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.     

pADPRT-2: a novel mammalian polymerizing(ADP-ribosyl)transferase gene related to truncated pADPRT homologues in plants and Caenorhabditis elegans.

Until recently, poly(ADP-ribosyl)ation was supposed to be confined only to polymerizing(ADP-ribosyl)transferase/(ADP-ribose)polymerase (E.C. 2.4.2.30). Here, we present novel polymerizing(ADP-ribosyl)transferase homologues from mouse and man that lack all of the N-terminal DNA binding and BRCA1 C-terminus domains and will be designated polymerizing(ADP-ribosyl)transferase-2 as distinguished from the classical polymerizing(ADP-ribosyl)transferase (polymerizing(ADP-ribosyl)transferase-1). The murine polymerizing(ADP-ribosyl)transferase-2 gene shares three identical intron positions with its Caenorhabditis elegans (EMBL nucleotide sequence database Z47075) and one with the Arabidopsis thaliana homologue ('APP', GenBank database AF069298). Expression of the murine polymerizing(ADP-ribosyl)transferase-2 gene was elevated in spleen, thymus and testis and the corresponding poly(ADP-ribosyl)ation activity might account for most of the residual poly(ADP-ribosyl)ation observed in polymerizing(ADP-ribosyl)transferase-1(-/-) mice.     

ADP-Ribosylation of Highly Purified Rat Brain Mitochondria

Highly purified synaptic and nonsynaptic mitochondria were prepared from rat brain, and their ADP-ribosyl transferase and NAD glycohydrolase activities were investigated. Data show that there is no significant difference in ADP-ribosyl transferase activity between these two types of subcellular preparations. However, NAD glycohydrolase activity appeared to be much higher in nonsynaptic mitochondria. The specific activity of both enzymes was investigated in the presence of the inhibitor nicotinamide or its analogue 3-aminobenzamide or other adenine nucleotides, such as ATP or ADP-ribose. The inhibitory effect of nicotinamide or 3-aminobenzamide on ADP-ribosyl transferase appears rather weak compared with their effect on NAD glycohydrolase activity. However, ADP-ribose and ATP appeared more effective in inhibiting ADP-ribosyl transferase. Our results provide evidence for the existence of ADP-ribosyl transferase activity in rat brain mitochondria. When NAD glycohydrolase was inhibited totally by nicotinamide, the transfer of ADP-ribose from NAD to mitochondrial proteins still occurred. The chain length determinations show that the linkage of ADP-ribose to mitochondrial proteins is oligomeric.     

Nuclear ADP-ribosyl transferase activity correlates with induction of P-450 monooxygenases by phenobarbital in rat liver microsomes

The effect of phenobarbital treatment on the nuclear ADP-ribosyl transferase activity has been studied in parallel with microsomal cytochrome P-450 concentration and related mono-oxygenase activities, in rat liver. A marked activation of the ADP-ribosyl transferase was observed 24 h after phenobarbital administration. The chronological study performed between 0-6 days after phenobarbital treatment showed a sharp increase in this nuclear enzyme activity, to approximately equal to 270% of the control value produced in 48 h. The administration of 5'-methylnicotinamide in vivo, an inhibitor of ADP-ribosyl transferase activity in vitro, produced a decrease both of the induction of liver microsomal cytochrome P-450 mono-oxygenases and nuclear ADP-ribosyl transferase activity. The role of nuclear ADP-ribosyl transferase in the adaptative response of the liver cell to phenobarbital is discussed.     

Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin

We have purified from Clostridium spiroforme strain 246 an heterogeneous population of proteins (Sa) ranging from 43 to 47 kilodaltons exhibiting ADP-ribosyl transferase activity as do C. botulinum C2 toxin component I or the ia chain of C. perfringens E iota toxin. C. spiriforme Sa had alone no activity upon injection in mice or inoculated to Vero cells. When spiroforme ADP ribosyl transferase were mixed with a trypsin activated protein (Sb) separated from C. spiroforme bacterial supernatant, a lethal effect in mice and cytotoxicity on Vero cells were recorded. The Sa cross-reacted immunologically with either the light chain of C. perfringens E iota toxin or the ADP-ribosyl transferase from C. difficile 196 strain. No immunological relatedness was observed between Sa and C2 toxin component I. C. spiroforme toxin is thus another binary toxin close to iota.     

ADP-ribosyl transferase and NAD glycohydrolase activities in rat liver mitochondria

ADP-ribosyl transferase and NAD glycohydrolase activities have been estimated in mitochondria in mitoplasts as well as in other submitochondrial fractions. A high activity of these two enzymes was present in mitoplasts as compared to the outer membrane preparation or intermembrane compartment. Inhibitor studies provide strong evidence for the involvement of ADP-ribosyl transferase in the process of ADP-ribosylation of mitochondrial proteins. When NAD glycohydrolase was blocked by nicotinamide or 3-aminobenzamide, the incorporation of ADP-ribose into mitochondrial proteins still occurs. ADP-ribosyl transferase activity could also be detected when NAD glycohydrolase was separated by hydroxylapatite chromatography. The protein-linked ADP-ribose moiety appears to be an oligomer in mitochondria.     

Amino acid specific ADP-ribosylation: substrate specificity of an ADP-ribosylarginine hydrolase from turkey erythrocytes

An ADP-ribosylarginine hydrolase, which catalyzes the degradation of ADP-ribosyl[14C]arginine to ADP-ribose plus arginine, was separated by ion exchange, hydrophobic, and gel permation chromatography from NAD:arginine ADP-ribosyltransferases, which are responsible for the stereospecific formation of alpha-ADP-ribosylarginine. As determined by NMR, the specific substrate for the hydrolase was alpha-ADP-ribosylarginine, the product of the transferase reaction. The ADP-ribose moiety was critical for substrate recognition; (phosphoribosyl) [14C]arginine and ribosyl[14C]arginine were poor substrates and did not significantly inhibit ADP-ribosyl[14C]arginine degradation. In contrast, ADP-ribose was a potent inhibitor of the hydrolase and significantly more active than ADP greater than AMP greater than adenosine. In addition to ADP-ribosyl[14C]arginine, both ADP-ribosyl[14C]guanidine and (2'-phospho-ADP-ribosyl)[14C]arginine were also substrates; at pH greater than 7, ADP-ribosyl[14C]guanidine was degraded more readily than the [14C]arginine derivative. Neither arginine, guanidine, nor agmatine, an arginine analogue, was an effective hydrolase inhibitor. Thus, it appears that the ADP-ribosyl moiety but not the arginine group is critical for substrate recognition. Although the hydrolase requires thiol for activity, dithiothreitol accelerated loss of activity during incubation at 37 degrees C. Stability was enhanced by Mg2+, which is also necessary for optimal enzymatic activity. The findings in this paper are consistent with the conclusion that different enzymes catalyze ADP-ribosylarginine synthesis and degradation. Furthermore, since the hydrolase and transferases possess a compatible stereospecificity and substrate specificity, it would appear that the two enzymatic activities may serve as opposing arms in an ADP-ribosylation cycle.     

Effect of an arginine-specific ADP-ribosyltransferase inhibitor on differentiation of embryonic chick skeletal muscle cells in culture.

Primary cultures of embryonic chick skeletal myogenic cells were used as an experimental model to examine the possible role of mono(ADP-ribosyl)ation reactions in myogenic differentiation. Initial studies demonstrated arginine-specific mono(ADP-ribosyl)transferase activity in the myogenic cell cultures. We then examined the effect of a novel inhibitor of cellular arginine-specific mono(ADP-ribosyl)transferases, meta-iodobenzylguanidine (MIBG), on differentiation of cultured embryonic chick skeletal myoblasts. MIBG reversibly inhibited both proliferation and differentiation of embryonic chick myoblasts grown in culture. Micromolar (15-60 microM) concentrations of MIBG blocked myoblast fusion, the differentiation-specific increase in creatine phosphokinase activity, and both DNA and protein accumulation in myogenic cell cultures. Meta-iodobenzylamine, an analog of MIBG missing the guanidine group, had no effect. Low concentrations of methylglyoxal bis-guanylhydrazone, a substrate for cholera toxin with a higher Km than MIBG, also had no effect, but higher concentrations reversibly inhibited fusion. These findings suggest a possible role for mono(ADP-ribosyl)ation reactions in myogenesis. In addition, the total arginine-specific mono(ADP-ribosyl)transferase activity increased with differentiation in the myogenic cell cultures, and this increase was also blocked by MIBG treatment. Because high levels of activity were found in the membrane fraction derived from later, myotube cultures, the membrane fraction from 96-h cultures was incubated with [32P]NAD+ and subjected to electrophoresis and autoradiography. Three proteins, migrating at 21, 20, and 17 kDa, that were ADP-ribosylated in the absence, but not the presence, of MIBG were identified. These proteins may be endogenous substrates for this enzyme.     

Isolation and properties of an NAD- and guanidine-dependent ADP-ribosyltransferase from turkey erythrocytes

An NAD- and guanidine-dependent ADP-ribosyltransferase has been purified more than 500,000-fold from turkey erythrocytes with an 18% yield. The enzyme in the 100,000 X g supernatant fraction was bound to phenyl-Sepharose, eluted with 50% propylene glycol, and further purified by sequential chromatographic steps on carboxymethylcellulose, NAD-agarose and concanavalin A-agarose. The transferase was specifically eluted from concanavalin A-agarose with alpha-methylmannoside. The enzymatic activity was extremely labile following the first purification step. Both propylene glycol and NaCl stabilized the transferase; significant increases in enzyme recovery were obtained by conducting the NAD- and concanavalin A-agarose chromatography in buffer containing propylene glycol. The purified protein exhibits one predominant protein band on SDS-polyacrylamide gels with an estimated molecular weight of 28,300. On Ultrogel AcA54 chromatography, single coincident peaks of ADP-ribosyltransferase activity and protein were observed. Enzyme activity was independent of DNA; the highly purified transferase was inhibited by thymidine, nicotinamide, and theophylline. The specific activity of the purified enzyme (350 mumol of ADP-ribose transferred from NAD to arginine methyl estermin-1mg-1) is comparable to that reported for purified NAD glycohydrolases and poly(ADP-ribosyl)transferases.     

Changes in topoisomerase I activity after irradiation of lymphoid cells

The activity of topoisomerase I in nuclear extracts increased about three-fold 5 min after gamma-irradiation (840-2500 rads) of human peripheral blood lymphocytes or cultured lymphoblastoid cells. The change may reflect modification of the enzyme by nuclear ADP-ribosyl transferase, which is known to be activated by DNA breaks.     

Involvement of poly (ADP-ribose) in the radiation response of mammalian cells

The evidence implicating poly (ADP-ribose) in the radiation response of mammalian cells is reviewed. It is concluded that the apparently conflicting results using inhibitors of ADP-ribosyl transferase (ADPRT) can be explained by a working hypothesis. This hypothesis maintains that poly (ADP-ribose) is required for repair of radiation damage (presumably to facilitate ligation). In most cells the synthesis of poly (ADP-ribose) is not rate limiting for repair and therefore, an almost complete inhibition of ADPRT activity is required to potentiate the radiation response. In radiation-sensitive cells (e.g. resting lymphocytes, L5178Y-S cells) with a deficient poly (ADP-ribose) metabolism, its synthesis can become rate limiting for repair. In such cells even a partial inhibition of ADPRT activity may enhance radiation-induced cell killing. It is suggested that if such differences exist between normal and cancer cells, they can be utilized to improve the therapeutic ratio of radiotherapy.     

Assay for ADP-ribosyl cyclase by reverse-phase high-performance liquid chromatography.

Cyclic ADP-ribose (cADPR), a natural metabolite of beta-NAD(+), is a second messenger for Ca(2+) signaling in T cells. As a tool for purification and identification of ADP-ribosyl cyclase(s) in T cells, a sensitive and specific enzymatic assay using 1,N(6)-etheno-NAD(+) as substrate was developed. A major problem-the sensitivity of 1,N(6)-etheno-cADPR toward the extraction medium perchloric acid-was solved by replacing the perchloric acid extraction procedure of nucleotides by a filtration step. Standard compounds for the HPLC analysis of ADP-ribosyl cyclases and NAD(+)-glycohydrolases, e.g., 1,N(6)-etheno-cADPR, 1,N(6)-etheno-ADPR, and 1,N(6)-etheno-AMP, were produced by ADP-ribosyl cyclase from Aplysia californica and dinucleotide pyrophosphatase. The assay was applied to subcellular fractions prepared from human Jurkat T cells. As a result ADP-ribosyl cyclase and NAD(+)-glycohydrolase activity could be detected and precisely quantified in different subcellular fractions indicating the presence of different isoenzymes in T cells.     

Poly(ADP-ribosylation) at successive stages of rooster spermatogenesis. Levels of polymeric ADP-ribose in vivo and poly(ADP-ribose) polymerase activity and turnover of ADP-ribosyl residues in vitro

Rooster testis cells were separated by sedimentation at unit gravity and the in vivo levels of polymeric ADP-ribose were determined both in intact cells and isolated nuclei by fluorescence methods. Poly(ADP-ribose) polymerase activity was assayed after cell permeabilization or after isolation of nuclei. The turnover of ADP-ribosyl residues was determined in isolated nuclei using benzamide. The content of poly(ADP-ribose), the poly(ADP-ribose) polymerase activity, and the turnover of ADP-ribosyl residues, decreased during the differentiation of the germinal cell line, especially at the end of spermiogenesis. Treatment of cells with 1 mM dimethyl sulfate for 1 h resulted in a marked stimulation of poly(ADP-ribose) polymerase activity in meiotic and premeiotic cells and also in round and late spermatids. The enzymatic activity was not detected and could not be induced in mature spermatozoa. These cells, however, still contained polymeric ADP-ribose with a 2% of branched form.     

Poly(ADP-ribosyl)ation of terminal deoxynucleotidyl transferase in vitro

The activity of purified bovine thymus terminal deoxynucleotidyl transferase was markedly inhibited when the enzyme was incubated in a poly(ADP-ribose)-synthesizing system containing purified bovine thymus poly(ADP-ribose) polymerase, NAD+, Mg2+ and DNA. All of these four components were indispensable for the inhibition. The inhibitors of poly(ADP-ribose) polymerase counteracted the observed inhibition of the transferase. Under a Mg2+-depleted and acceptor-dependent ADP-ribosylating reaction condition [Tanaka, Y., Hashida, T., Yoshihara, H. and Yoshihara, K. (1979) J. Biol. Chem. 254, 12433-12438], the addition of terminal transferase to the reaction mixture stimulated the enzyme reaction in a dose-dependent manner, suggesting that the transferase is functioning as an acceptor for ADP-ribose. Electrophoretic analyses of the reaction products clearly indicated that the transferase molecule itself was oligo (ADP-ribosyl)ated. When the product was further incubated in the Mg2+-fortified reaction mixture, the activity of terminal transferase markedly decreased with increase in the apparent molecular size of the enzyme, indicating that an extensive elongation of poly(ADP-ribose) bound to the transferase is essential for the observed inhibition. Free poly(ADP-ribose) and the polymer bound to poly(ADP-ribose) polymerase were ineffective on the activity of the transferase. All of these results indicate that the observed inhibition of terminal transferase is caused by the poly(ADP-ribosyl)ation of the transferase itself.     

Farnesyl protein transferase inhibition interferes with activation of MAP kinase family members in human peripheral blood monocytes

BACKGROUND: Farnesyl protein transferase inhibitors have emerged as promising novel agents for combating cancerous disease. Nevertheless, the importance for farnesyl protein transferase enzymatic activity for cellular physiology of untransformed cells remains poorly investigated. MATERIALS AND METHODS: Peripheral blood monocytes, isolated from the blood of eight healthy volunteers, were treated with a farnesyl protein transferase inhibitor (FTI 744,832) or vehicle control for 16 hr. Subsequently cells were challenged with different concentrations of lipopolysaccharide (LPS), colony stimulating factor-1 (CSF-1), or phorbol esters for 10 min, after which the activation state of p42/p44 MAP kinase, p38 MAP kinase, and Jun-N-terminal kinase was investigated using Western blotting and phosphospecific antibodies. RESULTS: We observed that farnesyl protein transferase inhibition abrogated activation of p38 MAP kinase by LPS, CSF-1, and phorbol esters. Also the activation of Jun-N-terminal kinase by LPS was not seen after farnesyl protein transferase inhibition. Finally, stimulation of p42/p44 MAP kinase with CSF-1 was strongly reduced by farnesyl protein transferase inhibition, whereas activation of p42/p44 MAP kinase by phorbol ester was only slightly effected. CONCLUSIONS: Farnesyl protein transferase enzymatic activity is required for proper activation of all major members of the MAP kinase family. The observation that activation the p38 MAP kinase and Jun-N-terminal kinase is sensitive to farnesyl protein transferase inhibition raises the possibility that, in addition to cancerous disease, farnesyl protein transferase inhibitors may be useful compounds in combating inflammatory disease.     

PolyADP-ribosyl polymerase (adenosine diphosphate ribosyl transferase) in bovine crystalline lens; modulation of its activity during aging

PolyADP-ribose polymerase activities were measured in bovine lens. Activities similar to those in brain were found in the epithelial cells; none activity was detected in the fiber cells. During aging ADP-ribosyl transferase activity of epithelial cells raised, the number of polyADP-ribose chains increased while the average chain length decreased. A possible correlation between ADP-ribosylation and cell proliferation or repair is discussed.     

Deficient regulation of DNA double-strand break repair in Fanconi anemia fibroblasts

Fibroblasts from patients with Fanconi anemia (FA) display genomic instability, hypersensitivity to DNA cross-linking agents, and deficient DNA end joining. Fibroblasts from two FA patients of unidentified complementation group also had significantly increased cellular homologous recombination (HR) activity. Results described herein show that HR activity levels in patient-derived FA fibroblasts of groups A, C, and G were 10-fold greater than those seen in normal fibroblasts. In contrast, HR activity in group D2 fibroblasts was identical to that in normal cells. Western blot analysis revealed that the RAD51 protein was elevated 10-fold above normal levels in group A, C, and G fibroblasts, but was not altered in group D2 fibroblasts. HR activity levels in these former cells could be restored to near-normal levels by electroporation with anti-RAD51 antibody, whereas similar treatment of normal and complementation group D2 fibroblasts had no effect. These findings are consistent with a model in which FA proteins function to coordinate DNA double-strand break repair activity by regulating both recombinational and non-recombinational DNA repair. Interestingly, whereas positive regulation of DNA end joining requires the combined presence of all FA proteins thus far tested, suppression of HR, which is minimally dependent on the FANCA, FANCC, and FANCG proteins, does not require FANCD2.