Transcription of the c-myc oncogene is altered in spontaneously immortalized rodent fibroblasts.

Cellular immortalization seems generally to be a necessary, but not sufficient step in tumourigenesis. We have analysed the role of the cellular oncogene, c-myc in the process of in vitro cellular ageing and spontaneous cellular immortalization using rodent fibroblasts. The steady-state level of c-myc of mouse and rat fibroblasts does not change significantly during cellular ageing in vitro. By contrast, the steady state level of c-myc mRNA increases 3- to 20-fold upon spontaneous establishment of these rodent fibroblasts. The increase in the steady-state level of this mRNA is essentially due to an increase in the transcriptional rate. Not all oncogenes respond in this way; the mRNA levels of both c-fos and c-K-ras do not show the same alteration. The changes in the steady-state level of c-myc mRNA are not due to gene amplification nor to gross gene rearrangements or translocations. However, the response of the myc gene to growth factor stimulation is present apparently equally in both mortal and immortal cells; a difference is seen in an increased maintenance of high c-myc mRNA levels after growth stimulation in established cell lines. Both young and senescent mortal cells, as well as immortal cells, respond to mitogen stimulation with a sharp increase in c-myc mRNA levels. Thus, senescent cells are able to see mitogen signals, but do not go on to initiate DNA synthesis. We also demonstrated that the c-myc mRNA levels do not respond to serum concentration above a minimum level, nor do they respond to factors in the conditioned medium of immortal cell cultures.

NAD turnover during early development of Xenopus laevis.

The NAD pools of Xenopus laevis oocytes and early embryos can be radioactively labelled by microinjection of [adenine-3H]NAD. This technique is used to study the metabolism of NAD in oocytes and during early development. The rate at which NAD is degraded in vivo has been monitored by determining the rate of transfer of adenine residues from the NAD pool into other nucleotides and polynucleotides. In oocytes, NAD turnover is extremely slow, with a half-life of about 400 h. NAD turnover increases dramatically after fertilisation, and the half-life of the compound decreases to 37 h in 5-h-old embryos and to 10 h in 40-h-old embryos. 2 mM 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) polymerase, reduces the NAD turnover rate by about 20%, whereas 5 mM isonicotinic acid hydrazide, a specific inhibitor of NAD glycohydrolase, produces no significant inhibition. This indicates that a significant fraction of the considerable NAD turnover observed involves poly(ADP-ribose) polymerase. Our results indicate that poly(ADP-ribose) polymerase is active during early development and suggest that this activity may be involved in one or more aspects of the nuclear metabolism of the embryo.

Effect of DNA intercalators on poly(ADP-ribose) glycohydrolase activity.

Proflavine, ethacridine (2-ethoxy-6,9-acridine diamine), ellipticine, daunomycin and Tilorone R10,556 DA (2,7-bis(piperidinobutyryl)-9H-fluoren-9-one) inhibit poly(ADP-ribose) glycohydrolase activity. The Ki values for proflavine and Tilorone R10,556 DA are 36 microM and 7.3 microM, respectively. The inhibition by intercalators is relieved by DNA but not by DNA-histone complexes. On the contrary, DNA-histone complexes increase the inhibition of some intercalators. Ethidium bromide is not inhibitory by itself. However, in the presence of DNA-histone complexes it strongly inhibits the enzyme activity. m-AMSA (4'-(9-acridinylamino)methanesulphon-m-anisidide) and chloroquine have no effect on the enzyme activity, even in the presence of DNA-histone complexes.

The effects of detergents and phospholipids on a glycolipid galactosyltransferase.

1. Phospholipids activated the enzyme, lactosylceramide: UDP-galactose alpha-galactosyltransferase in hamster cells (NIL 2E clone 8) when assayed in the presence of neutral detergents. 2. Phosphatidylserine and phosphatidylinositol were the most effective phospholipids in activating the enzyme. Other phospholipids were also effective, but sphingomyelin and lysophosphatidylcholine were inhibitory. 3. Considerable enzyme activity was obtained in the absence of any detergent. Most of this activity was due to glycosylation of endogenous acceptors. 4. There was a complex effect of detergents on the enzyme activity. Very low concentrations were sharply inhibitory, but higher concentrations, above the critical micelle concentration for detergent, caused regeneration of activity. 5. The phospholipids, in the absence of a detergent, are required to maintain the lipid substrate, lactosylceramide, in a suitable dispersion where it can be acted upon by the enzyme. In the presence of detergents, it is proposed that the phospholipids also act by affecting the state of the lipid substrate.

Changes in mRNA levels of poly(ADP-ribose) polymerase during activation of human lymphocytes.

The level of mRNA encoding the nuclear enzyme poly(ADP-ribose) polymerase (ADP-ribosyltransferase, EC 2.4.2.30) was found to be very low in quiescent human lymphocytes and to increase at least 10-fold between 1 and 2 dyas after stimulation with the mitogen phytohaemagglutinin, staying high for several days thereafter. This increase was inhibited by 3-methoxybenzamide (a competitive inhibitor of poly(ADP-ribose) polymerase) but was not affected significantly by aphidicolin. Incubation of activated cells with cycloheximide for 2 h increased the expression slightly. These data demonstrate that, during lymphocyte activation, the level of mRNA of the poly(ADP-ribose) polymerase gene correlates with, and hence is presumably responsible for, the increase in poly(ADP-ribose) polymerase protein detectable by enzyme assay or immunochemistry.

The effect of gamma radiation and neocarzinostatin on NAD and ATP levels in mouse leukaemia cells.

When mouse leukemia cells are treated with gamma-radiation or neocarzinostatin the intracellular NAD and ATP levels fall rapidly. We have shown that the ATP response is a consequence of the decreased NAD level. We suggest that this low NAD level results in decreased glycolytic activity and that there is a subsequent accumulation of phosphorylated sugars associated with the fall in ATP. Under these extreme conditions, therefore, the NAD level probably regulates the rate of glycolysis in cells which are utilising a rapidly metabolisable sugar as their energy source.

Sequence of a chicken erythroblast mono(ADP-ribosyl)transferase-encoding gene and its upstream region.

We have cloned the MADPRT gene encoding the 300-amino-acid mono(ADP-ribosyl)transferase (MADPRT) from chicken erythroblasts. The protein has homology to the rabbit and human skeletal muscle (50% identity) and two chicken heterophil (52% identity) NAD+:arginine MADPRT. The active site region is particularly conserved. The upstream region of the MADPRT gene from erythroblasts has several features characteristic of promoter sequences.

The dynamic nature of DNA-strand breaks present in differentiating muscle cells and quiescent lymphocytes.

Cellular differentiation in a number of eukaryotic systems is associated with changes in the number of DNA-strand breaks and involves the activity of adenosine diphosphoribosyl transferase (ADPRT). DNA-strand breaks are essential for activation of nuclear ADPRT, the activity of which is required for efficient religation of DNA-strand breaks. In this study we demonstrate the dynamic nature of DNA-strand breaks formed in the genome of differentiating avian skeletal muscle cells and quiescent human lymphocytes. Inhibition of ADPRT activity blocks DNA-strand ligation in both cell types and leads to the accumulation of a higher number of strand breaks.

Werner's syndrome T lymphocytes display a normal in vitro life-span.

Werner's syndrome (WS) is an autosomal recessive disorder displaying many features consistent with accelerated ageing. Fibroblasts from WS patients show a distinct mutator phenotype (characterised by the production of large chromosomal deletions) and a profound reduction in proliferative capacity. The disorder results from a mutation in a novel ReqQ helicase. Recently, we demonstrated that the proliferative defect was corrected by the ectopic expression of telomerase. From these data, we propose that mutations in the wrn gene lead to deletions at or near the telomere which reduce the cells replicative life-span. This hypothesis predicts that cell types which retain the ability to upregulate telomerase as part of their response to a proliferative stimulus would fail to show any significant effect of wrn gene mutations upon life-span. Human T lymphocytes represent a well-characterised example of such a cell type. To test the hypothesis, WS T lymphocytes were cultured until they reached replicative senescence. These cultures displayed life-spans which did not differ significantly from those of normal controls. These findings are consistent with the hypothesis that the effects of wrn mutations on replicative life-span are telomere-mediated.

ADP-ribosylation reactions.

ADP-ribosylation reactions have been studied now for over 30 years. They came to light originally in studies of some bacterial toxins, which turned out to be mono-ADP-ribosyl transferases. Subsequently, endogenous mono-ADP-ribosyltransferases were discovered. Although the substrates of the toxins are always so-called G-proteins, the substrate for a muscle mono-ADP-ribosyl transferase has been shown to be an extra-cellular cell adhesion molecule. A new pathway of NAD catabolism is the recently described cyclic ADP-ribose; this seems to be involved in calcium metabolism. Just under 30 years ago, poly(ADP-ribose) polymerase was discovered. From protein studies as well as from recent molecular biology, some amino acids essential for the enzyme activity or for binding to DNA have been identified. I suggest that poly(ADP-ribose) polymerase has several related functions in maintaining the integrity of the genome in eukaryotic cells. The highly-charged polymer, poly(ADP-ribose), is always made when free, ie naked DNA ends appear, in order to ensure the correct processing of these DNA breaks. In particular, the polymer may act to prevent DNA recombination reactions that would interfere with DNA repair. In addition, the polymer may protect the free DNA end from exonuclease action, and thirdly, it may unravel the chromatin structure. This suggests that this enzyme is not a necessary component of the process of DNA excision repair, but that this enzyme is required for correct and efficient DNA excision repair.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP-ribosyl transferase activity in Trypanosoma brucei.

Nuclear adenosine diphosphoribosyl transferase (ADPRT) catalyses the covalent modification of chromatin proteins by (ADP-ribose)n. This activity, which is entirely dependent on DNA containing strand breaks, is required for efficient DNA excision repair possibly because it regulates DNA ligation. ADPRT activity is also required for cytodifferentiation in a number of different cell types. We report here the presence of ADPRT activity in the blood-stream form of Trypanosoma brucei and its activation by DNA strand breaks formed by exposure to, either exogenously supplied deoxyribonuclease I, or treatment with the methylating agent, dimethylsulphate. 3-Aminobenzamide, but not its chemical analogue 3-aminobenzoic acid, is a competitive inhibitor of ADPRT activity in T. brucei. Intact trypanosomes are readily permeable to this competitive inhibitor of ADPRT activity.

Effect of 3-aminobenzamide on antigenic variation of Trypanosoma brucei.

African trypanosomes, like Trypanosoma brucei, depend on antigenic variation to evade the immune response of the vertebrate host. An antigenic switch corresponds to the activation of a variable surface glycoprotein (VSG) gene from a large silent repertoire. Most switches require the duplicative transposition of a VSG gene, which involves strand breaks in DNA and subsequent repair. The nuclear enzyme adenosine-diphosphoribosyl transferase (ADPRT), which is dependent on the presence of DNA strand breaks for its activity, might be involved in this process because it has a regulatory role in DNA repair in all eukaryotic cells studied so far. In previous work, the presence of ADPRT activity was demonstrated in T. brucei. Moreover, it was also shown in isolated trypanosomes the ADPRT activity, which is stimulated by the induction of DNA strand breaks, could be blocked by the competitive inhibitor 3-aminobenzamide. Here we report experiments using rats which were infected with small numbers of T. brucei expressing VSG gene 118. After two days, the rats were coupled to a continuous intraperitoneal infusion system administrating 3-aminobenzamide in 0.9% NaCl (81.4 mM) at a rate of 0.65 ml/hr/rat for a period of up to five days. Control rats received only a 0.9% NaCl infusion. At days 1, 3 and 5, 250 microliters blood was obtained from a tail artery. Plasma 3-aminobenzamide was determined using a new high performance liquid chromatography method, developed for these experiments. In most rats the plasma concentrations were maintained between 0.8 and 1.2 mM. The rate of antigenic switching was determined by quantitating the fraction of trypanosomes that had lost their VSG 118 coat, using antibody against VSG 118 and a limiting dilution in mice. The average switching rate found was 2.0 X 10(-6) in controls and 1.3 X 10(-7) in drug-treated rats (15-fold reduction). This suggests that ADPRT is required for completing most antigenic switching events. We discuss the possibility that drug-resistant switching only involves non-duplicative VSG gene activation.

Human monoclonal antibodies to phenolic glycolipid-1 from leprosy patients cross react with poly(ADP-ribose), polynucleotides and tissue bound antigens.

Antibodies which bind to poly(ADP-ribose) have been described in Systemic Lupus Erythematosus (SLE) and a variety of infectious diseases. Two IgM kappa human monoclonal antibodies (MAbs), TH3 and PR4, produced from the fusion of peripheral blood lymphocytes of leprosy patients with the GM4672 lymphoblastoid cell line, were found to bind to poly(ADP-ribose) in direct binding and inhibition ELISAs. Significant inhibition of binding of these MAbs to poly(ADP-ribose) occurred with phenolic glycolipid-1, the M. leprae specific glycolipid, ssDNA, dsDNA, poly(dT), as well as poly(ADP-ribose) itself. Up to 80% of binding of TH3, and 90% of binding of PR4, to poly(ADP-ribose) was inhibited by 10 mcg of ssDNA suggesting that there may be sharing of some conformational determinants. Although the serological binding profiles of TH3 and PR4 are similar, only PR4 was found to bind to basal keratinocytes of normal human interfollicular epidermis and astrocyte cytoplasm in normal brain tissue. These results support the concept that an antibody binding site may accommodate more than one epitope. Furthermore, small differences in antigen binding potential may distinguish relatively innocuous antibodies from those which may be more pathogenic.

Disease specificity of antibodies to poly (ADP-ribose); their relationships to anti-DNA antibodies and to disease activity in lupus.

In this study we have measured the level of anti poly (ADP-ribose) antibodies in the sera of a number of patients with SLE and their relatives, patients with a wide variety of other autoimmune and infectious diseases, and a group of normal healthy controls. It was found that these antibodies were not disease specific but were present in nine out of thirteen groups tested in significant numbers. The levels of anti poly (ADP-ribose) antibodies and anti DNA antibodies in SLE patients bled serially were also measured. The level of these antibodies fluctuated in parallel in many of these patients, although the anti poly (ADP-ribose) antibodies reflected disease activity more accurately in some.

Direct radioactive labelling of poly(ADP-ribose) in developing Xenopus laevis embryos.

It has not previously been possible to label the nuclear protein modification poly(ADP-ribose) directly from NAD because of the impermeability of the cell membrane. We have overcome this important problem by micro-injection of radioactively labelled NAD into Xenopus laevis early embryos. The polymer was identified and then quantified by its insensitivity to DNAase, RNAase, and spleen phosphodiesterase and by the chromatographic mobility of the products of digestion with snake-venom phosphodiesterase. The quantity of poly(ADP-ribose) present after 25 h of development (129 ng/mg DNA) is lower than that found in fully differentiated tissue.

Poly(ADP-ribose) polymerase-1: what have we learned from the deficient mouse model?

Poly (ADP-ribose) polymerase (113 kDa; PARP-1) is a constitutive factor of the DNA damage surveillance network developed by the eukaryotic cell to cope with the numerous environmental and endogenous genotoxic agents. This enzyme recognizes and is activated by DNA strand breaks. This original property plays an essential role in the protection and processing of the DNA ends as they arise in DNA damage that triggers the base excision repair (BER) pathway. The generation, by homologous recombination, of three independent deficient mouse models have confirmed the caretaker function of PARP-1 in mammalian cells under genotoxic stress. Unexpectedly, the knockout strategy has revealed the instrumental role of PARP-1 in cell death after ischemia-reperfusion injury and in various inflammation process. Moreover, the residual PARP activity found in PARP-1 deficient cells has been recently attributed to a novel DNA damage-dependent poly ADP-ribose polymerase (62 kDa; PARP-2), another member of the expanding PARP family that, on the whole, appears to be involved in the genome protection. The present review summarizes the recent data obtained with the three PARP knockout mice in comparison with the chemical inhibitor approach.

A mammalian cell variant in which 3-aminobenzamide does not potentiate the cytotoxicity of dimethyl sulphate.

Variants of mouse leukaemia L1210 cells have been isolated in which cytotoxicity to dimethyl sulphate is not fully potentiated by ADP-ribosyl transferase inhibitor 3-aminobenzamide, as occurs in normal L1210 cells. These variants were selected after mutagenesis by growing the cells in dimethyl sulphate and 3-aminobenzamide. The characterisation of one of these variants is described. Variant 3 cells repair low doses of DNA damage in the presence of ADP-ribosyl transferase inhibitors. The Vmax of the ADP-ribosyl transferase enzyme in these cells is only increased 35% compared to normal wild-type L1210 cells. The basal DNA ligase I activity is increased 66% above wild-type whereas DNA ligase II activity appears to be unchanged. The most striking observation, however, is that the DNA ligase II activity is not increased after dimethyl sulphate treatment as occurs in wild-type L1210 cells. It seems that by increasing DNA ligase I levels these cells can survive DNA damage in the presence of 3-aminobenzamide. This variant (mutant) provides genetic evidence for our previously published hypothesis that (ADP-ribose)n biosynthesis is required for efficient DNA repair after DNA damage by monofunctional alkylating agents, because ADP-ribosyl transferase activity regulates DNA ligase activity. This variant is the first mammalian cell reported in which DNA ligase activity is altered, as far as we are aware. In yeast, a DNA ligase mutant has a cell division cycle (cdc) phenotype. Presumably, DNA ligase is essential for DNA synthesis, repair and recombination. The present variant provides further evidence that in mammalian cells, DNA ligase II activity is related to ADP-ribosyl transferase activity.

Purification, characterisation, and molecular cloning of a chicken erythroblast mono(ADP-ribosyl)transferase.

We have purified an arginine-specific mono(ADP-ribosyl)transferase from chicken erythrocytes. The purified transferase was free from poly (ADP-ribose) polymerase activity. The molecular weight of the purified enzyme was estimated to be 27.5 kDa by gel filtration through Sephadex G-75 in a non-denaturing solvent. Activity gel experiments indicate that the active enzyme has an apparent molecular weight in SDS gels of about 28 kDa. The optimum pH of the reaction is about 8.0. The K(m) value for NAD+ of the purified enzyme is about 130 microM. Small molecular weight inhibitors of poly (ADP-ribose) polymerase have no significant effect on the mono ADP-ribosyl transferase enzyme activity. A number of inhibitors of the arginine-specific mono(ADP-ribosyl)transferase activity have been identified. Among the more effective inhibitors are 1,4 naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, 4-amino-1-naphthol and 1,2-naphthoquinone. We have also cloned a mono(ADP-ribosyl)transferase from chicken erythroblasts. This gene has been expressed in E. coli and ADP-ribosylation activity has been demonstrated using histones as substrate. The activity is shown to be arginine-specific by the use of poly-L-arginine as substrate. Use of a specific inhibitor has shown that this enzyme is indeed a mono(ADP-ribosyl)transferase and not a NAD glycohydrolase activity. The sequence of this gene is very similar to several other mono(ADP-ribosyl)transferase genes. There are thus at least three different chicken mono(ADP-ribosyl)transferase genes in the blood system alone; this suggests that there is a quite large family of mono(ADP-ribosyl)transferase genes in animals. We have also isolated the promoter region of this chicken gene and are able to identify several standard motifs in this promoter.