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1.
A selection strategy to obtain cells deficient in poly(ADP-ribose) polymerase was developed based on the fact that treatment with high levels of N-methyl-N'-nitro-N-nitrosoguanidine results in sufficient activation of poly(ADP-ribose) polymerase to cause NAD and ATP depletion leading to cessation of all energy-dependent processes and rapid cell death. In contrast, cells with low levels of poly(ADP-ribose) polymerase should not consume their NAD and might therefore be more likely to survive the DNA damage. Using this approach, we have cloned a number of cell lines containing 37-82% enzyme activity. The apparent decrease in poly(ADP-ribose) polymerase activity is not due to increases in NAD glycohydrolase, poly(ADP-ribose) glycohydrolase, or phosphodiesterase activities. Further characterization of the poly(ADP-ribose) polymerase-deficient cells indicates that they have prolonged generation times and increased rates of spontaneous sister chromatid exchanges.  相似文献   

2.
We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.  相似文献   

3.
The biological function of poly(ADP-ribose) polymerase in DNA repair, cell-cycle regulation and cellular differentiation has yet to be defined. Isolation of cells which are deficient in poly(ADP-ribose) synthesis would greatly facilitate the determination of the biological role of this enzyme. A method is described for isolating Chinese hamster ovary (CHO) cells deficient in the poly(ADP-ribose) polymerase activity by direct screening of colonies for enzyme activity. Colonies with decreased production of poly(ADP-ribose) are recovered from nylon replicas for further analysis. Using this method we have isolated a series of CHO cells which have 50% or less poly(ADP-ribose) polymerase activity. These mutants have normal generation times and are 20% more sensitive to the effects of DNA (m)ethylating agents than the parental cell. However, these mutants display normal sensitivity to gamma-rays.  相似文献   

4.
Postirradiation incubation of V79 Chinese hamster cells with inhibitors of poly(ADP-ribose) synthesis was found to potentiate the killing of cells by X rays. Potentiation increased with incubation time and with concentration of the inhibitor. Preirradiation incubation had only a small effect. The enhanced response correlated well with the known extent of the inhibition of poly(ADP-ribose) synthesis. A radiation-sensitive line, V79- AL162 /S-10, was affected to a lesser extent than the normal cells. Cells repaired the radiation damage with which the inhibitors interacted within 1 hr, a process that has similar kinetics to what is observed when a postirradiation treatment with hypertonic buffer is used [H. Utsumi and M. M. Elkind , Radiat . Res. 77, 346-360 (1979)]. However, the sectors of damage affected by inhibitors of poly(ADP-ribose) synthesis and hypertonic buffer do not entirely overlap. The inhibitor nicotinamide enhanced the killing mainly of late S-phase cells and did not affect cells at the G1/S border. It is concluded that the repair process(es) involving poly(ADP-ribose) synthesis is important for cell survival in repair-competent cells and that the radiation-sensitive cells that were examined are partially deficient in a repair pathway in which poly(ADP-ribose) participates.  相似文献   

5.
The activity of tyrosine aminotransferase (TAT) (EC 2.6.1.5) was enhanced 3-fold after a 5-h exposure of cultured rat liver cells (RLC) to streptozotocin (SZ) at concentrations higher than 100 microgram/ml (0.38 mM) in the presence of 10 nM dexamethasone, a potent glucocorticoid inducer for the enzyme. The structurally related carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) also enhanced the aminotransferase in the presence of the glucocorticoid, but its optimal concentration was at 100 ng/ml (0.68 microM). While the cellular NAD (NAD+ + NADH) concentration was reduced to 60% of the control levels, the rate of poly(ADP-ribose) formation in the isolated cell nuclei was unaffected by treating the cells with SZ. The enhancement of tyrosine aminotransferase by SZ and MNNG was effectively prevented by nicotinamide. Using nicotinamide and its derivatives such as 1-methyl-, N'-methyl- or 6-amino-derivatives it was found that the degree of enzyme induction is almost inversely proportional to the cellular NAD content, though the activity of nuclear poly(ADP-ribose)polymerase remains unchanged. The results indicate that SZ or MNNG, in combination with dexamethasone, stimulate the induction of tyrosine aminotransferase through their NAD lowering action.  相似文献   

6.
Poly(ADP-ribose) polymerase is a chromatin enzyme which adds long chains of ADP-ribose to various acceptor proteins in response to DNA strand breaks. Its primary function is unknown; however, a role in DNA repair and radiation resistance has been postulated based largely on experiments with enzyme inhibitors. Recent reports of mutant cell lines, deficient in poly(ADP-ribose) polymerase activity, have supported previous studies with inhibitors, which suggests the involvement of poly(ADP-ribose) polymerase in maintaining baseline levels of sister chromatid exchanges. Mutant cells with even slightly depressed enzyme levels show large elevation of baseline sister chromatid exchanges. Since intracellular poly(ADP-ribose) polymerase levels can vary greatly between different nonmutant cell lines, we surveyed levels of baseline sister chromatid exchange in normal and tumor human cell lines and compared them with endogenous levels of poly(ADP-ribose) polymerase. Despite 10-fold differences in poly(ADP-ribose) polymerase, the baseline level of sister chromatid exchanges remained relatively constant in the different cell lines (0.13 +/- 0.03 SCE/chromosome), with no indication of a protective effect for cells with high levels of the enzyme.  相似文献   

7.
Nicotinamide-adenine dinucleotide (NAD+) is the substrate used by cells in poly(ADP-ribose) synthesis. X-irradiation of log-phase Chinese hamster cells caused a rapid decrease in NAD+ levels which was linearly dependent on radiation dose. The activity of ADP-ribosyl transferase ( ADPRT ) also increased linearly with radiation dose. The decrease of NAD+ was slower, and the increase in ADPRT activity was less pronounced, in a radiation sensitive line, V79- AL162 /S-10. An inhibitor of ADPRT , m-aminobenzamide, largely prevented the depletion of cellular NAD+ and reduced the rate at which ADPRT activity disappeared during post-irradiation incubation. Post-irradiation treatment with hypertonic buffer or with medium containing D2O--which inhibit repair of radiation-induced potentially lethal damage--enhanced the depletion of NAD+ and prevented the reduction in ADPRT activity following irradiation. The characteristics of the effects of treatment with hypertonic buffer on NAD+ metabolism were qualitatively similar to the effects that such treatment has on radiation-induced cell killing. These results suggest that poly(ADP-ribose) synthesis after irradiation plays a role in the repair of potentially lethal damage.  相似文献   

8.
Poly(ADP-ribose) polymerase activity in nuclei isolated from differentiating cardiac muscle of the rat has been characterized and its activity measured during development. Optimum enzyme activity is observed at pH 8.5. Poly(ADP-ribose) polymerase is inhibited by ATP, thymidine, nicotinamide, theophylline, 3-isobutyl-1-methylxanthine and caffeine and stimulated by actinomycin D. The activity measured under optimal assay conditions increases during differentiation of cardiac muscle and is inversely related to the rate of DNA synthesis and to the activities of DNA polymerase alpha and thymidine kinase. When DNA synthesis and the activity of DNA polymerase alpha are inhibited in cardiac muscle of the 1-day-old neonatal rat by dibutyryl cyclic AMP or isoproterenol, the specific activity of poly(ADP-ribose) polymerase measured in isolated nuclei is increased. The concentration of NAD+ in cardiac muscle increases during postnatal development. In the adult compared with the 1-day-old neonatal rat the concentration of NAD+ relative to fresh tissue weight, DNA or protein increased 1.7-fold, 5.2-fold or 1.4-fold respectively. The concentration of NAD+ in cardiac muscle of the 1-day-old neonatal rat can be increased by approx. 20% by dibutyryl cyclic AMP. These data suggest that NAD+ and poly(ADP-ribose) polymerase may be involved with the repression of DNA synthesis and cell proliferation in differentiating cardiac muscle.  相似文献   

9.
The sensitivities (Do-values) of the cytotoxic effect of MNU on four rodent cell lines were: mouse L1210, 0.07 mM; rat Yoshida sarcoma, 0.52 mM; Chinese hamster V79A, 0.70 mM and the UV sensitive, X-ray sensitive V79/79, 0.35 mM. The abilities of maximum non-toxic doses of the poly-(ADP-ribose) polymerase inhibitors, 5-methyl nicotinamide (5MeN), 3-methoxybenzamide (3MBA) and caffeine to potentiate this cytotoxicity and that of UV light in V79A and V79/79 was measured. The degree of potentiation (ratio Do without inhibitor/Do with inhibitor) was both agent and cell line dependent. In general the lymphoid cell lines L1210 and YS showed greater potentiation, up to 4-fold, than did the fibroblast lines V79A and V79/79. The use of inhibitors in pairs suggested that 5MeN and 3MBA affect one process whereas caffeine affects additional processes. The data provide further support for a role for poly(ADP-ribose) in DNA repair, but indicate that metabolic factors may modify the effectiveness of individual inhibitors of poly(ADP-ribose) polymerase in different cell lines.  相似文献   

10.
1-Methylnicotinamide, a direct methylation product of nicotinamide, stimulates the DNA synthesis and proliferation of rat liver cells (RLC) in culture at concentrations higher than 20 μM. The effect of nicotinamide, which is a potent inhibitor of DNA synthesis and proliferation, is counteracted by 1-methylnicotinamide. The intracellular NAD concentration decreases within 2 h under 1-methylnicotinamide, whereas it increases in the presence of nicotinamide. The poly(ADP-ribose) synthesizing activity in the isolated nuclei remained unchanged. These results suggest a physiological role of 1-methylnicotinamide in the cell growth through a lowering of intracellular NAD level.  相似文献   

11.
Poly(ADP-ribose) polymerase specifically recognizes DNA strand breaks by its DNA-binding domain. DNA binding activates the enzyme to catalyze the formation of poly(ADP-ribose) utilizing NAD as substrate. By a molecular genetic approach we set out to inhibit this enzyme activity in a highly specific manner, thus avoiding the inherent side effects of NAD analogs which have been used extensively as enzyme inhibitors. cDNA sequences coding for the human poly(ADP-ribose) polymerase DNA-binding domain were subcloned into eucaryotic expression plasmids and transiently transfected into monkey cells. Cells were fixed with ethanol followed by incubation with NAD. Indirect double immunofluorescence to detect both overexpressed protein and poly(ADP-ribose) in situ revealed that overexpression of the DNA-binding domain greatly inhibited poly(ADP-ribosyl)ation catalyzed by the resident enzyme during NAD postincubation. The same inhibition was observed when transfected cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine to induce DNA strand breaks in vivo and subjected to trichloroacetic acid/ethanol fixation and subsequent immunofluorescence analysis, a novel method we developed for the in situ detection of polymer synthesis in intact cells. This molecular genetic approach may prove to be a selective and efficient tool to investigate possible functions of poly(ADP-ribosyl)ation in living cells.  相似文献   

12.
Zhou Y  Feng X  Koh DW 《Biochemistry》2011,50(14):2850-2859
We previously demonstrated that the absence of poly(ADP-ribose) glycohydrolase (PARG) led to increased cell death following DNA-damaging treatments. Here, we investigated cell death pathways following UV treatment. Decreased amounts of PARG-null embryonic trophoblast stem (TS) cells were observed following doses of 10-100 J/m2 as compared to wild-type cells. In wild-type cells, caspase-cleaved poly(ADP-ribose) polymerase-1 (PARP-1) and activated caspase-3 were detected 12-24 h after UV treatment. Surprisingly, both were detected at decreased levels only after 24 h in PARG-null TS cells, indicating a decreased level and delayed presence of caspase-mediated events. Further, a time- and dose-dependent accumulation of poly(ADP-ribose) (PAR) levels after UV was observed in PARG-null TS cells and not in wild-type cells. Determination of the levels of nicotinamide adenine dinucleotide (NAD+), the substrate for PAR synthesis and a coenzyme in cellular redox reactions, demonstrated a UV dose-dependent decrease in the level of NAD+ in wild-type cells, while NAD+ levels in PARG-null TS cells remained at higher levels. This indicates no depletion of NAD+ in PARG-null TS cells following increased levels of PAR. Lastly, cell death mediated by apoptosis-inducing factor (AIF) was analyzed because of its dependence on increased PAR levels. The results demonstrate nuclear AIF translocation only in PARG-null TS cells, which demonstrates the presence of AIF-mediated cell death. Herein, we provide compelling evidence that the absence of PARG leads to decreased caspase-3 activity and the specific activation of AIF-mediated cell death. Therefore, the absence of PARG may provide a strategy for specifically inducing an alternative apoptotic pathway.  相似文献   

13.
A possible role of poly(ADP-ribose) synthesis in modulating the response of V79 cells to DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) was investigated. Inhibition of [3H]thymidine (dThd) incorporation into DNA and lowering of NAD+ levels in intact cells were employed as parameters of DNA-synthesis inhibition and poly(ADP-ribose) synthesis, respectively. Dose responses of these parameters were studied in cells 2 and 24 h after treatment with the methylating agents in medium with or without dThd. The initial inhibition of DNA synthesis was uniformly associated with stimulation of poly(ADP-ribose) synthesis whether the cells were treated with MNNG or MMS, incubated with or without 20 microM dThd which did not inhibit poly(ADP-ribose) synthesis, or incubated with 3 mM dThd which did inhibit the latter synthesis. By contrast, the DNA-synthesis inhibition detected 24 h after treatment with MNNG was not associated with poly(ADP-ribose) synthesis. These data suggest that (i) the mechanism of this later inhibition of DNA synthesis is different from that of the initial inhibition, (ii) DNA-synthesis inhibition does not stimulate poly(ADP-ribose) synthesis, and (iii) single-strand breaks, resulting from N-methylation of the DNA, stimulate poly(ADP-ribose) synthesis, which may produce the initial inhibition of DNA synthesis. The initial inhibition of DNA synthesis was not uniformly associated with mutagenesis and dThd facilitation of MNNG-induced cytotoxicity and mutagenesis. This indicates that O-methylation of DNA does not stimulate poly(ADP-ribose) synthesis. Our data suggest that, in V79 cells treated with methylating agents, poly(ADP-ribose) synthesis is stimulated by single-strand breaks, inhibits DNA synthesis, and thereby serves to allow time for repair of the DNA prior to replication.  相似文献   

14.
DNA strand breaks, NAD metabolism, and programmed cell death   总被引:16,自引:0,他引:16  
An intimate relationship exists between DNA single-strand breaks, NAD metabolism, and cell viability in quiescent human lymphocytes. Under steady-state conditions, resting lymphocytes continually break and rejoin DNA. The balanced DNA excision-repair process is accompanied by a proportional consumption of NAD for poly(ADP-ribose) synthesis. However, lymphocytes have a limited capacity to resynthesize NAD from nicotinamide. An increase in DNA strand break formation in lymphocytes, or a block in DNA repair, accelerates poly(ADP-ribose) formation and may induce lethal NAD and ATP depletion. In this way, the level of DNA single-strand breaks in the lymphocyte nucleus is linked to the metabolic activity of the cytoplasm. The programmed removal of lymphocytes (and perhaps of other cells) with damaged DNA, may represent a novel physiologic function for poly(ADP-ribose)-dependent NAD cycling.  相似文献   

15.
NAD is a critical cofactor for the oxidation of fuel molecules. The exposure of human PBL to agents that cause DNA strand breaks to accumulate can deplete NAD pools by increasing NAD consumption for poly(ADP-ribose) formation. However, the pathways of NAD synthesis and degradation in viable PBL have not been carefully documented. The present experiments have used radioactive labeling techniques to trace the routes of NAD metabolism in resting PBL. The cells could generate NAD from either nicotinamide or nicotinic acid. PBL incubated with [14C]nicotinic acid excreted [14C]nicotinamide into the medium. Approximately 50% of a prelabeled [14C]NAD pool was metabolized during 6 to 8 hr in tissue culture. Basal NAD turnover was prolonged threefold to fourfold by 3-aminobenzamide (3-ABA), an inhibitor of poly(ADP-ribose) synthetase. Supplementation of the medium with 3-ABA also prevented the accelerated NAD degradation that ensued after exposure of PBL to deoxyadenosine plus deoxycoformycin at concentrations previously shown to cause DNA strand break accumulation. These results demonstrate that quiescent human PBL continually produce NAD and utilize the nucleotide for poly(ADP-ribose) synthesis.  相似文献   

16.
J L Sims  S J Berger  N A Berger 《Biochemistry》1983,22(22):5188-5194
Inhibitors of poly(ADP-ribose) polymerase stimulated the level of DNA, RNA, and protein synthesis in DNA-damaged L1210 cells but had negligible effects in undamaged L1210 cells. The poly(ADP-ribose) polymerase inhibitors stimulated DNA repair synthesis after cells were exposed to high concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (68 and 136 microM) but not after exposure to low concentrations (13.6 and 34 microM). When the L1210 cells were exposed to 136 microM N-methyl-N'-nitro-N-nitrosoguanidine, the activation of poly(ADP-ribose) polymerase resulted in the rapid depletion of oxidized nicotinamide adenine dinucleotide (NAD+) levels and subsequent depletion of adenosine 5'-triphosphate (ATP) pools. After low doses of N-methyl-N'-nitro-N-nitrosoguanidine (13.6 microM), there were only small decreases in NAD+ and ATP. Poly(ADP-ribose) polymerase inhibitors prevented the rapid fall in NAD+ and ATP pools. This preservation of the ATP pool has a permissive effect on energy-dependent functions and accounts for the apparent stimulation of DNA, RNA, and protein synthesis. Thus, the mechanism by which poly(ADP-ribose) polymerase inhibitors stimulate DNA, RNA, and protein synthesis in DNA-damaged cells appears to be mediated by their ability to prevent the drastic depletion of NAD+ pools that occurs in heavily damaged cells, thereby preserving the cells' ability to generate ATP and maintain energy-dependent processes.  相似文献   

17.
18.
We have studied the clonogenic survival response to X-rays and MNNG of V79 Chinese hamster cells and two derivative cell lines, ADPRT54 and ADPRT351, deficient in poly(ADP-ribose) polymerase (PARP) activity. Under conditions of exponential growth, both PARP-deficient cell lines are hypersensitive to X-rays and MNNG compared to their parental V79 cells. In contrast, under growth-arrested, confluent conditions, V79 and PARP-deficient cells become similarly sensitive to X-rays and MNNG suggesting that PARP may be involved in the repair of X-ray or MNNG-induced DNA damage in logarithmically growing cells but not in growth-arrested confluent cells. This suggestion, however, creates a dilemma as to how PARP can be involved in DNA repair in only selected growth phases while it is functionally active in all growth phases. To explain these paradoxical results and resolve this dilemma we propose a hypothesis based on the consistent observation that inhibition of PARP results in a significant increase in sister chromatid exchange (SCEs). Thus, we propose that PARP is a guardian of the genome that protects against DNA recombination. We have extended this theme to provide an explanation for our results and the studies done by many others.  相似文献   

19.
NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADP-ribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule.  相似文献   

20.
Control of the rate of cardiac cell division by oxygen occurs most probably by altering the redox state of a control substance, e.g. NAD(+)right harpoon over left harpoonNADH. NAD(+) (and not NADH) forms poly(ADP-ribose), an inhibitor of DNA synthesis, in a reaction catalysed by poly(ADP-ribose) polymerase. Lower partial pressure of oxygen, which increases the rate of division, would shift NAD(+)-->NADH, decrease poly(ADP-ribose) synthesis, and increase DNA synthesis. Chick-embryo heart cells grown in culture in 20% O(2) (in which they divide more slowly than in 5% O(2)) did exhibit greater poly(ADP-ribose) polymerase activity (+83%, P<0.001) than when grown in 5% O(2). Reaction product was identified as poly(ADP-ribose) by its insensitivity to deoxyribonuclease, ribonuclease, NAD glycohydrolase, Pronase, trypsin and micrococcal nuclease, and by its complete digestion with snake-venom phosphodiesterase to phosphoribosyl-AMP and AMP. Isolation of these digestion products by Dowex 1 (formate form) column chromatography and paper chromatography allowed calculation of average poly(ADP-ribose) chain length, which was 15-26% greater in 20% than in 5% O(2). Thus in 20% O(2) the increase in poly(ADP-ribose) formation results from chain elongation. Formation of new chains also occurs, probably to an even greater degree than chain elongation. Additionally, poly(ADP-ribose) polymerase has very different K(m) and V(max.) values and pH optima in 20% and 5% O(2). These data suggest that poly(ADP-ribose) metabolism participates in the regulation of heart-cell division by O(2), probably by several different mechanisms.  相似文献   

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