Novikoff Hepatoma Research Articles

A role for DNA polymerase in the specificity of nucleotide incorporation opposite N-acetyl-2-aminofluorene adducts

Escherichia coli DNA polymerase I (Klenow fragment), DNA polymerase α from both calf thymus and human lymphoma cells and DNA polymerase β from calf thymus and Novikoff hepatoma cells can incorporate nucleotides opposite N-guanin-8-yl-acetyl-2-aminofluorene in DNA. The polymerases incorporate dCTP opposite some AAF-dG § § Abbreviations used: AAF, N-acetyl-2-aminofluorene; AAF-dG, N-guanin-8-yl-acetyl-2-aminofluorene; AMV, avian myeloblastosis virus; Poll (Kf), E. coli DNA polymerase I (Klenow fragment); Pol, DNA polymerase; ssB, E. coli single-stranded DNA binding protein; dNTP, deoxynucleoside triphosphate; dNMP, deoxynucloside monophosphate; dNTPαS, Sp diastereomer of 2′ deoxynucleotide 5′-O-(l-thiotriphosphate); u.v., ultraviolet light. lesions when Mg 2+ is the divalent cation. Substitution of Mn 2+ for Mg 2+ broadens the specificity of insertion: E. coli DNA polymerase I (Klenow fragment) also inserts A, and at specific sites G or T; DNA polymerase α inserts any of the four dNTPs with A and C incorporated preferentially to G and T. Polymerase β is specific, inserting mainly C even in the presence of Mn 2+. The K m for addition of dATP opposite a lesion by E. coli polymerase I (Klenow fragment) in the presence of Mn 2+ is about 0.5 m m. dNMPs increase the insertion of nucleotides opposite AAF-dG in the presence of Mg 2+ and increase both the rate and number of sites at which incorporation occurs in the presence of Mn 2+. dNTPαS and recA protein increase only the insertion of C. We suppose that the incorporation of dCTP reflects normal base-pairing with the AAF-deoxyguanine in the anti conformation, whereas insertion of the other nucleotides (including some of the C) reflects insertion opposite the AAF adduct in its preferred syn conformation. The fact that the DNA polymerase plays a role in determining the specificity of insertion opposite a lesion terminating DNA synthesis suggests that the spectrum of base substitution mutagenesis seen in vivo may reflect the properties of the protein components, including the polymerase, involved in bypass synthesis.

Silver staining, immunofluorescence, and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23.

Nucleolar organizer region (NOR)-specific silver staining and immunolocalization of nucleolar phosphoproteins B23 and C23 were compared in Novikoff hepatoma ascites cells. Silver staining and protein C23 immunostaining were both localized in the fibrillar shell surrounding the fibrillar center and in the fibrillar center. During mitosis, silver staining and protein C23 were localized at the NORs. Therefore, protein C23 and the silver-staining protein both seem to be associated with rDNA-containing structures (Mirre and Stahl 1981). A comparison of toluidine blue staining specific for RNA and B23 immunostaining demonstrated that protein B23 was associated with RNA-containing regions of the nucleolus and was absent from the fibrillar centers. Localization of these proteins and their functions are discussed in relation to the organization of the nucleolus.

Purification and partial characterization of a 19 KD/pI 4.5 nucleolar phosphoprotein

Two-dimensional PAGE analysis of proteins associated with the slowly sedimenting “fibrillar” structures of HeLa nucleoli revealed a protein with a M r of 19,000 and a pI of 4.5 which was highly labeled both with 32P-orthophosphate and 35S-methionine. The protein was isolated from Novikoff hepatoma nucleoli by extraction in 0.35 M NaCl and 5 mM DTT followed by chromatography in EDTA on DEAE-cellulose and Sephadex G-100. The protein was homogeneous with respect to two-dimensional PAGE, number of tryptic peptides and carboxyl terminal analysis. The protein contained an acidic/basic amino acid ratio of 2.1, 7 residues of methionine, 2 residues of cysteine, a blocked amino terminus and a carboxyl terminal lysylleucine.

Alterations in the maturation and structure of ribosomal precursor RNA in Novikoff hepatoma cells induced by 5-fluorocytidine.

The effects of 5-fluorocytidine on ribosomal RNA maturation and structure in Novikoff hepatoma cells were investigated. Like other nucleic acid base analogues that are incorporated into RNA, this compound inhibits maturation of the 45S ribosomal RNA precursor. The 45S RNA precursor produced in the presence of 5-fluorocytidine has an abnormal electrophoretic mobility compared with that of the control precursor under nondenaturing conditions, but the two have identical mobilities under denaturing conditions. Under the conditions of these experiments, 5-fluorocytidine inhibited cellular protein synthesis only slightly, whereas equimolar concentrations of 5-azacytidine resulted in nearly 75% inhibition of this process. Despite this difference in the effects of the two analogues as well as the greater chemical lability of the 5-azacytidine, their effects on ribosomal RNA maturation are identical.

Induction of hepatocyte stimulating activity by T3 and appearance of the activity despite inhibition of DNA synthesis by adriamycin.

A hepatocyte stimulating activity (HSA) has been extracted from rats that had received an injection of a pharmacological dose of T3 20 hours earlier. The injection of HSA from T3-treated rats into different recipient rats that had previously had 40% of their liver removed resulted in a significant increase in hepatic DNA synthesis. The injection of saline or HSA from normal rat liver had little or no effect on hepatic DNA synthesis in recipient rats. HSA from the T3-treated rats also stimulated DNA synthesis in Novikoff hepatoma cells and primary hepatocytes in culture, and in isolated normal rat liver nuclei in a nuclear incorporating system. In further experiments in which the increased DNA synthesis that follows partial hepatectomy was blocked by adriamycin, HSA appeared in these non-regenerating livers. This latter observation had indicated that the development of HSA is not merely an accompaniment of DNA synthesis.

Lipid peroxidation and lipid antioxidants in normal and tumor cells.

Lipid peroxidation is often low in tumor tissue as compared to the corresponding normal tissue and it has been postulated that lipid peroxidation may be associated with cell division. In this paper the various contributory factors which control the rate of microsomal lipid peroxidation in normal rat liver and in the Novikoff hepatoma have been carefully analyzed. The low rate of lipid peroxidation in the hepatoma seems to be due to a combination of factors: low levels of polyunsaturated fatty acids and of cytochrome P-450 and elevated levels of lipid-soluble antioxidant. This lipid-soluble antioxidant is principally alpha-tocopherol.

Immunoelectron microscopic localization of snRNPs.

Small nuclear ribonucleoprotein particles (snRNPs) were identified in nuclear sonicates of Novikoff hepatoma ascites cells and in intact Novikoff hepatoma and PtK2 cells by immunofluorescence and immunoelectron microscopy. Auto-antibodies (anti-Sm and anti-RNP) obtained from patients with systemic lupus erythematosus an autoimmune disease, were used to localize snRNP particles. The Sm antibody is specific for U1, U2, U4, U5 and U6 containing snRNPs. The RNP antibody is specific for only U1 containing snRNPs. Isolated particles, 120 +/- 10 A in diameter, were found to be associated with ferritin-conjugated goat anti-human antibodies coupled to Sm antibodies. In addition, these particles (snRNPs) were occasionally associated with larger particles measuring 230 +/- 10 A in diameter which are presumed to be hnRNP particles. Double label immunofluorescence and immunoelectron microscopy have shown Sm and RNP antibodies to colocalize in PtK2 cells. However, the perinucleolar chromatin and juxtanuclear envelope chromatin was devoid of RNP immunostaining. Therefore, U1 containing snRNPs do not appear to be in these regions. The Sm antibody localizes in a nuclear network including the perinucleolar chromatin and juxtanuclear envelope chromatin. Cells treated with the drug DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole), which inhibits hnRNA synthesis, show an altered pattern of Sm immunostaining. Such cells contain large clusters of snRNPs which do not extend to the perinucleolar chromatin or perinuclear lamina chromatin. Nuclear matrix preparations maintain an snRNP nuclear network as visualized by Sm immunofluorescence. It is notable that the size and density of the immunostained particles in the nuclear network during interphase, is similar to that of interchromatinic granules.

Effect of cesium and potassium salts on survival of rats bearing Novikoff hepatoma.

The effect of CsCl on the life span of female Sprague-Dawley rats innoculated with Novikoff's hepatoma was studied as a function of both pre- and post-treatment with CsCl and as a function of the inoculant dose. The effect of KCl on the CsCl treatment was also studied. Rats treated with CsCl for 12 consecutive days prior to or immediately after inoculation with 1.0 ml of viable hepatoma cell suspension showed an increase in mortality score from corresponding controls. Conversely, increases in the dose of the inoculant resulted in delaying the onset of toxicity in rats receiving the Cs-treatment after inoculation as evidenced by a decrease in mortality. Availability of KCl in drinking water ad lib further decreased total mortality when given alone but not when combined with CsCl. The results indicate a dose-dependent paradoxical effect of CsCl on Novikoff hepatoma cell toxicity and suggest a critical intercellular balance requirement between Cs+ and K+ on the effect studied.

Effect of cesium and ethanol on tumor bearing rats.

The effect of separate and combined administration of 15% ethanol and 0.2% CsCl solution on life span of rats with Novikoff hepatoma implants was studied as a function of time of initiation of treatment. Pretreatment with CsCl alone or combined with ethanol resulted in earlier onset on morbidity compared to the ethanol-treatment or to controls. As high as 87.5% of Cs-treated animals died 16 days post tumor implantation compared to 33% of rats receiving CsCl and ethanol combined. This protective action of ethanol against Cs-evoked toxicity in tumor-bearing rats persisted through the experiment. Animals subjected to drug treatment immediately after tumor transplantation displayed delayed onset of morbidity compared to drug pretreated rats. In both cases the Cs-treatment enhanced morbidity by approximately 2 folds from corresponding controls. Animals sacrificed 18 days post tumor inoculation showed an induction of hepatic alcohol dehydrogenase and an increase in Vmax without changes in the apparent Km by the Cs-treatment. There was an increase in liver mitochondrial aldehyde dehydrogenase of hepatoma-bearing rats from tumor-free controls which was associated with an increase in the apparent Km value. The results indicate potentiation of the hepatoma toxicity by CsCl which may be minimized by ethanol. A role for hepatic enzymes determined in the pathogenesis of tumor line studied and/or their use as a biochemical correlate is suggested.

Association of cytokeratin p39 with DNA in intact Novikoff hepatoma cells.

The relationship between the rat tumor-associated cytokeratin p39 (Mr 39,000) and cellular DNA has been studied in intact cells. Using a DNA-protein crosslinking technique, incubation with cis-dichlorodiammineplatinum, we present evidence for the association of p39 with DNA in intact Novikoff ascites hepatoma cells. The cells were treated with cis-dichlorodiammineplatinum and solubilized in NaDodSO4-containing buffer, and the DNA was pelleted by high-speed centrifugation. By immunotransfer analysis, the cytokeratin was found in the DNA pellet of the crosslinked samples while absent from the controls. This result was further substantiated by CsCl density-gradient centrifugation. Collectively, these results suggest a cytokeratin-DNA association at the filament binding sites on or near the nuclear lamina.

Detection of enzymatic activities in sodium dodecyl sulfate-polyacrylamide gels: DNA polymerases as model enzymes

Recent techniques for detecting the catalytic activity of enzymes in sodium dodecyl sulfate (SDS)-polyacrylamide gels have been hampered by lack of reproducibility associated with variability in commercial SDS preparations. Simple expedients which facilitate reproducible detection of DNA polymerase activity and which appear to be widely applicable to detection of other enzymes are reported here. It was observed that reproducibility of a reported procedure for DNA polymerase detection (Spanos, A., Sedgwick, S. G., Yarranton, G. T., Hübscher, U., and Banks, G. R. (1981) Nucl. Acids Res. 9, 1825–1839) depends on the SDS used for electrophoresis, and that sensitivity is markedly reduced if currently available SDS is substituted for the discontinued product specified by Spanos et al. A modified procedure yielding sensitivity with contemporary commercial SDS, which exceeds the sensitivity observed when using the protocol and the SDS originally specified, is described. The modifications employed, which presumably promote renaturation of enzymes, are (1) embedding fibrinogen in gels and (2) washing detergent from gels with aqueous isopropanol after electrophoresis. These expedients permit detection of picogram amounts of Escherichia coli DNA polymerase I and its Klenow fragment and nanogram amounts of calf thymus α and rat liver (Novikoff hepatoma) β polymerases. Finally, it is shown that sensitivity of DNA polymerase detection is reduced by lipophilic contaminants in contemporary commercial SDS, and that the expedients employed here mitigate the deleterious effect of these impurities.

Sequence homology between potato spindle tuber viroid and U3B snRNA

Sequence homology was found by computer analysis between potato spindle tuber viroid (PSTV) RNA and U3B snRNA of Novikoff hepatoma cells. This homology is colinear in arrangement, extends in length to 81% of the entire U3B snRNA molecule and is involved in the PSTV molecule unique sites which, if depicted in terms of the secondary structure of the circular PSTV molecule, reveal a conspicuous regularity in their location. A strong relation in primary structure between PSTV and U3B snRNA is demonstrated by statistical analysis.

Antibodies to dehistonized chromatin of normal and fetal rat liver

Antisera to dehistonized adult or fetal rat liver chromatin were treated with electrophoretically separated chromosomal proteins of adult and fetal liver, Novikoff hepatoma and adult rat kidney. Both types of antisera reacted with numerous antigens in both tissue chromatins. However, immunoabsorption experiments have shown that while adult rat liver shared most of its nuclear antigens with other tissues, antisera to dehistonized fetal liver chromatins were more specific. In terms of antigenic specificity, the fetal rat liver and Novikoff hepatoma chromatins were similar; however, the former contained several antigens which could not be absorbed with Novikoff hepatoma chromatin.

DNA-binding peptides from rat liver and Novikoff hepatoma cells: quantitative level and possible biochemical differences.

DNA isolated from rat liver by intensive deproteinization with chloroform/isoamyl alcohol and phenol contains low molecular weight peptides in a quantity of about 20 micrograms/mg DNA. These peptides show high specific activity in inhibiting transcription in a reconstituted cell-free system with prokaryotic and eukaryotic RNA polymerase. Their level is markedly decreased in DNA prepared from Novikoff hepatoma cells. Moreover the amino acid analysis and the pattern of analytical separation by high performance liquid chromatography (HPLC) show some biochemical differences between DNA-binding peptides extracted from rat liver and Novikoff hepatoma cells. The possibility that carcinogenesis may involve mechanisms which lead to selective removal of some components of the DNA-binding peptides, is discussed.

Identification of a La protein binding site in a RNA polymerase III transcript (4.5 I RNA).

Anti-La antibodies are frequently found in patients with autoimmune diseases; the antigen was reported to be a 50,000-Da protein (Rinke, J., and Steitz, J. A. (1982) Cell 29, 149-159). Because this protein was associated with many nascent RNA polymerase III transcripts, it was suggested to be an RNA polymerase III transcription factor. The present study was designed to analyze 4.5 I ribonucleoprotein, an RNA polymerase III transcript which contains the La antigen. It was found that the 3'-end 20-30-nucleotide portion was the most protected portion of 4.5 I RNA when 4.5 I ribonucleoprotein was digested with T1 RNase. When U2 RNA (an RNA polymerase II transcript) and 4.5 I RNA were incubated with the S-100 fraction of Novikoff hepatoma cells, the 4.5 I RNA bound La antigen but the U2 RNA did not. When partial and complete T1 RNase digestion fragments of 4.5 I RNA were incubated with the S-100 fraction, the 3'-end fragments bound preferentially to the La antigen. However, the fragments of 4.5 I RNA bound less efficiently to La antigen than whole 4.5 I RNA. These results indicate that the 3'-end of 4.5 I RNA is the La antigen binding site in this molecule and suggest that the overall conformation of RNA aids in the binding of La antigen.

A novel single-stranded DNA-binding protein from the Novikoff hepatoma which stimulates DNA polymerase beta. Purification and general characterization.

We have isolated and purified to homogeneity a novel single-stranded DNA-binding protein from the Novikoff hepatoma. This protein is distinguished from other eukaryotic DNA-binding proteins by binding weakly, but cooperatively, to single-stranded DNA, by its ability to partially destabilize a double helix at 37 degrees C, and by its ability to stimulate DNA polymerase beta. The protein exists as a globular monomer of Mr = 48,000 and is capable of binding 45-49 nucleotides. It does not form a complex with the polymerase, but binds the DNA template, allowing an increased rate and extent of DNA synthesis. The enhancement of synthesis is greatest with larger gap-sized templates and with low polymerase concentrations. The mechanism of stimulation is thought to be due largely to placing the template strand into a conformation that facilitates rapid polymerization rather than strand displacement in advance of the polymerase. This protein has been named SSB-48.

Phosphorylation of purified Novikoff hepatoma topoisomerase I

The purified Novikoff hepatoma nuclear phosphoprotein with a molecular weight of 110 kdalton and pI 8.4 was found to be a type I topoisomerase. When isolated from 32P-labeled Novikoff ascites cells or incubated in vitro with protein kinase, phosphoserine was found to be its major phosphorylated amino acid. The enzymatic activity of topoisomerase I was altered by changes in phosphorylation. Its activity was increased by protein kinase and it was decreased by alkaline phosphatase.

Tissue and cell-specific antigens in chromatin from cultured rat Sertoli cells

Cell-specific chromatin antigens have been detected in rat Sertoli cells. Antisera were raised in rabbits to dehistonized chromatin prepared from 5- to 6-day cultures of rat Sertoli cells and immunoreactivity was assessed with microcomplement fixation tests or immunoidentification of antigens separated electrophoretically and transferred to nitrocellulose sheets. Tissue specificity was confirmed further by immunoabsorption. These antisera recognized only components of Sertoli cell chromatin; chromatins prepared from rat liver, kidney, thymus, Novikoff hepatoma, a testes germinal cell fraction, or purified rat DNA showed little or no immunoreactivity. Nitrocellulose transfers of total chromosomal proteins revealed the presence of two high-molecular-weight antigens (greater than 200,000) and a broad range of weaker immunologic species ( M r about 50,000–200,000) in Sertoli cell chromatin but not liver or thymus chromatin. Deproteinization of Sertoli cell chromatin with concentrated salt and urea at pH 6 or 8 produced an increase in complement-fixing activity of the fraction sedimenting with DNA and micrococcal nuclease digestion studies showed these fractions to depend in part on DNA for antigenicity. Individual antigens in the protein-DNA pellets of pH 8 salt and urea fractionated chromatin could not be identified with antigens on nitro-cellulose sheets. Collectively, these observations suggest that at least two groups of cell-specific antigens exist in Sertoli cell chromatin; one group is detected after electrophoretic separation and transfer to nitrocellulose and the other group, reactive in complement fixation, cannot be detected through this method, apparently becoming antigenically inactive once the complex of protein and DNA is dissociated.

The effect of chemical mutagens on purine and pyrimidine nucleotide biosynthesis

Nucleotide biosynthesis in Novikoff hepatoma cells is markedly altered by a variety of chemical mutagens, whether the mechanism of mutagenesis is by base substitution, covalent binding (adduct formation), intercalation, or cross-linking of DNA. The compounds investigated ( N-methyl- N′-nitro- N-nitrosoguanidine, 4-nitroquinoline 2-oxide, 9-aminoacridine, and mitomycin C), at concentrations that cause some inhibition of RNA and DNA synthesis, bring about a large increase in the pool levels of all four nucleoside triphosphates. At the same time, reactions leading to the synthesis of CTP from exogenous uridine and GTP and ATP from exogenous hypoxanthine are severely inhibited. The formation of UTP from uridine and ATP from adenosine, by more direct phosphorylation reactions, appears relatively unaffected. The increase in nucleotide pool size cannot be accounted for by a corresponding increase in de novo purine and pyrimidine nucleotide synthesis, as experiments with labeled formate and aspartate show similar inhibitions by the mutagens. With the salvage precursors, [ 3H]uridine and [ 3H]hypoxanthine, the mutagens can produce a widely divergent reduction in the labeling of RNA-CMP versus RNA-UMP and of RNA-GMP versus RNA-AMP, mostly a result of these agents causing large differences in the specific activities of the respective triphosphate precursors. These observations suggest that, in addition to the reactions with DNA, nucleotide biosynthesis could be another important biochemical target of chemical mutagens.

Growth inhibitory activity of succinylacetone: studies with Walker 256 carcinosarcoma, Novikoff hepatoma and L1210 leukemia.

Succinylacetone (SA, 4,6-dioxoheptanoic acid) inhibits d-aminolevulinic acid dehydrase, the second enzyme of the heme biosynthetic pathway and thereby inhibits heme biosynthesis. In the present study SA is shown to inhibit the growth of the Walker carcinosarcoma (W256) in vitro and in vivo, the Novikoff hepatoma in vivo, and L1210 leukemia in vitro, but only slightly in vivo. Rats can tolerate significantly larger doses of SA for at least twice as long as were administered in the present study without gross evidence of toxicity. In contrast to findings in previously published studies with murine erythroleukemia cells, the inhibition of growth of L1210 and W256 cells by SA in vitro is not accompanied by a decrease in cellular heme and is not reversed by addition of hematin to the medium. This suggests a second mechanism of growth inhibition of tumor cells unrelated to heme biosynthesis. Although the growth of both W256 and L1210 cells was markedly inhibited by the same concentration of SA in culture, there was a great difference in responsiveness in vivo, in that much greater inhibition of the growth of the Walker tumor was produced by SA.