histone proteins, i.e. NHP1 and NHP2 eluted from hydroxyapatite allowed to suppose that protein of Mw 18,000 is specific for animal tumour cells. 2. However, the studies on cellular distribution of this polypeptide indicated that it is exclusively located in nuclei of hepatoma and normal liver as well. 3. The former observation seems to be the result of changes of the affinity of this protein to DNA during neoplastic transformation.

Identification of a nuclear antigen with molecular weight of 48,000 differentially expressed in tumour and normal cells.

A non-histone protein with mol. wt of 48,000 differentially expressed in normal and tumour cells was identified using immunological criteria. Antibodies were raised against a component specific for Kirkman-Robbins hepatoma of mol. wt about 48,000 separated from hepatoma non-histone proteins by preparative electrophoresis in polyacrylamide gel. It was demonstrated by immunoblotting that Morris hepatoma 7777 and Ehrlich ascites cells share an antigenic non-histone protein with Kirman-Robbins hepatoma. Tumour cells when compared with normal cells, i.e. hamster and rat liver, are characterized by significant enrichment of this component. Intracellular distribution of the polypeptide with mol. wt 48,000 suggests that this component may be a structural protein the biosynthesis of which increases or the antigenic determinants of which change in tumour cells.

Cellular distribution of the hamster liver specific nucleolar antigens.

The immunochemical localization of hamster liver nucleolar antigens in subcellular fractions (nuclei, 10,000 x g pellet, 100,000 x g pellet and supernatant), nuclear substructures (chromatin, nuclear matrix, nuclear envelope, nucleoli, RNP particles and nucleosomes), and three classes of nonhistone chromosomal proteins with different affinities to DNA (NHCP1, NHCP2 and NHCP3) from nuclease-sensitive and nuclease-resistant chromatin fractions of hamster liver were studied. Six main nucleolar antigens with mol. wts 27,000; 29,000; 30,000; 36,000; 45,000; and 46,000 were found in subcellular fractions, nuclear substructures and classes of non-histone proteins of hamster liver. The antigens with mol.wts of approx. 27,000; 29,000; and 36,000 which were absent in hamster pancreas, spleen and Kirkman--Robbins hepatoma nuclei, seem specific for liver tissue.

Molecular characterization of non-histone chromatin proteins from experimental tumours.

Using two-dimensional (2-D) electrophoresis, two non-histone chromatin protein fractions (NHCP1 and NHCP2) from three animal tumours (Kirkman-Robbins hepatoma, Morris hepatoma 7777 and Ehrlich ascites cells) and normal hamster liver were analyzed. Apart from many common components several tissue specific polypeptides of the NHCP1 and NHCP2 fractions were detected. It was found that some spots present in electropherograms of non-histone proteins of tumour cells (M X 10(-3)/pI): 17-24/4.9-6.5 (NHCP1 and NHCP2); 34-41/4.9-6.0 (HCP1 and NHCP2); 44-46/5.3-7.5 (HCP2); 46-49/5.0-7.5 (NHCP1); 49/5.9-7.5 (NHCP2) and 102-134/5.6-7.0 (NHCP1) were absent from normal liver.

Hamster liver chromatin immunospecific non-histone proteins.

Three antisera were prepared against non-histone protein classes named NHCP1, NHCP2 and dehistonized chromatin (with different affinity to DNA) from hamster liver. Two main antigenic bands of MW 17,000 and 36,000 were specific in the NHCP1 fraction and one antigen of MW 56,000 was specific for the NHCP2 fraction from nuclease-sensitive and especially nuclease-resistant chromatin. Other NHCP2 liver antigens of MW 22,000, 27,000, 30,000, 36,000, 37,000, 40,000, 45,000, 46,000, 51,000, 98,000 and 100,000 were present only in nuclease-resistant chromatin of hamster liver. Immunologically specific hamster liver non-histone proteins within the NHCP1 and NHCP2 fractions seem to be restricted to nuclease-resistant chromatin fraction of this tissue. The above mentioned liver specific antigens are absent or present only at trace amounts in analogous Kirkman-Robbins hepatoma fractions.

Growth-related changes of non-histone chromatin proteins from Kirkman-Robbins hepatoma.

1. Non-histone chromatin protein fractions NHCP1 and NHCP2 eluted from hydroxyapatite with 50 and 100 mM phosphate buffer (pH 6.8) from nuclei of Kirkman-Robbins hepatoma from 4th, 7th and 9th day of growth were analysed by one- and two-dimensional gel electrophoresis as well as Western blot technique in the presence of antibodies elicited against NHCP1, NHCP2 and dehistonized chromatin of hamster hepatoma and liver. 2. The presence of electrophoretically and immunologically specific components among NHCP1 and NHCP2 fractions during Kirkman-Robbins hepatoma growth was stated.

Diversity of non-histone protein fraction NHCP2 from hamster Kirkman-Robbins hepatoma and liver.

Non-histone protein fraction NHCP2 eluted from hydroxyapatite with 100 mM phosphate buffer (pH 6.8) of undigested, nuclease-sensitive and nuclease-resistant nuclei of hamster Kirkman-Robbins hepatoma and liver was studied by two-dimensional gel electrophoresis and microcomplement fixation test in the presence of antibodies elicited against NHCP2 of examined tissues. The NHCP2 of undigested nuclei as well as from two chromatin fractions with different susceptibility to nuclease of both tissues, besides many common components, showed some differences in their non-histone patterns especially within molecular weights of 17,000-24,000, 36,000-44,000 and 60,000-90,000. Immunological analysis confirmed the high specificity of hepatoma non-histone components of the NHCP2 fraction. However, these components appeared not to be exclusively localized either in nuclease-sensitive or nuclease-resistant part of chromatin of neoplastic tissue.

Molecular and functional diversity of non-histone protein fraction NHCP1 from hamster Kirkman-Robbins hepatoma and liver.

Non-histone protein fraction NHCP1 of micrococcal nuclease-sensitive and nuclease-resistant chromatin from Kirkman-Robbins hepatoma and hamster liver was studied by two-dimensional electrophoresis followed by Coomassie and silver staining and by microcomplement fixation technique in the presence of antibodies elicited against NHCP1 of both tissues. Apart from many common spots several tissue specific components associated with either nuclease-sensitive or nuclease-resistant chromatin were found. The presence of tissue specific components among NHCP1 from hepatoma and liver was confirmed by immunological analysis. It was stated that these components are exclusively localized in nuclease-resistant part of chromatin from neoplastic and normal tissues thus suggesting their structural function.

Chicken erythrocyte chromatin and nuclear envelope antigens.

Chromatin and inner layer nuclear envelope were isolated from chicken erythrocyte nuclei. Two antisera against dehistonized chromatin and nuclear envelope of chicken erythrocytes were obtained. Using the antiserum against dehistonized chromatin of erythrocytes we found: the presence of the antigens at approximate mol. wts of 56,000 and 77,000 tightly bound with DNA and characteristic of only erythrocyte chromatin; localized antigens at approximate mol. wts of 63,000, 68,000 and 92,000 tightly bound with DNA and common only for chromatin and nuclear envelope of chicken erythrocytes; heterogeneity of the antigens tightly bound with DNA. Using the antiserum against inner layer nuclear envelope we did not find antigens specific only for nuclear envelope and absent in erythrocyte chromatin. Some of the antigens were present in the control preparations of chicken liver chromatin and may be regarded as being species specific.

Immunological analysis of histone H2A from the slime mold Physarum polycephalum.

Specific antibodies against the histone H2A from calf thymus were generated by injecting rabbits with complexes: histone H2A-RNA with a protein to RNA ratio of 3:1. In the microcomplement fixation assay the antibodies against the histone H2A from calf thymus immuno-reacted with the histone H2A from calf thymus but not with H2A from Physarum polycephalum. The histone H2A from calf thymus therefore appears to have an immunological determinant(s) which does not exist in H2A from Physarum polycephalum.

High mobility group non-histone chromosomal proteins from chicken thrombocytes and erythrocytes.

The high mobility group non-histone chromosomal proteins (HMG) from chicken thrombocytes were compared with those from chicken erythrocytes. In terms of their extractability, electrophoretic behaviour in polyacrylamide gels containing 2.5 M-urea, and amino acid composition, the HMG proteins of these cells were indistinguishable. However, microcomplement fixation assay performed in the presence of antiserum against HMG-1 and HMG-2 revealed that these high molecular weight HMG proteins of chicken thrombocytes and erythrocytes are not cross-reactive. Electrophoretic analysis of HMG-1 and HMG-2 from these two types of cells in polyacrylamide gels containing 6.25 M-urea suggested differences in the HMG-2 patterns.

Comparison of non-histone proteins from hamster Kirkman-Robbins hepatoma and liver.

Three classes of non-histone proteins were obtained from hamster Kirkman-Robbins hepatoma and liver nuclei following separation of nucleic acids with the polyethylene glycol-dextran mixture and fractionation of nuclear proteins on hydroxylapatite in a salt-glycerol-phenylmethylsulphonyl fluoride system at increasing concentrations of Na+ and K+ phosphate buffer, pH 6.8. Two-dimensional polyacrylamide gel electrophoresis of these proteins documented their high heterogeneity; many spots were common but some spots specific only for neoplastic or normal tissue were also observed.

Specificity of Kirkman-Robbins hepatoma non-histone chromatin proteins: electrophoretic and immunological analyses.

The specificity of Kirkman-Robbins hepatoma and hamster liver non-histone chromatin proteins has been studied by comparing polypeptide patterns in polyacrylamide gel electrophoresis and by their immunological activity in the complement fixation test. Non-histone proteins were separated from DNA with a polyethylene glycol-dextran mixture and fractionated by hydroxylapatite chromatography into three classes named NHCP1, NHCP2, and NHCP3. Electrophoretic analysis indicated that among the non-histone proteins of Kirkman-Robbins hepatoma and hamster liver differences mainly of a quantitative nature can be observed. However, the polypeptides with molecular weight 25 000, 31 000, 36 000, 73 000 in NHCP1; 20 000, 40 000 in NHCP2 and 20 000, 23 000, 32 000, 38 000, 44 000, 75 000, 80 000 in NHCP3 were found to be specific for hepatoma chromatin. Application of antibodies against NHCP1, NHCP2 and dehistonized chromatin of Kirkman-Robbins hepatoma revealed that the highest specificity of NHCP2 eluted from hydroxylapatite with 100 mM phosphate buffer at pH 6.8. The NHCP1 of hepatoma shares some common antigenic determinants with analogous proteins of liver. On the other hand non-histone proteins specific for hepatoma dehistonized chromatin can be localized in the NHCP3 and partially in the NHCP1 fractions.

Chromatin proteins associated with micrococcal nuclease-sensitive and nuclease-resistant chromatin fractions of Kirkman-Robbins hepatoma and hamster liver.

Micrococcal nuclease-sensitive (SP) and nuclease-resistant (PP) chromatin fractions from Kirkman-Robbins hepatoma and hamster liver were obtained. The molecular distribution of three non-histone proteins (NHCP1, NHCP2 and NHCP3), histones, and chromatin-bound protease activity between SP and PP fractions of both tissues was compared. Differences, mainly of quantitative nature, among non-histone proteins of neoplastic and normal tissue were observed. Moreover, it was found that polypeptides with mol. wt 81 000 (NHCP1), 39 000 (NHCP2) and 21 000, 35 000, 37 000 (NHCP1), 70 000, 112 000, 141 000, 157 000 (NHCP2), 30 000-33 000 (NHCP3) were associated only with the nuclease-sensitive part of chromatin of hepatoma and normal tissue, respectively. A major difference in histone composition of hamster hepatoma and liver concerns histones H2A and H1. Furthermore, an enrichment of high mobility group proteins as well as other soluble non-histone proteins in an acid extract of the SP fraction was observed. Apparently chromatin-bound protease activity can be found in both fractions of chromatin.