ADP-ribose Transferase Research Articles

PARP1 associates with R-loops to promote their resolution and genome stability.

PARP1 is a DNA-dependent ADP-Ribose transferase with ADP-ribosylation activity that is triggered by DNA breaks and non-B DNA structures to mediate their resolution. PARP1 was also recently identified as a component of the R-loop-associated protein-protein interaction network, suggesting a potential role for PARP1 in resolving this structure. R-loops are three-stranded nucleic acid structures that consist of a RNA-DNA hybrid and a displaced non-template DNA strand. R-loops are involved in crucial physiological processes but can also be a source of genome instability if persistently unresolved. In this study, we demonstrate that PARP1 binds R-loops in vitro and associates with R-loop formation sites in cells which activates its ADP-ribosylation activity. Conversely, PARP1 inhibition or genetic depletion causes an accumulation of unresolved R-loops which promotes genomic instability. Our study reveals that PARP1 is a novel sensor for R-loops and highlights that PARP1 is a suppressor of R-loop-associated genomic instability.

ADP-ribose transferase PARP16 mediated-unfolded protein response contributes to neuronal cell damage in cerebral ischemia/reperfusion.

Ischemic stroke is known to cause the accumulation of misfolded proteins and loss of calcium homeostasis, leading to impairment of endoplasmic reticulum (ER) function and activating the unfolded protein response (UPR). PARP16 is an active (ADP-ribosyl)transferase known tail-anchored ER transmembrane protein with a cytosolic catalytic domain. Here, we find PARP16 is highly expressed in ischemic cerebral hemisphere and oxygen-glucose deprivation/reoxygenation (OGD/R)-treated immortalized hippocampal neuronal cell HT22. Using an adeno-associated virus-mediated PARP16 knockdown approach in mice, we find PARP16 knockdown decreases infarct demarcations and has a better neurological outcome after ischemic stroke. Our data indicate PARP16 knockdown decreases ER stress and neuronal death caused by OGD/R, whereas PARP16 overexpression promotes ER stress-mediated cell damage in primary cortical neurons. Furthermore, PARP16 functions mechanistically as ADP-ribosyltransferase to modulate the level of ADP-ribosylation of the corresponding PERK and IRE1α arm of the UPR, and such modifications mediate activation of PERK and IRE1α. Indeed, pharmacological stimulation of the UPR using Brefeldin A partly counteracts PARP16 knockdown-mediated neuronal protection upon OGD/R treatment. In conclusion, PARP16 plays a crucial role in post-ischemic UPR and PARP16 knockdown alleviates brain injury after ischemic stroke. This study demonstrates the potential of the PARP16-PERK/IRE1α axis as a target for neuronal survival in ischemic stroke.

PARPs and ADP-ribosylation in RNA biology: from RNA expression and processing to protein translation and proteostasis.

ADP-ribosylation (ADPRylation) is a posttranslational modification of proteins discovered nearly six decades ago, but many important questions remain regarding its molecular functions and biological roles, as well as the activity of the ADP-ribose (ADPR) transferase enzymes (PARP family members) that catalyze it. Growing evidence indicates that PARP-mediated ADPRylation events are key regulators of the protein biosynthetic pathway, leading from rDNA transcription and ribosome biogenesis to mRNA synthesis, processing, and translation. In this review we describe the role of PARP proteins and ADPRylation in all facets of this pathway. PARP-1 and its enzymatic activity are key regulators of rDNA transcription, which is a critical step in ribosome biogenesis. An emerging role of PARPs in alternative splicing of mRNAs, as well as direct ADPRylation of mRNAs, highlight the role of PARP members in RNA processing. Furthermore, PARP activity, stimulated by cellular stresses, such as viral infections and ER stress, leads to the regulation of mRNA stability and protein synthesis through posttranscriptional mechanisms. Dysregulation of PARP activity in these processes can promote disease states. Collectively, these results highlight the importance of PARP family members and ADPRylation in gene regulation, mRNA processing, and protein abundance. Future studies in these areas will yield new insights into the fundamental mechanisms and a broader utility for PARP-targeted therapeutic agents.

The Role of Sirt6 in Obesity and Diabetes.

Sirt6 is one of the sirtuin family members, a kind of NAD+-dependent histone deacetylase and ADP-ribose transferase enzyme. It has an important role in physiological and pathological processes, regulating aging, cancer, obesity, insulin resistance, inflammation, and energy metabolism. Recent studies have suggested that reduced Sirt6 action is related to obesity and diabetes. Aging and overnutrition, two major risk factors for obesity and diabetes, lead to decreased Sirt6 level and function, which results in abnormal glucose and lipid metabolism. Whole-body ablation of Sirt6 in mice results in severe hypoglycemia. Sirt6 deficiency leads to liver steatosis and promotes diet-induced obesity and insulin resistance. Sirt6 has a protective effect on obesity and diabetes. This review surveys evidence for an emerging role of Sirt6 as a regulator of metabolism in mammals and summarizes its major functions in obesity and diabetes.

Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae

The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC(50) = 4.6 +/- 0.4 ng/ml) and crustaceans (Artemia nauplii LD(50) = 10 +/- 2 mug/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-A resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.

Toward the Elucidation of the Catalytic Mechanism of the Mono-ADP-Ribosyltransferase Activity of Pseudomonas aeruginosa Exotoxin A

The catalytic mechanism for the mono-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A was investigated by steady-state and stopped-flow kinetic analyses. The rate constants for binding of the NAD(+) substrate to the enzyme were found to be 4.7 +/- 0.4 microM(-1) s(-1) and 194 +/- 15 s(-1) for k(on) and k(off), respectively. The k(on) and k(off) rate constants for the eEF-2 substrate binding to the enzyme were 320 +/- 39 microM(-1) s(-1) and 131 +/- 22 s(-1), respectively. A potent, competitive inhibitor against the enzyme, 1,8-naphthalimide, bound the enzyme with k(on) and k(off) rates of 82 +/- 9 microM(-1) s(-1) and 51 +/- 6 s(-1), respectively. Furthermore, the binding on and off rates for the reaction products, ADP-ribose and nicotinamide, were too rapid for detection with the stopped-flow technique. Investigation of the pre-steady-state kinetics for the ADP-ribose transferase activity of the toxin-enzyme showed that there is no pre-steady-state complex formed during the catalytic cycle. Binding of NAD+ and smaller compounds representing the various parts of this substrate were investigated by the fluorescence quenching of the intrinsic toxin fluorescence. The binding data revealed a significant structural change in the enzyme upon NAD+ binding that could not be accounted for on the basis of the sum of the structural changes induced by the various NAD+ constituents. Product inhibition studies were conducted with nicotinamide and eEF-2-ADP-ribose, and the results indicate that the reaction involves a random-order ternary complex mechanism. Detailed kinetic analysis revealed that the eEF-2 substrate shows sigmoidal kinetic behavior with the enzyme, and fluorescence resonance energy transfer measurements indicated that wheat germ eEF-2 is oligomeric in solution.

Cholera toxin B subunit activates arachidonic acid metabolism.

Cholera toxin (CT) increases intestinal secretion of water and electrolytes and modulates the mucosal immune response by stimulating cellular synthesis of arachidonic acid (AA) metabolites (e.g., prostaglandin E2), as well as the intracellular second messenger cyclic AMP (cAMP). While much is known about the mechanism of CT stimulation of adenylate cyclase, the toxin's activation of phospholipase A2, which results in increased hydrolysis of AA from membrane phospholipids, is not well understood. To determine whether CT activation of AA metabolism requires CT's known enzymatic activity (i.e., ADP-ribosylation of GSalpha), we used native CT and a mutant CT protein (CT-2*) lacking ADP-ribose transferase activity in combination with S49 wild-type (WT) and S49 cyc- murine Theta (Th)1.2-positive lymphoma cells deficient in GSalpha. The experimental results showed that native CT stimulated the release of [3H[AA from S49 cyc- cells at a level similar to that for S49 WT cells, indicating that GSalpha is not essential for this process. Further, levels of cAMP in the CT-treated cyc- cells remained the same as those in the untreated control cells. The ADP-ribosyltransferase-deficient CT-2* protein, which was incapable of increasing synthesis of cAMP, displayed about the same capacity as CT to evoke the release of [3H]AA metabolites from both S49 WT and cyc- cells. We concluded that stimulation of arachidonate metabolism in S49 murine lymphoma cells by native CT does not require enzymatically functional CT, capable of catalyzing the ADP-ribosylation reaction. These results demonstrated for the first time that stimulation of adenylate cyclase by CT and stimulation of AA metabolism by CT are not necessarily coregulated. In addition, the B subunits purified from native CT and CT-2* both simulated the release of [3H]AA from S49 cyc- cells and murine monocyte/macrophage cells (RAW 264.7), suggesting a receptor-mediated cell activation process of potential importance in enhancing immune responses to vaccine components.

Inhibitors of poly (ADP-ribose) synthetase protect rat renal proximal tubular cells against oxidant stress.

Inhibitors of poly (ADP-ribose) synthetase protect rat proxiIschemia followed by reperfusion of the kidney inmal tubular cells against oxidant stress. volves a complex and interrelated sequence of events Background. The generation of reactive oxygen species (ROS) that result in the injury of renal cells and eventual cell has been implicated in the pathogenesis of renal ischemiadeath due to apoptosis and necrosis [1, 2]. Although reperfusion injury. ROS produce DNA strand breaks that lead reperfusion is essential for the survival of ischemic tissue, to the activation of the DNA-repair enzyme poly (ADP-ribose) synthetase (PARS). Excessive PARS activation results in the there is good evidence that reperfusion itself causes addidepletion of its substrate, nicotinamide adenine dinucleotide tional cell injury (reperfusion-injury) [3], which has been (NAD) and subsequently of adenosine 59-triphosphate (ATP), attributed to calcium overload, neutrophil infiltration leading to cellular dysfunction and eventual cell death. The and generation of reactive oxygen species (ROS) [1–3]. aim of this study was to investigate the effect of various PARS inhibitors on the cellular injury and death of rat renal proximal ROS–mediated cell injury and death has been implicated tubular (PT) cells exposed to hydrogen peroxide (H2O2). in the pathogenesis of renal ischemia-reperfusion injury Methods. Rat PT cell cultures were incubated with H2O2 and associated renal failure [1, 2]. Within the kidney, (1 mm) either in the presence or absence of the PARS inhibitors the situation is particularly complex, as ischemia itself 3-aminobenzamide (3-AB, 3 mm), 1,5-dihydroxyisoquinoline can cause early irreversible damage, which appears to (0.3 mm) or nicotinamide (Nic, 3 mm), or increasing concentrations of desferrioxamine (0.03 to 3 mm) or catalase (0.03 to 3 be mediated by ROS [2]. Furthermore, the proximal U/ml). Cellular injury and death were determined using the tubule (PT) appears to be particularly susceptible to MTT and lactate dehydrogenase (LDH) assays, respectively. reperfusion-injury [1, 2]. H2O2-mediated PARS activation in rat PT cells and the effects ROS produce cellular injury and necrosis via several of PARS inhibitors on PARS activity were determined by mechanisms including peroxidation of membrane lipids, measurement of the incorporation of [H]NAD into nuclear proteins. protein denaturation and DNA damage [3]. Evidence obResults. Incubation of rat PT cells with H2O2 significantly tained using cultured cells demonstrate that ROS produce inhibited mitochondrial respiration and increased LDH restrand breaks in DNA, which triggers energy-consuming lease, respectively. Both desferrioxamine and catalase reduced DNA repair mechanisms and activates the nuclear enH2O2-mediated cellular injury and death. All three PARS inhibitors significantly attenuated the H2O2-mediated decrease zyme poly (ADP-ribose) synthetase (PARS) [EC 2.4.2.30, in mitochondrial respiration and the increase in LDH release. also referred to as poly (ADP-ribose) polymerase Incubation with H2O2 produced a significant increase in PARS (PARP) or poly (ADP-ribose) transferase (pADPRT)] activity that was significantly reduced by all PARS inhibitors. [4]. PARS is an abundant, chromatin-bound enzyme con3-Aminobenzoic acid (3 mm) and nicotinic acid (3 mm), strucstitutively expressed in numerous cell types [5], which tural analogs of 3-AB and Nic, respectively, which did not inhibit PARS activity, did not reduce the H2O2-mediated injury when activated catalyzes the transfer of ADP-ribose and necrosis in cultures of rat PT cells. moieties from NAD to nuclear proteins including hisConclusion. We propose that PARS activation contributes tones and onto PARS itself (automodification) with the to ROS-mediated injury of rat PT cells and, therefore, to the concomitant formation of nicotinamide [6]. However, cellular injury and cell death associated with conditions of oxidant stress in the kidney. there is now good evidence that exposure of cells to oxidant stress in the form of superoxide anions, hydrogen peroxide (H2O2) and hydroxyl radicals causes strand

Identification of Domains of Poly(ADP-ribose) Polymerase for Protein Binding and Self-association

Cellular proteins extracted from normal and cancer cells bind polymerizing ADP-ribose transferase (pADPRT) on nitrocellulose membrane transblots. Histones at 1 mg/ml concentration completely prevent the binding of pADPRT to cellular proteins, indicating that the binding of histones to pADPRT sites competitively blocks the association of pADPRT to proteins other than histones. The direct binding of pADPRT to histones is shown by cross-linking with glutaraldehyde. The COOH-terminal basic histone H1 tail binds to the basic polypeptide domain of pADPRT. The basic domain present in the NH2-terminal part of core histones is the probable common structural feature of all core histones that accounts for their binding to pADPRT. Two polypeptide domains of pADPRT were identified, by way of CNBr fragments, to bind histones. These two domains are located within the 64-kDa fragment of pADPRT and are contiguous with the polypeptide domains that were shown to participate in self-association of pADPRT, ending at the 606th amino acid residue. The polypeptide domains of pADPRT which participate in DNA binding are thus shown to associate also with other proteins. Intact pADPRT binds to both the zinc-free or zinc-reconstituted basic polypeptide fragments of pADPRT. Histones activate auto-poly(ADP)-ribosylation of pADPRT by increasing the number of short oligomers on pADPRT. This reaction is also dependent in a biphasic manner on the concentration of pADPRT. Histones in solution are only marginally poly(ADP)-ribosylated but are good polymer acceptors when incorporated into artificial nucleosome structures.

ADP-Ribosylation of Neurofilaments by a Cytoplasmic ADP-Ribose Transferase Associated with Free mRNP

ADP-ribosylation of neurofilaments by an ADP-ribose transferase isolated from cytoplasmic ribonucleoprotein particles is demonstrated. The 150 kDa neurofilament subunit appears to be the main ADP-ribose acceptor with the transfer of ADP-ribose dimers or monomers. A binding of about 1 mole ADP-ribose per 8 moles of neurofilament subunits has been recorded. An interaction between neurofilaments′ ADP-ribosylation and their phosphorylation state is demonstrated.

Destabilization of Zn 2+ coordination in ADP-ribose transferase (polymerizing) by 6-nitroso-1,2-benzopyrone coincidental with inactivation of the polymerase but not the DNA binding function

6-Nitroso- 1,2-benzopyrone, an oxidation product of 6-amino- 1,2-benzopyrone, binds to the DNA-recognizing domain of the ADP-ribose transferase protein and preferentially destabilizes Zn 2+ from one of the two zinc finger polypeptide complexes present in the intact enzyme, as determined by the loss of 50% of 65Zn 2+ from the 65Zn 2+-isolated protein molecule, coincidental with the loss of 99% of enzymatic activity. The 50% zinc-deficient enzyme still binds to a DNA template. consisting of a 17-mer DNA primer annealed to M 13 positive strand, resulting in the blocking of DNA synthesis by the Klenow fragment of Pol I, Auto-poly-ADP-ribosylated ADP-ribose transferase, which is the probable physiological state of this protein in intact cells, does not bind to primer-template DNA and does not block DNA synthesis by the Klenow fragment. On the basis of this in vitro model it is proposed that molecules which inhibit or inactivate ADP-ribose transferase in intact cells can induce significant alteration in DNA structure and replication.

DNA strand breaks and DNA repair response in lymphocytes after chronic in vivo exposure to very low doses of ionizing radiation in mice

In contrast to the well-documented negative effects of high-dose oxidant exposure, accumulating evidence supports a positive, perhaps essential physiologic role for very low-level oxidant stress. For example, low-level oxidant exposure, within or below the physiologic range, has been reported to stimulate membrane signal transduction, proliferation, antioxidant defense and DNA repair. In the present study, we have examined whether whole-body exposure to low-dose radiation (LDR) results in an alteration in constitutive (steady state) levels of DNA-strand breaks and whether an adaptive increase in DNA-repair response is induced. C57B1/6J mice were exposed to 0.04 Gy (4 cGy) of γ-radiation as a model of low level oxidant stress. End points measured after chronic in vivo LDR included: (1) constitutive expression of DNA-strand breaks in quiescent spleen cells; (2) sensitivity to DNA damage after high-dose radiation exposure in vitro; (3) repair of constitutive and radiation-induced DNA strand breaks after mitogen stimulation: (4) activity of the DNA-repair associated enzyme, poly(ADP-ribose)transferase (ADPRT) and its substrate, NAD. The results indicated that the constitutive expression of DNA-strand breaks is significantly decreased chronic LDR; however, DNA-repair capacity after high-dose radiation exposure is not increased above that observed in sham-irradiated mice. Associated with the reduction in consitutive DNA-strand break accumulation was a decrease in resting levels of the DNA-repair-associated enzyme poly(ADP-ribose) transferase (ADPRT). These results are consistent with the interpretation that cumulative DNA damage and associated DNA-repair activity in unstimulated cells are both reduced after chronic LDR exposure.

Abnormal sister-chromatid exchange induction by 3-aminobenzamide in an SV40-transformed Indianamuntaj cell:line: Relationships with DNA maturation and DNA-strand breakage

In SVM cells, an SV40-transformed line of Indian muntjac fibroblasts, levels of sister-chromatid exchanges are known to be abnormally high after UV-irradiation or alkylation. The SVM line is also known to have a defect in the processing of DNA-strand breaks. Sister-chromatid exchange in other cells is known to be stimulated by the poly(ADP-ribose) transferase inhibitor, 3-aminobenzamide, which also retards DNA-break sealing. Sister-chromatid exchanges in SVM cells are found to be hypersensitive to 3-aminobenzamide, or to nicotinamide deprivation which similarly inhibits poly(ADP-ribosyl)ation; DNA-strand breaks are likewise induced by 3-aminobenzamide. Bromodeoxyuridine, needed to detect sister-chromatid exchanges, is more toxic to SVM cells and itself induces sister-chromatid exchanges to a greater extent than it does in normal muntjac cells. However, in contrast to the situation reported for other cell types prone to sister-chromatid exchange (the Chinese hamster ovary mutant EM9 and human Bloom's Syndrome cells), SVM cells do not show an abnormal delay in DNA maturation when, under the influence of bromodeoxyuridine and 3-aminobenzamide, they show a high level of sister-chromatid exchange. The mechanism by which BrdU exerts its effects can largely be explained in terms of familiar effects on deoxyribonucleotide pools and DNA integrity. 3-Aminobenzamide, however, induces sister-chromatid exchanges in SVM cells by another mechanism.

Residual Activation Events Functional after Irradiation of Mouse Splenic Lymphocytes

The radioresistance of lymphocytes increases after mitogenic stimulation, suggesting that a radiosensitive activation event contributes to the overall radiosensitivity of lymphocytes. We have sought to identify this activation event by determining the extent of activation of mitogen-stimulated lymphocytes previously exposed to growth-inhibiting doses of radiation. Mouse splenic lymphocytes were exposed to 0-15 Gy 137Cs radiation, and structural and functional damage were assayed. Although damage to cellular thiols and nonprotein thiols was modest, there was a significant loss of viability by 6 h as determined by uptake of propidium iodide (PI). Since cells did not die immediately after irradiation, the activation events which remained were evaluated. Growth-inhibiting doses of radiation left cells partially responsive to mitogen, in that cells were able to exit G0 phase, but they could progress no further into the cell cycle than G1a phase. It is important to note that assessment of viability by uptake of PI indicated substantial cell death after 15 Gy (45%, 6 h; 90%, 24 h); however, cell cycle analysis at 24 h indicated no significant decrease in progression from G0 to G1a phase. The LPS-stimulated response of B cells was more radiosensitive than the Con A-stimulated response of T cells. Further analysis of the Con A response indicated that production of interleukin-2 (IL-2) was unaffected, but expression of the IL-2 receptor was inhibited. Inhibition of poly-ADP-ribosylation and damage to lipids did not prevent the lack of mitogen responsiveness, since neither the ADP-ribose transferase inhibitor 3-aminobenzamide nor lipid radical scavengers had restorative effects on the mitogenic response. Nor was Con A-stimulated incorporation of [3H]thymidine restored with inhibitors of prostaglandin or leukotriene synthesis, suggesting that inhibition was due to direct effects on the Con A responders, and not indirect effects mediated by arachidonate metabolites. These results indicate that growth-inhibiting doses of radiation trigger the process in lymphocytes that culminates in apoptosis, yet leave the cells partially responsive to mitogenic stimuli.

Poly(ADP-ribose) Metabolism in Proliferating Versus Quiescent Cells and Its Relationship to Their Radiation Responses

In the murine tumour cell lines 66 and 67 growing in vitro, quiescent (Q; unfed plateau-phase) cells are more sensitive to X-ray-induced cell killing than are proliferating (P) cells, while St4 cells (Q cells that have been re-fed and returned to 37 degrees C for 4h) are similar to P cells in radiosensitivity. We have been investigating parameters of poly(ADP-ribose) metabolism in order to determine whether such factors contribute to the variations in radiosensitivity of these growth states. These parameters were cellular NAD content, the activity of poly(ADP-ribose) transferase (ADPRT) in permeabilized cells and the activity of poly(ADP-ribose)-degrading enzymes. The results suggest that in line 66, but not 67, a reduced ability to regenerate NAD following irradiation was associated with the reduced survival of Q cells. However, neither the baseline activity of ADPRT nor the degree of stimulation of ADPRT by X-rays was found to correlate with survival, or with the induction and repair of DNA strand breaks. Stimulation of ADPRT by X-rays was dependent on dose and was greatest for a 2-min incubation with 3H-NAD. For a 2-min incubation the stimulation of ADPRT following a dose of 50 Gy was 7- and 10-fold in 66 and 67 P cells, respectively, versus 3-4-fold in Q cells. Detectable stimulation was observed in 66 P and Q cells for doses as low as 5 Gy. P and Q cells did not differ in the rate of degradation of the poly(ADP-ribose) polymers.

Inhibitor of poly(ADP-ribose)transferase potentiates the recombinogenic but not the mutagenic action of alkylating agents in somatic cells in vivo in Drosophila melanogaster

The effect on somatic mutations and recombination by 3-aminobenzamide (3-ABA), an inhibitor of poly(ADP-ribose) transferase, was studied in the somatic wing spot test in Drosophila melanogaster, 3-ABA did not exert any effect by itself, but caused a strong increase of wing spots in combination with methyl methanesulphonate (MMS) and ethylnitrosourea (ENU). MMS and ENU differ in their mutagenic effects, ENU causing a much higher frequency of point mutations than MMS. The enhancement by 3-ABA of chemically induced wing spots was totally eliminated by the introduction of a heterozygous inversion. It can be concluded that 3-ABA interacted with the chemical mutagens for the induction of somatic recombination, but had no effect on point mutations or intragenic events.

Substituted benzamides as poly(adp-ribose) transferase inhibitors: development of new derivatives by using computer graphics techniques

Substituted benzamides as poly(adp-ribose) transferase inhibitors: development of new derivatives by using computer graphics techniques

Chromium effects on chondrocytic differentiation in vitro.

A tissue culture study was conducted on the effects of chromium on chondrocytic differentiation. Mesenchymal cells from stage 22-24 chick limb buds were dispersed and cultured as micromasses, where they differentiated into chondrocytes. Addition of chromium(VI) to the cultures indicated that the production of proteoglycans (as detected by Alcian blue staining) was more sensitive to chromium's effects than was cell proliferation. Whereas Alcian blue nodule formation was inhibited by 1 microM Cr(VI), cell proliferation (as detected by cell counts) was not. Chromium (VI) was added to cultures at daily intervals, and these studies indicated that the interval of d 1-2 was the most sensitive period. ADP-ribose transferase activity in these cultures was measured; the pattern of enzyme activity in control cultures was high 1 and 24 h after the start of culture, decreased abruptly between 24 and 48 h, and then decreased more gradually. In the presence of Cr(VI), elevated ADP-ribose transferase levels were maintained throughout the culture period. We suggest that, in presence of 1.0 microM chromium(VI) or higher concentrations, the balance of events favors nucleolytic action rather than repair of damage.

Non-histone chromosomal protein acceptors for poly(ADP)-ribose in phorbol-12-myristate-13-acetate treated mouse embryo fibroblasts (C3H10T1/2).

The tumor promoter phorbol-12-myristate-13-acetate (PMA) increases the level of poly ADP-ribosylation of chromosomal proteins in mouse embryo fibroblasts C3H10T1/2. The poly ADP-ribosylated nuclear proteins fall into the following molecular weight classes: 40, 48, 61, 77, 92, 158, 200 kd. Preincubation with catalase reduced the poly ADP-ribose (ADPR) substitution of all these proteins essentially to control levels. Western blot analysis with antibody directed against ADPR transferase indicates that the major acceptors are ADP-ribose transferase (116 kd) itself and its proteolytic degradation products of 20-25, 45 and 72-95 kd. Poly ADP-ribosylation of these proteins is suppressed by cycloheximide, 3-aminobenzamide, antipain and catalase. The latter three inhibitors possess anti-promotional activities in certain in vitro cell culture systems. Auto-poly ADP-ribosylation of ADPR transferase and its proteolytic cleavage as well as the poly ADP-ribosylation of other chromosomal proteins may play a role in the modulation of gene expression by PMA.

The effect of inhibitors of ADP-ribose transferase on the concentration of (ADP-ribose)n in vivo

The effect of inhibitors of ADP-ribose transferase on the concentration of (ADP-ribose)<i>n</i> <i>in vivo</i>