Lectin-binding proteins in nuclear preparations from rat liver and malignant tumors.

Lectin binding [concanavalin A, biotinylated ricinus communis agglutinin, and biotinylated succinylated wheat germ agglutinin (B-SWGA)] was used to detect the glycosylated proteins associated with a residual protein fraction [insoluble in 4% sodium dodecyl sulfate and termed the nuclear residual fraction (NRF)] or with nuclear matrix preparations from normal rat liver, azo dye (3'-MeDAB)-induced rat hepatoma, and Walker 256 transplantable carcinosarcoma. One- and two-dimensional gel electrophoresis were used with lectins, polyclonal antisera, and monoclonal antibody binding to characterize some of the glycoconjugates. Two polypeptide bands with approximate molecular weights of 95,000 and 55,000, shown previously to be present only in the induced tumor cells and the Walker 256 tumor, were reactive with lectins.

Comparison of chicken erythroid cell nuclear isolation methods using morphological, immunochemical and biochemical criteria.

Chicken erythroid nuclei were prepared using four published methods. Our findings indicate that nuclei prepared by nitrogen cavitation are less likely to be contaminated with plasma membrane fragments than those made by procedures involving cell disruption by hypotonic lysis. However, globin gene sequences were much less sensitive to DNase I digestion in nuclei prepared by nitrogen cavitation.

Specificity of non-histone protein antigens in chicken liver nuclei.

1. Chicken liver nuclei were fractionated by disruption with ultrasound and subsequent precipitation with divatent cations. A small, protein-rich fraction (CS), representing less than 5% of the total nuclear DNA reacted strongly with antisera to dehistonized chicken liver chromatin.

In vivo DNA-protein cross-linking by cis- and trans-diamminedichloroplatinum(II).

When Novikoff hepatoma-bearing rats were given injections of a therapeutic dose of cis-diamminedichloroplatinum(II) (cis-DDP) (7 mg/kg), DNA-protein cross-links could be detected by using antisera to dehistonized chromatin, nuclear matrix, or Novikoff hepatoma cytoskeletal preparation. The extent of cross-linking increased in time up to 24 h after the injection, after which time the DNA-protein cross-links were gradually repaired, with no cross-links detectable at 72 h. trans-DDP in equitoxic (40 mg/kg) dose was very efficient in forming DNA-protein cross-links.

DNA-protein crosslinking by heavy metals in Novikoff hepatoma.

Crosslinking of proteins to DNA was studied in live intact Novikoff ascites hepatoma cells exposed in vitro to salts of chromium VI, III, and II, nickel II, cadmium II, and to CoCl2, As2O3, and AlK(SO4)2. DNA-protein complexes were separated by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and assayed by polyacrylamide gel electrophoresis. Hexavalent chromium compounds formed DNA-protein complexes very efficiently.

Modified method of silver staining of proteins in polyacrylamide gels.

The very sensitive and reliable silver staining method to visualize proteins in polyacrylamide gels described by Wray et al. (Anal. Biochem.

Poly(ADP-ribosyl)ation studies in situ.

We have studied the poly(ADP-ribosyl)ation of nuclear proteins in situ by examining the incorporation of [3H]NAD-derived ADP-ribose into polymers. We have devised a way to deliver [3H]NAD to cells growing in vitro, and we have determined the kinetics of uptake and incorporation into nuclear proteins using this delivery system. Incorporation into the histone fraction, known acceptors of poly(ADP-ribose), was examined and shown to be sensitive to the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide.

Properties of a nuclear protein marker of human myeloid cell differentiation.

The Mr 55,000 nuclear antigen present in the human promyelocytic cell line HL-60 is a basic protein that is extracted from nuclei or chromatin by 0.35 M NaCl. The antigen is confined to the nucleus of the interphase HL-60 cell as judged by immunocytochemical localization but disperses throughout the cell during mitosis.

In vitro translation of rat liver and Novikoff hepatoma cytokeratin mRNAs.

Cytoplasmic poly(A)+ RNA was isolated from normal rat liver and Novikoff ascites hepatoma cells, translated in vitro using rabbit reticulocyte lysate system and the translational products were assayed by immunoprecipitation with antibodies specific for Novikoff hepatoma principal cytokeratins p39, p49 (a group of hepatic cytokeratins C, D, and E) and p56. The identity of the precipitated antigens was further confirmed by two-dimensional polyacrylamide gel electrophoresis. Only the Novikoff hepatoma poly(A)+ RNA contained translatable mRNA coding for the p39 cytokeratin while the p49 and p56 cytokeratins were translated from both the normal rat liver and Novikoff hepatoma poly(A)+ RNAs.

The in vivo cross-linking of proteins and DNA by heavy metals.

Cross-linking of proteins to DNA in live, intact Novikoff ascites hepatoma cells exposed in vitro to different concentrations of CuSO4, Pb(NO3)2, HgCl2, and AlCl3 was studied. Protein-DNA complexes were separated by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and assayed by electrophoretic separation of proteins associated with the DNA-containing pellets. Concentration dependence experiments showed that the optimal cross-linking occurred at metal concentration of 0.

Cross-linking of cytokeratins to DNA in vivo by chromium salt and cis-diamminedichloroplatinum(II).

The in vivo cross-linking of cytokeratins to DNA in intact Novikoff ascites hepatoma cells exposed to the chromium salt K2CrO4 and cis-diamminedichloroplatinum(II) (cis-DDP) was studied. Cytokeratin-DNA complexes were obtained by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate. The cytokeratins were identified electrophoretically and immunologically by use of polyclonal and monoclonal antibodies.

Effects of hemin on a lymphoblastoid cell line that expresses the human epsilon- and gamma-globin genes.

Northern blot analysis using probes specific for each of the human embryonic (epsilon), fetal (gamma), and adult (beta) globin genes indicates that the human lymphoblastoid F-265 cells express the embryonic and fetal globin genes. Unlike the erythroid cell line K562, in which globin RNA levels increase during treatment with hemin in culture, globin RNA levels decrease in F-265 cells in the presence of hemin. This effect is reversible after passage of F-265 cells in fresh medium without hemin.

Cell specificity of rat cytokeratin p39 during azo dye-induced hepatocarcinogenesis.

Monoclonal or affinity-purified antibodies specific to Novikoff hepatoma cytokeratin p39 were employed to study the origin and fate of p39-containing cell types during hepatocarcinogenesis induced with N,N-dimethyl-p(m-tolylazo)aniline. Frozen sections were obtained from the livers of animals autopsied temporally during carcinogen feeding and were assayed immunohistochemically. In normal, untreated liver or in liver from animals fed the hepatotoxin alpha-naphthyl-isothiocyanate, the localization of p39 was restricted to bile duct epithelial cells while hepatocytes were non-reactive.

Cross-linking of Novikoff ascites hepatoma cytokeratin filaments.

We have investigated the structure of solubilized cytokeratins from Novikoff ascites hepatoma using the cleavable cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) in the presence of 6 M urea to effect partial complex melting. By two-dimensional gel electrophoresis, in which the protein cross-links were broken in the second dimension, we have identified two major complexes as a p39-p56 dimer and a (p39-p56)2 tetramer, p39 and p56 being two of the major cytokeratins in Novikoff ascites hepatoma. Experiments investigating possible relationships between the dimer and tetramer employed immunoblots and two monoclonal antibodies which recognized either p56 or p39 cytokeratins.

Nuclear antigens in the HeLa cell cycle.

Antisera to 0.35 M NaCl extracts and residues of S phase HeLa nuclei were reacted with electrophoretically separated proteins from the nuclei or nuclear material of HeLa cells synchronized in G1, S, G2 or M phases of the cell cycle. Quantitative evaluation of the peroxidase-antiperoxidase stained nitrocellulose transfers (Western blots) revealed significant changes in the quantities of nuclear non-histone proteins during the cell cycle.

Chromium-induced cross-linking of nuclear proteins and DNA.

The in vivo cross-linking of proteins to DNA in intact Novikoff ascites hepatoma cells exposed to the chromium salt K2CrO4 was studied. DNA-protein complexes were assayed by high speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and by electrophoretic identification of proteins associated with DNA-containing pellets. Further evidence of DNA-protein complexes, not dissociable in this buffer, was obtained by CsCl gradient centrifugation.

cis-Diamminedichloroplatinum-mediated crosslinking of nuclear proteins to DNA is cell cycle specific.

Immunochemical analysis was employed to investigate the cell cycle-dependent protein-DNA crosslinking by cis-diamminedichloroplatinum II (cis-DDP), in HeLa-S3 cells. Cells synchronized by double thymidine block or hydroxyurea were released into S phase and incubated at 2-h intervals with cis-DDP as they progressed through S1, G2, M, and then into G1 and S phases of the subsequent cycle. Immunoblots of the DNA-crosslinked antigens reacted with antisera to 0.

Enzymatic modification of Novikoff hepatoma lamins A and C.

A significant increase in the molecular weights of lamin A and more so of lamin C was observed when isolated Novikoff hepatoma chromatin was incubated in the presence of Ca2+. This increase did not occur to any significant degree in similar preparations of normal rat liver nuclei. Although detectable in Coomassie Brilliant Blue stained gels, this increase to a higher molecular weight (by approximately 2000 Mr) was much more visible when the electrophoretically separated lamins were transferred to nitrocellulose sheets and stained (using peroxidase-antiperoxidase) with polyclonal antiserum to the three major lamin proteins.

Characterization of monoclonal antibodies to novikoff hepatoma cytokeratin antigen p39.

Three stable monoclonal antibody-producing mouse hybridoma lines have been developed which produce high-titer, immunoglobulin M antibodies specific for the Novikoff ascites hepatoma (NAH) Mr 39,000 cytokeratin antigen (p39). Immunotransfer assays of cytoskeletal protein-enriched fractions indicated p39 to be present in a range of rat tissues, including colon, breast, lung, and uterus. Two-dimensional gel immunoblots confirmed that immunoreactivity in the latter tissues was for polypeptides with similar isoelectric points to those of NAH p39; however, reactivity in the colon contained a wide range of additional isomeric forms.

Nuclear matrix antigens in azo dye-induced primary rat hepatomas.

Polyvalent antisera, monoclonal antibodies, and immunotransfer methodology have been used to identify and characterize a group of chromosomal protein antigens which appear during azo dye hepatocarcinogenesis. Experiments were designed to probe for the location and placement of antigens in chromatin according to solubility and possible DNA-binding properties. The majority of nuclear antigens were associated with high-speed DNA-containing pellets after ultracentrifugation of chromatin solubilized with denaturing buffers containing 6 M guanidine-HCl:2% sodium dodecyl sulfate, or 2 M NaCl:5 M urea.

Two human colon tumor cell lines with similar nuclease sensitivities have different ethidium bromide binding characteristics.

Chromatin from two human colon adenocarcinoma cell lines (HT-29 and LoVo) showed similar digestion kinetics when sensitivities to DNase I and micrococcal nuclease were examined. Chromatin conformations were probed by examining the binding of ethidium bromide. A Scatchard plot revealed that both chromatins bound the same amount of ethidium bromide per mole of DNA, but the DNA from LoVo cells was more accessible to the intercalator.

Antigenic similarity of nonhistone chromosomal proteins in cultured rat liver epithelial cells, fetal liver, and transplantable tumors.

The cell and tissue specificity of antisera prepared to chromatin fractions from a nontumorigenic adult rat liver-derived clonal epithelial cell line (ARL-15Cl1) was characterized with immunotransfer analysis and immunoabsorption experiments. These antisera reacted with a range of high-molecular-weight chromosomal proteins greater than Mr 100,000. Extensive immunoabsorption-immunoblocking studies indicated antigenic homologies between the chromatins of the nontumorigenic ARL-15Cl1 cell line, a transformed, tumorigenic cell line (ARL-16T2), fetal liver, and several transplantable malignant tumors.

cis- and trans-Diamminedichloroplatinum(II)-mediated cross-linking of chromosomal non-histone proteins to DNA in HeLa cells.

The cross-linking of chromosomal non-histone proteins to DNA in isolated nuclei or intact HeLa cells exposed to different concentrations of cis- and trans-diamminedichloroplatinum(II) (cis- and trans-DDP) for various time intervals was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical methods. Both the cis- and the trans-DDP cross-linked significant numbers of chromosomal non-histone proteins to the DNA. The quantity and the types of the cross-linked proteins depended on the time of incubation as well as on the concentrations of the drugs.

Nonhistone protein antigen profiles of five leukemic cell lines reflect the extent of myeloid differentiation.

The human leukemic cell lines, K562, KG-1, and HL-60, and the blast subclones, KG-1a and HL-60 blast, were utilized to relate differences in nonhistone protein antigens to stages of myeloid cell differentiation. Chromatin proteins were separated on SDS-polyacrylamide gels, transferred electrophoretically to nitrocellulose sheets, and visualized by the peroxidase-antiperoxidase method of Sternberger. Screening with antisera raised against total and dehistonized chromatin and a nuclear extract from these cells revealed quantitative as well as qualitative differences between the cell lines.

Variations in the organization and expression of human histone genes in normal diploid and tumor cell lines.

The organization and expression of human histone genes were examined in W138 normal human diploid fibroblasts, SV40 transformed W138 cells, A549 epithelial lung carcinoma cells, two adeno-carcinoma cell lines (LOVO and HT29) and three leukemia cell lines (HL60, KG1 and K562). Analysis of the restriction enzyme digests of total genomic DNAs by hybridization with a series of cloned human histone sequences indicated polymorphic organization of at least a subset of the moderately reiterated human histone genes in these cells. Quantitative and qualitative differences were also observed in the representation of histone mRNAs by Northern blot analysis using cloned human histone genes as hybridization probes.