Biochemical effects of bleomycin A2 on Novikoff hepatoma ascites cells.

Purified nucleolar DNA was markedly degraded at a concentration of 13 mug/ml by bleomycin A2; bleomycin concentrations 20-30 times greater were required to degrade nucleoplasmic DNA. Whole nuclear DNA was degraded to only a small extent at 13 mug/ml but was markedly degraded at higher bleomycin concentrations. Treatment of the various types of DNA with high concentrations of bleomycin A2 produced low molecular weight (approximately 6S) fragments that were no longer sensitive to degradation by bleomycin A2. Hybridization studies demonstrated a loss of ribosomal DNA sequences from nucleolar DNA treated with bleomycin A2 in vitro. Studies on RNA synthesis in Novikoff hepatoma ascites cells in vitro showed there was a decreased uptake of 32Pi into high molecular weight nuclear RNA in the presence of bleomycin A2. These results indicate that nucleolar function is inhibited by a direct effect of bleomycin A2 on nucleolar DNA.     

A comparison of the utilization of thymine and thymidine for the synthesis of DNA-thymine in novikoff hepatoma cells

A comparison was made between the utilization of thymine and thymidine for the synthesis of DNA in Novikoff hepatoma cells growing in suspension culture. When the cell cultures were switched from exponential growth to a relatively non-growing condition, by resuspending them in culture media minus serum for 18 h, there was an 85% decrease in the rate of thymidine incorporation but only a 15% decrease in the rate of thymine incorporation. Exposure to an alkylating agent (methyl methane sulfonate) resulted in a 79% decrease in thymidine incorporation, while thymine incorporation was decreased only 35%. Thymidine at a concentration equal to its Km for incorporation into DNA (4 × 10⁻⁷ M) had virtually no effect on thymine incorporation. It was not until a thymidine concentration of ten times the K_m was employed that appreciable (40%) decreases in the rate of thymine incorporation were observed. Examination of total cellular DNA or nuclear DNA gave similar results. These studies are interpreted as indicating the presence of multiple precursor pools for the synthesis of DNA-thymine in Novikoff hepatoma cells.     

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. When very low protein concentrations were cross-linked, the dimer was the predominant product. As protein concentration increased, we noted a decrease in dimers and a corresponding increase in tetramers, suggesting that the dimer may be a precursor to the tetramer. In support of the cross-linking experiments, two-dimensional gel electrophoresis using 4 M urea in the first dimension indicated a predominant association of p56 and p39 in the Novikoff ascites hepatoma cytokeratin complexes.     

Metabolism of 5-fluorouracil in sensitive and resistant Novikoff hepatoma cells.

N1-S1/FdUrd Novikoff hepatoma cells, which lack thymidine kinase activity, are resistant to 5-fluorouracil (FUra) as well as 5-fluorodeoxyuridine (FdUrd), suggesting that the pathway, FUra leads to FdUrd leads to FdUMP, is utilized for the conversion of FUra to FdUMP. However, the inhibition of thymidylate synthetase activity, the presumed target of FdUMP, by 1 X 10(-4) M FUra in intact N1-S1 Novikoff hepatoma cells, which have significant levels of thymidine kinase activity, is completely eliminated by 5 X 10(-4) M hydroxyurea, which is a potent inhibitor of ribonucleotide reductase. These results imply that the formation of ribonucleotides and does not involve thymidine kinase. This apparent dichotomy can be explained by the fact that, in addition to the well known lack of thymidine kinase activity, [14C]FUra conversion to ribonucleotides is greatly depressed in the N1-S1/FdUrd cells. Hence, the formation of FdUMP from FUra in Novikoff hepatoma cells apparently proceeds primarily via the intermediate formation of ribonucleotides. The decreased conversion of FUra to ribonucleotides in N1-S1/FdUrd cells decreases not only the ability of the analog to inhibit DNA synthesis, but also its effect on RNA metabolism. FUra, at a concentration of 1 X 10(-5) M, inhibits rRNA maturation in N1-S1 cells, but not in N1-S1/FdUrd cells. Since N1-S1/FdUrd cells are completely resistant to 1 X 10(-5) M FUra, whereas N1-S1 cells are completely inhibited by 1 X 10(-5) M FUra, even in the presence of 1 X 10(-4) M thymidine, the effects of FUra on RNA metabolism appear to contribute significantly to the cytotoxicity of the analog at higher drug concentrations.     

Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells.

The effects of exogenous proteins on the incorporation of [3H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A2, 5123C, and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10-20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100-400 microgram/ml. In experiments with lower cell concentrations, a 60% or greater increase in [3H]-thymidine incorporation could be obtained with total calf thymus histone and with F1 and arginine-rich histones from rat liver. At concentrations of 1-2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80-90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10 microgram/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis.     

Effects of phenethyl alcohol on transport reactions, nucleotide pools and macromolecular synthesis in Novikoff rat hepatoma cells growing in suspension culture

At cytostatic concentrations, phenethyl alcohol has immediate and reversible effects on multiple metabolic processes of Novikoff rat hepatoma cells growing in suspension culture. These include an inhibition of the transport of various low molecular weight substances into the cell, an inhibition of DNA and protein synthesis and the processing of ribosomal RNA, and a degradation of ribosomal RNA. All effects might be explained as resulting from an interaction of the chemical with cellular membranes. Phenethyl alcohol does not have an immediate effect on RNA synthesis per se. The immediate failure of phenethyl alcohol-treated cells to incorporate uridine from the medium into RNA is due to an inhibition of the uridine transport reaction.     

Thymidine transport in cultured mammalian cells. Kinetic analysis, temperature dependence and specificity of the transport system.

The transport of thymidine has been characterized kinetically and thermodynamically in Novikoff rat hepatoma cells grown in culture and, less extensively, in mouse L cells, Chinese hamster ovary cells, P388 murine leukemia cells and HeLa cells. That the characterizations pertained to the transport system per se was ensured, (i) by employing recently developed methods for rapid sampling of cell/substrate mixtures in order to follow isotope movements within a few seconds after initial exposure of cells to substrate; (ii) by utilizing cells rendered, by genetic or chemical means, incapable of metabolizing thymidine; and (iii) by demonstrating conformity of the transport data to an integrated rate equation derived for a simple, carrier-mediated system. The results indicate that thymidine is transported into mammalian cells by a functionally symmetrical, non-concentrative system for which the carrier : substrate dissociation constant ranges from about 100 microM in Chinese hamster ovary cells, to 230 microM in Novikoff hepatoma cells. In all cell lines investigated, the velocity of transport was sufficient to nearly completely equilibrate low concentration of thymidine across the membrane membrane within 15 s. Temperature dependence of transport velocity and substrate : carrier dissociation were continuous (EA = 18.3 kcal/mol, delta H0' = 9.3 kcal/mol, respectively), and showed no evidence of abrupt transitions. Several natural and artificial nucleosides and nucleic acid bases inhibited influx of radiolabeled thymidine, apparently by competing with thymidine for the transport carrier.     

Synthesis and biological activity of canavanine hydrazide derivatives

Summary. The canavanine derivatives L-canavanine hydrazide (CH), L-canavanine-bis-(2-chloroethyl)hydrazide (CBCH) and L-canavanine phenylhydrazide (CPH) were synthesized and evaluated for biological activity in microorganisms, plants and tumor cells using canavanine as a positive control. (1) In microbial systems, the compounds exerted activity, as assessed in 14 bacterial strains. The effect of canavanine was easily removed by equimolar concentrations of arginine or ornithine, while the effect of CBCH or CPH was abolished by 10-fold excess of arginine or 10- to 100-fold excess of ornithine. (2) In plants, the activity of CH and CBCH were relatively low, whereas the inhibitory potential of CPH was comparable or even superior to that of canavanine, resulting at 1 mM concentration in a nearly complete block of tomato cell growth, and reducing by up to 80% the length of radicles of cress, amaranth, cabbage and pumpkin. (3) In pumpkin seeds, CPH or canavanine induced the synthesis of four small heat shock proteins of hsp-17 family in the pH range of 6 to 7.5. The proteins exhibited in both cases a similar profile, but differed in the timing of their expression and/or accumulation. With canavanine, the highest hsp-17 expression was found after 48 h of drug treatment, while with CPH this maximum was shifted to 24 h. (4) CPH proved to be highly cytotoxic against Friend leukemia cells in culture, exceeding by one order of magnitude the cytotoxicity of canavanine. The effect of canavanine was completely removed in the presence of equimolar amounts of arginine, while a 20-fold excess of arginine failed to abolish the cytotoxicity of CPH. Thus, a proper hydrazide modification of canavanine may lead to a significant increase in its growth-inhibitory activity and to a change in the mode of action of the parent compound. Received October 5, 1999, Accepted January 27, 1999     

Canavanine-lnduced inhibition of growth and heterocyst differentiation inAnabaena doliolum and isolation of a canavanine-resistant mutant

The effect of the arginine analogue, canavanine on growth and heterocyst differentiation in the nitrogen-fixing algaAnabaena doliolum has been studied. The analogue inhibited growth and heterocyst differentiation at a concentration as low as 1 μM. The treated algal cells lacked conspicuous granular inclusions, whereas treatment with chloramphenicol led to increased synthesis of granules (probably cyanophycin granules). Exogenously added arginine completely reversed the effect of the analogue but lysine could only partially relieve the effect. A time course study with canavanine indicated inhibition of fresh protein(s) synthesis at all steps where a new class of proteins is synthesized so that the action of the analogue does not seem to be specific for a particular kind of protein. A mutant resistant to this analogue has been successfully isolated indicating that this alga does not show mutational immunity at least to the amino acid analogues unlike in the observation with different antibiotics. Our observations indicate that canavanine either directly inhibits protein synthesis or forms defective protein(s) which produces all the observed effects.     

Agglutination of Trypanosoma cruzi by Concanavalin A*

Epimastigotes of Trypanosoma cruzi obtained in culture agglutinate readily with low concentrations of concanavalin A (Con A). Agglutination was linear with time up to 10 min providing that the initial cell density was greater than 1 × 10⁸ cells/ml. Under these conditions, the percentage agglutination was dependent on the Con A concentration. Agglutination was inhibited by α-methyl D-mannoside, α-D-mannose, and α-D-glucose. Pretreatment of cells with trypsin had no effect on the epimastigote agglutinations. Blood forms (trypomastigotes) of T. cruzi did not agglutinate even in the presence of 100 times more Con A. Results suggest differences in membrane structure between blood forms and cultured epimastigotes of T. cruzi. These membrane differences might be related to the different pathogenic properties of both cell forms of T. cruzi.     

Uridine uptake inhibition in Novikoff hepatoma cells by perturbants of intracellular Ca2+ and K+ levels

The rate of uptake of uridine into the acid-soluble fraction of Novikoff hepatoma cells is inhibited by low concentrations of the ionophores A23187 and gramicidin and other perturbants of intracellular cation levels. Inhibition of uridine uptake by A23187 is dependent on Ca2+ and is reduced by serum and high levels of Mg2+. The effectiveness of A23187 is dependent on the Ca2+/Mg2+ ratio rather than the absolute concentration of either ion. Inhibition of uridine uptake by gramicidin is not significantly affected by serum or divalent cations. Other effectors of monovalent cation flux such as ouabain and valinomycin also inhibit uridine uptake. These results indicate that net uptake of uridine may be influenced by intracellular levels of certain monovalent and divalent inorganic cations.     

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.     

Monovalent carboxylic ionophores inhibit transport of carbamoyl-phosphate synthetase I into mitochondria in Reuber hepatoma H-35 cells and cause accumulation of enzyme precursor

Transport of the precursor for carbamoyl-phosphate synthetase I into mitochondria in Reuber hepatoma H-35 cells was inhibited by adding monensin or nigericin to the culture medium at a concentration of 0.5 microM, and the enzyme precursor accumulated, mainly in the cytosolic fraction. Accumulated precursor was degraded slowly with a half-life of more than 16 min. Valinomycin, nonactin, A23187, X-537A (lasalocid), bromo-lasalocid, and carbonyl cyanide m-chlorophenylhydrazone did not exhibit these effects at concentrations at which they did not inhibit protein synthesis of the cells.     

Inhibition of transport systems in cultured rat hepatoma cells by colcemid and ethanol

The transport of uridine, hypoxanthine, and choline in cultures of Novikoff rat hepatoma cells is competitively inhibited by colcemid with apparent K_i values of 135, 60, and 250 μM respectively, whereas the transport of 2-deoxy-D-glucose is not affected. Ethanol at high concentrations inhibits the transport of all four substrates in an apparent competitive manner.     

Inhibition of mitogen-induced lymphocyte transformation by local anesthetics.

Chlorpromazine (CPZ) and lidocaine were added to cultures of mouse spleen cells stimulated by concanvalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and lipopolysaccharide (LPS). Concentrations of CPZ greater than 5 x 10(-6)M and concentrations of lidocaine greater than 2 x 10(-3)M totally inhibited the mitogenic responses to all four mitogens. Minimal inhibitory concentrations of neither drug interferred with cell viability as determined by trypan blue uptake or 51Cr release. The effects were totally reversed by the removal of the drugs from the culture. Addition of the drug at intervals after mitogen exposure demonstrated that the inhibited event occurred relatively soon after exposure to the mitogen. For example, the addition of lidocaine or CPZ more than 24 hr after Con A stimulation had no effect on tritiated thymidine incorporation. Elevated concentrations of cyclic AMP, cyclic GMP (or their derivatives) or calciunown membrane active actions of these drugs and the rapid reversibility of the effect strongly support the idea that the local anesthetics act on the surface membrane of lymphocytes. Binding of radiolabeled Con A or LPS to lymphocyte membranes in the presence of lidocaine or CPZ was not inhibited. The possibility exists that CPZ and lidocaine disorganized cell membranes so as to interfere with the surface membrane elaboration or action of a second messenger, or interfere with cell-cell interactions.     

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. Time dependence experiments showed that detectable cross-linking occurred after cells were exposed to K2CrO4 for at least 4 h, and the amount of keratin-DNA complexes increased with the incubation time. Each of the three Novikoff ascites hepatoma cytokeratins (p39, p49, and p56) showed a different apparent rate of cross-link formation with DNA. Cytokeratin-DNA complexes were detectable in our system only with K2CrO4 concentrations of 200 microM or greater, and saturation in cross-linking was effected at approximately 2 mM. Higher K2CrO4 concentrations (up to 5 mM) did not produce further significant increases in the amount of cross-linked cytokeratins. Chromium and cis-DDP cross-linked the same cytokeratins at approximately the same ratios; however, both agents cross-linked the major cytokeratins selectively, since not all cytokeratins present in Novikoff hepatoma cell lysates could be cross-linked to DNA. Further evidence of DNA-cytokeratin complexes was obtained by CsCl gradient centrifugation. Our results document the ability of chromate and cis-DDP to produce DNA-cytokeratin cross-links in vivo and show that in live Novikoff hepatoma cells some, but not all, of the components of intermediate filaments are within cross-linking distance of DNA.     

Insulin-mimetic actions of wheat germ agglutinin and concanavalin A on specific mRNA levels

Insulin has pleiotropic effects on sensitive cells, including the regulation of specific mRNA accumulation initiated by the binding of insulin to its plasma membrane receptor. Lectins, such as wheat germ agglutinin (WGA) and concanavalin A (Con A), are known to be insulin mimetic. It is thought that WGA and Con A interact with the insulin receptor or associated membrane glycoproteins which, when activated, lead to insulin-mimetic responses. We attempted to determine whether WGA and Con A could induce the accumulation of a specific messenger RNA (p33-mRNA). Insulin treatment of H4IIE (H4) hepatoma cells increased the concentration of p33-mRNA within 30 min after addition, with a maximum effect of 10- to 15-fold. WGA and Con A also exhibited time- and dose-dependent stimulatory effects on p33-mRNA accumulation with maximal effects of 30- to 40-fold. The effect of insulin was maximal by 1 h and plateaued thereafter, whereas lectins had maximal effects at 2 h after addition to cell cultures. Insulin, WGA, and Con A did not significantly alter the stability (half-life) of p33-mRNA. The addition of RNA synthesis inhibitors blocked the ability of insulin, WGA, and Con A to induce the amount of p33-mRNA. These data suggest that lectins, as well as insulin, induce the synthesis of p33-mRNA in acutely treated H4 hepatoma cells.