In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.     

Biological properties of a hepatocyte growth factor from rat platelets

In an accompanying communication we demonstrated that about half of the potency of rat serum to stimulate DNA synthesis in cultured adult rat hepatocytes resides in a polypeptidelike substance from the platelets. A lysate of rat platelets was able to restore the potency of platelet-poor rat serum, whereas a lysate of human platelets inhibited thymidine incorporation by the hepatocytes. Moreover, addition to these cultures of either highly purified human platelet-derived growth factor (PDGF) or human platelet factor 4 (PF-4) failed to influence DNA synthesis either alone or in the presence of rat or human platelet-poor serum, which is required for expression of PDGF activity. Unlike the human platelet factors, rat platelet lysate (RPL) was moderately active by itself and was augmented equally well by platelet-poor serum from either source. At concentrations below 5%, platelet-poor serum from hypophysectomized rats was as potent as that from normal rats in augmenting RPL activity. This suggests that, unlike PDGF, which is not activated by hypophysectomized rat serum, the hepatotrophic component of RPL does not require the presence of exogenous somatomedins for activity, but interacts instead with other plasma constituents or with somatomedins produced by the hepatocytes in vitro. Rat platelets do, however, appear to contain PDGF or its rat equivalent in addition to the hepatocyte growth factor, since if they are heated to 100 degrees C for 10 min, their ability to stimulate nuclear labeling in confluent BALB/c 3T3 cells is not impaired, while their ability to stimulate DNA synthesis in rat hepatocytes is destroyed. These studies indicate that the hepatocyte growth factor from rat platelets differs from PDGF in its biological as well as physical characteristics, but that rat platelets also contain PDGF or an equivalent substance.     

Stimulation of collagen production in growth-arrested myocytes and fibroblasts in culture by growth factor(s) from platelets

Human fibroblasts, rabbit aortic fibroblasts and rabbit aortic myocytes were growth-arrested in serum-free media and stimulated with material spontaneously released from human platelets in vitro. The cells increased their collagen production 2- to 3-fold with increasing platelet factor concentration, whereas the cell mass, measured by total DNA, was unaffected. An increase in [³H]thymidine incorporation into DNA was noted, however. Thus, platelets can release factors stimulating collagen synthesis, without necessarily inducing concomitant cell division.     

Characterization of a yeast mitochondrial locus necessary for tRNA biosynthesis. Deletion mapping and restriction mapping studies

Yeast mitochondrial DNA codes for a complete set of tRNAs. Although most components necessary for the biosynthesis of mitochondrial tRNA are coded by nuclear genes, there is one genetic locus on mitochondrial DNA necessary for the synthesis of mitochondrial tRNAs other than the mitochondrial tRNA genes themselves. Characterization of mutants by deletion mapping and restriction enzyme mapping studies has provided a precise location of this yeast mitochondrial tRNA synthesis locus. Deletion mutants retaining various segments of mitochondrial DNA were examined for their ability to synthesize tRNAs from the genes they retain. A subset of these strains was also tested for the ability to provide the tRNA synthesis function in complementation tests with deletion mutants unable to synthesize mature mitochondrial tRNAs. By correlating the tRNA synthetic ability with the presence or absence of certain wild-type restriction fragments, we have confined the locus to within 780 base pairs of DNA located between the tRNAMetf gene and tRNAPro gene, at 29 units on the wild-type map. Heretofore, no genetic function or gene product had been localized in this area of the yeast mitochondrial genome.     

DNA synthesis in isolated mitochondria and mitochondrial extracts from wheat embryos

DNA synthesis was studied using purified wheat embryo mitochondria as well as mitochondrial lysates deprived of endogenous DNA. The optimal conditions for DNA synthesis are very similar in both systems: ATP stimulates dramatically mitochondrial DNA synthesis and magnesium is a better co-factor than manganese, contrary to what has been reported in animal mitochondrial systems. Wheat mitochondrial DNA synthesis is resistant to aphidicolin and strongly inhibited by dideoxythymidine triphosphate and ethidium bromide. Thus, the DNA polymerase involved in this system seems to be the same as that previously purified and characterized from wheat embryo mitochondria (Christopheet al., Plant Science Letters 21: 181, 1981). Two different approaches: restriction endonuclease digestion followed by electrophoresis, and autoradiography and cesium chloride equilibrium centrifugation of mitochondrial DNA, where BrdUTP has been incorporated instead of TTP, show that long stretches of the mitochondrial genome have been synthesized.     

DNA synthesis rate and unlabeled cells with S-phase DNA content in P388 leukemic cells in vivo studied by flow cytometry and cell sorting

DNA synthesis kinetics of P388 leukemic cells growing in ascites form in BDF1 hybrid mice were investigated during the periods of exponential growth and growth restriction. Incorporation of tritiated thymidine, and in some instances tritiated uridine, was studied by autoradiography in cells sorted from S-phase fractions during DNA flow cytometry. During exponential growth continuous labeling with tritiated thymidine indicated a growth fraction of unity, whereas the growth fraction was about 30% during growth restriction. At this growth phase the majority of cells with S phase DNA content remained unlabeled after pulse labeling with tritiated thymidine or uridine, indicating that both the "salvage" and the "de novo" DNA synthesis pathways were blocked in most S-phase cells. After pulse labeling with tritiated thymidine the DNA synthesis rate pattern was investigated by sorting of consecutive fractions of cells throughout the S phase followed by quantitative autoradiography. With exception of a reduced rate in the middle of S phase, the DNA synthesis rate increased as the cells progressed through S phase during exponential growth. In contrast, the DNA synthesis rate pattern had a relative peak in the middle of S phase during growth restriction, which is otherwise characterized by a low mean DNA synthesis rate.     

Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue in the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation.     

Thymidylate nucleotide supply for mitochondrial DNA synthesis in mouse L-cells. Effect of 5-fluorodeoxyuridine and methotrexate in thymidine kinase plus and thymidine kinase minus cells.

The effects of 5-fluorodeoxyuridine and methotrexate on [3H]thymidine and 32P labeling of mtDNA were studied in two lines of mouse L-cells. LMTK- cells, which lack the major cellular thymidine kinase (EC 2.7.1.21) but contain a genetically distinct mitochondrial enzyme, were compared to LA9 cells, which contain both thymidine kinase activities. LMTK- cells were resistant to 5-flurodeoxyuridine by a factor of 200 in comparison to LA9 cells. In both cells lines appropriate drug treatment increased utilization of exogenous thymidine for mtDNA synthesis. The maximum enhancement was 10- to 12-fold for LA9 cells and approximately 20-fold for LMTK- cells when treated with 10 muM methotrexate. The rates of mtDNA and nuclear DNA synthesis during drug treatment were analyzed with 32P labeling and 5-bromo-2'-deoxyuridine density labeling experiments. Synthesis of both mtDNA and nuclear DNA were strongly inhibited by drug treatment of either LA9 or LMTK- cells in the absence of exogenous thymidine. The rate of mtDNA synthesis substantially exceeded that of nuclear DNA in LA9 cells treated with 4 muM 5-fluorodeoxyuridine and less than 5 muM thymidine. Both synthetic rates approached those of untreated LA9 control cultures if 20 muM thymidine was present during 5-fluorodeoxyuridine treatment. In contrast, in LMTK- cells treated with 10 muM methotrexate and 20 muM thymidine, mtDNA synthesis continued at 50 to 60% of the control rate for at least 10 hours while nuclear DNA synthesis was 96% inhibited. Synthesis of mtDNA mass-labeled in both strands with 5-bromouracil occurred when LMTK- cells were incubated for 30 hours with 10 muM methotrexate and 20 muM 5-bromodeoxyuridine. These results indicate that mtDNA synthesis is resistant to a limitation of the thymidine triphosphate supply and is not strictly dependent upon concomitant nuclear DNA synthesis in these cells.     

Mapping and identification of the vaccinia virus thymidine kinase gene

The thymidine kinase gene of vaccinia virus (VV) was mapped on the viral genome by using cloned fragments of the viral DNA to hybridize to early viral mRNA. Individual DNA fragments that represented about half of the viral genome were assayed, both for their ability to arrest the cell-free synthesis of active VV thymidine kinase and for their ability to select functional mRNA for the viral enzyme. Both activities were located in HindIII fragment J, which maps near the middle of VV DNA and contains about 2.6% of the genome (4,800 base pairs). This DNA fragment encodes four known early polypeptides, and to determine which of these was thymidine kinase, early VV mRNA was fractionated by sucrose gradient centrifugation and used to direct cell-free synthesis of the active enzyme. The thymidine kinase mRNA cosedimented with several species that encoded polypeptides in the molecular weight range 15,000 to 25,000. Hybridization of these mRNAs to HindIII-J DNA selected a message that directed the synthesis of thymidine kinase and a single polypeptide with an apparent molecular weight of 19,000. The native molecular weight of VV thymidine kinase is about 80,000, so these data indicate that, unlike thymidine kinase from several other sources, the active VV enzyme is probably a tetramer of 19,000-molecular-weight subunits.     

Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue into the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of the unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

Mitochondrial DNA topoisomerase I from human platelets.

An anucleated cell system has been used for the first time to study mitochondrial topoisomerase activity. Mitochondrial extracts from human blood platelets contained type I topoisomerase. The type I classification was based on ATP-independent activity, inhibition by ATP or camptothecin, and the lack of inhibition by novobiocin. Platelet mitochondrial topoisomerase I relaxation activity was inhibited linearly by increasing concentrations of EGTA. Topoisomerase activity greater than 90% inhibited by 175 microM EGTA was partially restored to 16 and 50% of the initial level of activity by the subsequent addition of 50 and 100 microM Ca2+, respectively. Additionally, results from studies of partially purified platelet mitochondrial topoisomerase I were consistent with the crude extract data. This work supports the hypothesis that platelet mitochondria contain a type I topoisomerase that is biochemically distinct from that previously isolated and characterized from cell nuclei.     

Effects of Ribonucleosides on Thymidine Incorporation: Selective Reversal of the Inhibition of Deoxyribonucleic Acid Synthesis in Thymineless Auxotrophs of Escherichia coli

Addition of certain ribonucleosides to exponentially growing cultures of Escherichia coli increased the extent of thymidine incorporation. The prolonged uptake of thymidine was correlative with the ability of these ribonucleosides to prevent the degradation of thymidine. In addition to protecting thymidine, uridine reversed partially (70 to 80%) the inhibition of deoxyribonucleic acid (DNA) synthesis in thymineless auxotrophs by cytosine arabinoside, hydroxyurea, and nalidixic acid. This reversal was selective for auxotrophic strains since no reversal of inhibition by uridine was observed in any of the prototrophic strains examined. In the presence of uridine, the rapid assimilation of thymidine by prototrophic and auxotrophic strains was prevented and the rate of DNA synthesis became a function of the available exogenous thymidine. Under these conditions, prototrophic strains accumulated equivalent amounts of thymidine into the acid-soluble (pool) and acid-insoluble (DNA) cell fractions. In contrast, 95 to 98% of the thymidine taken up by auxotrophs was found in the acid-insoluble (DNA) cell fraction. The results suggest that different mechanisms for DNA synthesis exist in auxotrophs and prototrophs. Based on these observed differences, some possible mechanisms for the selective reversal of the inhibition of DNA synthesis in auxotrophs are discussed.     

Stimulation of fibroblast proliferation by thrombospondin

Thrombospondin purified from human platelets was examined for its ability to promote proliferation of human dermal fibroblasts. The results show that thrombospondin could stimulate the incorporation of [3H]thymidine by quiescent fibroblasts in a dose-dependent manner without stimulating protein or collagen synthesis. The effect was observed even in the total absence of serum, although the degree of stimulation was substantially lower than that in the presence of 0.4% fetal calf serum, but higher than that in the presence of 4% serum. The effect was specific and not due to contaminants as demonstrated by the ability of antibodies to thrombospondin to specifically inhibit this stimulation. Three monoclonal antibodies directed at different epitopes in the thrombospondin molecule were equally effective in inhibiting this effect. This stimulation of fibroblast proliferation by thrombospondin suggests a potential role for this matrix protein in the mesenchymal cell response in tissue injury and repair.     

Sulfated glycolipids are the platelet autoantigens for human platelet-binding monoclonal anti-DNA autoantibodies

The human monoclonal autoantibody HF2-1/17, produced by a human-human hybridoma derived from lymphocytes of a lupus patient with thrombocytopenia, reacts with single stranded DNA and platelets. To determine the chemical nature of the autoantigen against which this antibody is directed on platelets, this platelet antigen was purified by the lipid extraction of sonicated platelets, DEAE-Sephadex chromatography, and high performance liquid chromatography. The purified glycolipids, a trace component in platelets, demonstrated high reactivity with the HF2-1/17 antibody using a competition enzyme-linked immunosorbent assay system or immunostaining of thin layer chromatograms. The purified glycolipids co-migrated with bovine sulfatides by thin layer chromatography. The purified glycolipids contain sulfate and galactose but not sialic acid or phosphate. Fast atom bombardment-mass spectrometry revealed these sulfatides to be sulfated monohexyl ceramides. The dominant species has a molecular weight of 794 while a minor form has a molecular weight of 812 due to an extra hydroxyl group and loss of a double bond. These results indicate that the platelet autoantigen against which the human monoclonal anti-DNA antibody is directed represents a family of novel monogalactosyl sulfatides.     

Platelet-collagen interaction. Inhibition by a monoclonal antibody raised against collagen receptor

Monoclonal antibodies to the purified platelet type I collagen receptor were produced to study platelet receptor function. The antibody specifically reacted with the platelet receptor in immunoblot experiments. The IgG purified from the monoclonal antibodies and isolated Fab' fragments inhibited the binding of radiolabeled alpha 1(I) chain to washed platelets competitively. Soluble and fibrillar type I collagen-induced platelet aggregations were inhibited by purified IgG suggesting that soluble and fibrillar collagens shared a common receptor. The adhesion of platelets to an artificial collagen matrix was also inhibited by the monoclonal antibody. However, adenosine diphosphate-induced platelet aggregation was not inhibited by the same amount of IgG that inhibited collagen-induced platelet aggregation. The results suggest that collagen-induced platelet aggregation is mediated through the interaction of collagen with the platelet receptor.     

Changes in macromolecular synthesis in Xanthomonas oryzae infected with bacteriophage XP-12.

Phage XP-12, which has complete substitution of the cytosine residues in its DNA with 5-methylcytosine residues, was shown to inhibit incorporation of uracil into host DNA and RNA during the latent period. This apparent inhibition of host macromolecular synthesis was not accompanied by extensive degradation of the host chromosome. Phage DNA synthesis in infected cells occurred at a faster rate than host DNA synthesis in analogous uninfected cells. However, phage DNA synthesis could not be accurately monitored by incorporation of [methyl-3H]thymidine into DNA because, soon after infection, there was a marked inhibition of utilization of exogenous thymidine for DNA synthesis. Phage infection conferred upon a thymine auxotrophic host the ability to synthesize thymine nucleotides for phage DNA synthesis. It is suggested that a phage-induced thymidylate synthetase activity is partially responsible for the inhibition of thymidine incorporation.     

Platelet adhesiveness and aggregation: the collagen:glycosyl, polypeptide:N-acetylgalactosaminyl and glycoprotein:galactosyl transferases of human platelets

Four glycoprotein:glycosyl transferases were identified, purified, and characterized from human platelets. The enzymes present were collagen:glc, collagen:gal, polypeptide:galNAc, and glycoprotein:gal; the fetuin:glcNAc transferase was absent. Each of the 4 transferases was found to be almost exclusively bound to the platelet plasma membrane. Incubation of platelet homogenates without exogenous acceptors yielded no transfer of monosaccharide, indicating the complete lack of endogenous acceptors. These results lead to the interesting speculation that the transferases may not be responsible in the mature platelet for glycoprotein synthesis at all, but rather that they may function for intercellular adhesion and the primary step in hemostasis, the adhesion of collagen to platelets.     

Modulation of platelet activation by native DNA.

Native DNA (dsDNA) was found to induce the aggregation of isolated human platelets and the release of platelet 5HT; this activation was inhibited by both theophylline and TYA, suggesting a role for cAMP and metabolic products formed from arachidonate. By contrast, nonaggregating amounts of dsDNA inhibited platelet activation induced by collagen or thrombin. This inhibition, which could be overcome by use of greater amounts of the stimulatory agents, was not associated with the loss of platelet viability. Activation of platelets by dsDNA was not observed in plasma or in isolated platelet systems to which small amounts of cell-free plasma were added. However, dsDNA maintained in plasma its ability to inhibit platelet aggregation induced by collagen and thrombin. RNA and single-stranded DNA failed to induce platelet aggregation or release of 5HT and to block the platelet activation stimulated by dsDNA. Further, dsDNA did not significantly inhibit platelet aggregation in platelet-rich plasma stimulated by ADP or epinephrine. These data implicate dsDNA as a selective and potentially important activator and modulator of platelet responsiveness.     

The role of blood platelets in nucleoside metabolism: regulation of platelet thymidine phosphorylase

Blood platelets are the smallest cellular elements in mammalian blood. Because of their small size, platelets have an usually large surface area: volume ratio and are exquisitely sensitive to a multitude of physiologica and environmental stimuli. Platelets lack nuclei, but most possess functional mitochondria and remain capable of both anaerobic and aerobic energy metabolism, for which they utilise a variety of substrates including many which are cytotoxic and genotoxic for other (nucleated) cells. Nucleic acid precursors are amongst the potentially genotoxic compounds for which platelets have an apparently insatiable appetite. In particular platelets actively scavenge adenine and adenosine, which they convert to nucleotides and use in energy metabolism, but they also rapidly phosphorylyse thymidine and liberate thymine into the extracellular medium. In addition, platelets contain non-metabolisable membrane-bound pools of adenine nucleotides which they secrete in response to strong agonists. Taken together, these observations suggest that blood platelets play an important role in nucleic acid precursor metabolism.

In the previous paper we have shown that most thymidine phosphorylase activity present in normal human blood resides in the cytoplasm of platelets. Here we demonstrate that this enzyme activity can be modulated in a dose-dependent fashion, not only by substances recognised as platelet agonists and antagonists, but also by some compounds which are considered to be toxic, mutagenic and/or carcinogenic. The data which we present provide additional support for our previous suggestion that platelets regulate thymidine homeostasis and further imply that this is the normal, physiological, platelet function. Preliminary results suggest that assays of blood platelet thymidine metabolism may provide data with a wide variety of applications.     

Effect of N-trifluoroacetyladriamycin-14-valerate on [3H]thymidine uptake and DNA synthesis of human lymphoma cells

The effects of N-trifluoroacetyladriamycin-14-valerate on the uptake of [3H]thymidine and its incorporation into DNA of human P3HR-1 lymphoma cells were studied. In the absence of the drug, at 0 degrees C, [3H]thymidine was transported into the cells but not incorporated into DNA, as determined by both the trichloroacetic acid-soluble and -precipitable counts obtained with the cells. At 37 degrees C, [3H]thymidine was readily transported into the cells and incorporated into DNA. In the presence of the drug, both [3H]thymidine uptake (as shown by acid-soluble counts) and the amount of its incorporation into acid-precipitable materials were markedly reduced. However, the uptake of [3H]thymidine at 0 degrees C was found to be equally sensitive to drug inhibition as at 37 degrees C. The incorporation at 37 degrees C of [3H]thymidine into acid-precipitable materials of the cells, which had been prelabeled at 0 degrees C with [3H]thymidine, was found to be insensitive to inhibition by the drug. The in vitro activities of DNA polymerases alpha and beta purified from human P3HR-1 cells were also found not to be susceptible to inhibition. Nuclei purified from cells pretreated with the drug continued to synthesize DNA. The cytofluorograms of the cells treated with the drug indicated that the treated cells accumulated at the G2/M phase, whereas the S phase of the cells was not arrested. These results suggest that N-trifluoroacetyladriamycin-14-valerate inhibits [3H]thymidine uptake but not cellular DNA synthesis in human P3HR-1 lymphoma cells.