NAD and poly(ADP-ribose) regulation of proteins involved in response to cellular stress and DNA damage.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
S J Berger
Publications and source records attributed to S J Berger.
Topoisomerase I activity in squamous cell carcinoma of the head and neck.
In recent preclinical and clinical trials, topoisomerase I inhibitors have shown great promise as antitumor agents. These agents are most effective against tumors with high topoisomerase I activity. Therefore, determining topoisomerase I activity in advance may predict response to topoisomerase I inhibitors. Squamous cell carcinoma of the head and neck and normal tissue samples were obtained from 12 patients. Cellular extracts were prepared, and topoisomerase I activity assays were performed. The results suggest that topoisomerase I activity in squamous cell carcinoma of the head and neck is increased approximately sixtyfold compared to normal tissue. Increased activity often correlates with clinical responsiveness; these results predict that topoisomerase I inhibitors should be effective and selective against squamous cell carcinoma of the head and neck.
Aged↗
Drastic reduction of topoisomerase II alpha associated with major acquired resistance to topoisomerase II active agents but minor perturbations of cell growth.
V511 and V513 cell lines, derived from Chinese hamster V79 cells following alkylating agent mutagenesis and subsequent selection with VP-16, showed resistance to cytotoxicity and DNA strand breaks induced by topoisomerase (topo) II inhibitors and were resistant to VP-16-induced sister chromatid exchanges. They showed no amplification of the multidrug-resistant p-glycoprotein. In a kinetoplast-DNA decatenation assay, V511 and V513 showed 51% and 49% topo II activity relative to parental V79 cells, respectively. By western-blot analysis all three logarithmically growing cell lines showed similar levels of topo II beta (M(r) 180,000), which increased as cells progressed to quiescence. In contrast, immunoreactive levels of topo II alpha (M(r) 170,000) were 6.8% in V511 and 62.4% in V513 relative to V79. V511 showed drastically decreased topo II alpha in both log growth and quiescence. In a second approach, immunoreactive topo II was analyzed in different phases of the cell cycle in logarithmically growing cells fractionated by fluorescence-activated cell sorting. All cell lines demonstrated relatively stable topo II beta throughout the cell cycle. Topo II alpha showed little cell cycle variation in V79 or V513. However, in V511, it was only detectable at low levels in G2/M phase. When cell growth parameters were measured, V511 and V513 showed a 17% increase in cell doubling time relative to V79. These studies indicate that cells with a drastic reduction in topo II alpha (V511) or mutant topo II alpha (V513) but with normal levels of topo II beta show only minor perturbations of cell growth.
Alkylating Agents↗
Induction of M(r) 78,000 glucose-regulated stress protein in poly(adenosine diphosphate-ribose) polymerase- and nicotinamide adenine dinucleotide-deficient V79 cell lines and its relation to resistance to the topoisomerase II inhibitor etoposide.
Cell lines deficient in poly(ADP-ribose) synthesis due to enzyme deficiency (ADPRT54 and ADPRT351) or substrate deficiency (N2, N3, and N4) are resistant to topoisomerase II-directed agents, including etoposide (VP-16), N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulfonamide, and Adriamycin, relative to the effect of these agents on parental V79 Chinese hamster cells. Resistance is stable in the ADPRT54 and ADPRT351 cell lines, whereas resistance in the N2, N3, and N4 cell lines occurs when the cells are grown in nicotinamide-deficient medium to produce a state of NAD deficiency. However, sensitivity to VP-16 reverts to normal when cellular NAD levels return to control levels during growth in nicotinamide-containing complete medium. Poly(ADP-ribose) polymerase-deficient cell lines show constitutively increased levels of a protein at M(r) 78,000 on Coomassie blue-stained, sodium dodecyl sulfate-polyacrylamide gels that was subsequently confirmed with monoclonal antibodies to be M(r) 78,000 glucose-regulated stress protein (GRP78). Similarly, N2, N3, and N4 cells show induction of GRP78 under nicotinamide-deficient conditions. Induction of GRP78 is associated with elevated levels of GRP78 mRNA and appears to be regulated at the transcriptional level. When N3 cells with deficiency of poly(ADP-ribose) synthesis due to NAD deficiency are shifted to complete, nicotinamide-containing medium, they restore their NAD content, undergo a decrease in GRP78 levels, and regain sensitivity to VP-16. When V79 cells are shifted to nicotinamide-deficient medium they undergo a reduction in NAD content, followed by a progressive elevation in GRP78 levels, and they subsequently become increasingly resistant to VP-16. These studies demonstrate a clear association between deficiency of the NAD-poly(ADP-ribose) synthesis system, induction of GRP78 synthesis, and resistance to VP-16.
Animals↗
Combined depletion of O6-alkylguanine-DNA alkyltransferase and glutathione to modulate nitrosourea resistance in breast cancer.
MCF-7 human breast cancer cells possess high levels of O6-alkylguanine-DNA alkyltransferase and moderate levels of glutathione, and are more resistant to chloroethylnitrosoureas (CNUs) than cells with low levels of either molecule. The role of each as a component of CNU resistance was assessed using O6-benzylguanine (O6-bG) or O6-methylguanine (O6-mG) to deplete the alkyltransferase and L-buthionine sulfoxamine (L-BSO) to deplete glutathione. O6-bG and O6-mG potentiated 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) cytotoxicity, resulting in a dose modification factor of 5.4 and 2.3, respectively, which reflected the more potent inhibitory effect of O6-bG. L-BSO alone had little effect on the survival of MCF-7 cells following BCNU exposure, but when combined with O6-mG, BCNU cytotoxicity was additive, yielding a dose modification factor of 3.2. O6-bG or O6-mG and L-BSO acted independently, as neither class of inhibitor affected the other's mechanism of CNU resistance. Furthermore, MCF-7 cells overexpressing GST mu were not more resistant to BCNU than the parent cell line in either the presence or absence of O6-bG or L-BSO. These results indicate that on a relative basis in MCF-7 cells, the alkyltransferase is the cell's first line of defense against CNUs. This suggests that therapeutic trials based on O6-bG-induced biochemical modulation of CNU resistance may increase the efficacy of these chemotherapeutic agents against human malignant cells and that L-BSO may have little additive effect when used with these agents.
Breast Neoplasms↗
Sensitive enzymatic cycling assay for glutathione: measurements of glutathione content and its modulation by buthionine sulfoximine in vivo and in vitro in human colon cancer.
We have measured glutathione content in small tissue samples derived from biopsies of primary and metastatic human colon tumors and from colon cancer cell lines in tissue culture and xenografts in athymic mice. Measurements were performed using an enzymatic cycling assay designed to quantitate extremely low levels of glutathione (GSH) (down to 10(-14) mol) from perchlorate extracts of tissue samples weighing less than 1 mg wet weight. Glutathione was stable in these acid extracts for at least 6 months when stored at -80 degrees C. A survey of normal tissues in mice, rats, and some human tissues showed considerable variation in GSH content of different tissues but generally similar levels were identifiable for the same tissues from different species. The highest GSH level was 56.9 nmol/mg protein in rat liver and the lowest was 1.8 nmol/mg protein in rat skeletal muscle. High GSH levels were also determined in mouse and human liver, while low GSH levels were detected in mouse muscle. Human colon cancer cell lines showed slightly higher GSH levels than did colon cancer tumor samples obtained from biopsies. These studies revealed a marked inter-individual difference in tumor GSH content, as well as a difference in GSH content between tumor deposits at different metastatic sites in the same individual. These results indicate the importance of direct tumor measurements of GSH content in clinical trials designed to modulate tumor glutathione content to try to increase sensitivity to chemotherapy or radiation therapy. Buthionine sulfoximine, an inhibitor of gamma-glutamyl cysteine synthetase, was shown to produce almost complete depletion of GSH in four different human colon cancer cell lines in 24 h. Buthionine sulfoximine was also shown to be capable of producing drastic depletion of GSH in human colon cancer grown as xenografts in athymic animals.
Animals↗
Alkylating agent hypersensitivity in poly(adenosine diphosphate-ribose) polymerase deficient cell lines.
Starting with the V79 cell line, two poly(ADP-ribose) polymerase deficient mutants, designated ADPRT 54 and ADPRT 351, had been shown to be hypersensitive to x- and UV-irradiation and to topoisomerase I inhibitors but to be resistant to topoisomerase II inhibitors (Chatterjee, S.; Cheng, M. F.; Berger, N. A. Hypersensitivity to clinically useful alkylating agents and radiation in poly(ADP-ribose) polymerase-deficient cell lines. Cancer Commun. 2:401-407;1990). We now report that these mutants were hypersensitive to a series of different alkylating agents, including alkylsufonates, alkylnitrosoureas, and nitrosoguanidine. In addition, they were hypersensitive to the UV-mimetic agent 4-nitroquinoline-1-oxide. Our findings provide strong evidence that poly(ADP-ribose) polymerase was involved in the repair of alkylating agent induced DNA damage as well as in the damage induced by UV- and x-irradiation and radiomimetic agents. The poly(ADP-ribose) polymerase deficient cell lines showed a marked decrease in the shoulder region of their survival curves, suggesting that poly(ADP-ribose) polymerase was involved in the repair of alkylating agent induced sublethal damage.
4-Nitroquinoline-1-oxide↗
Mutant cells defective in poly(ADP-ribose) synthesis due to stable alterations in enzyme activity or substrate availability.
We used two different approaches to develop cell lines deficient in poly(ADP-ribose) synthesis to help determine the role of this reaction in cellular functions. One approach to this problem was to develop cell lines deficient in enzyme activity; the other approach was to develop cell lines capable of growing with such low nicotinamide adenine dinucleotide (NAD) levels so as to effectively limit substrate availability for poly(ADP-ribose) synthesis. The selection strategy for obtaining cells deficient in activity of poly(ADP-ribose) polymerase was based on the ability of this enzyme to deplete cellular NAD in response to high levels of DNA damage. Using this approach, we first obtained cell lines having 37-82% enzyme activity compared to their parental cells. We now report the development and characterization of two cell lines which were obtained from cells having 37% enzyme activity by two additional rounds of further mutagenization and selection procedures. These new cell lines contain 5-11% enzyme activity compared to the parental V79 cells. In pursuit of the second strategy, to obtain cells which limit poly(ADP-ribose) synthesis by substrate restriction, we have now isolated spontaneous mutants from V79 cells which can grow stably in the absence of free nicotinamide or any of its analogs. These cell lines maintain NAD levels in the range of 1.5-3% of that found in their parental V79 cells grown in complete medium. The pathway of NAD biosynthesis in these NAD-deficient cells is not yet known. Further characterization of these lines showed that under conditions that restricted poly(ADP-ribose) synthesis, they all had prolonged doubling times and increased frequencies of sister chromatid exchanges.
Animals↗
Induction of the pyridine nucleotide synthesis pathway in mitogen-stimulated human T-lymphocytes.
Mitogen stimulation of purified human T-lymphocytes with the phorbol ester 12-O-tetradecanoyl, phorbol-13-acetate (TPA) and a monoclonal antibody to the T3 cell surface antigen caused a 6-11-fold increase in cellular levels of poly(ADP-ribose) polymerase, a 6-20-fold amplification of cellular NAD+ levels and a 3-21-fold increase in NADP+ levels. Treatment of the cells with a combination of the two mitogenic signals also caused a 5-20-fold increase in NMN pyrophosphorylase activity, a 3-14-fold increase in ATP-NMN adenylyl transferase activity, and a 5-13-fold increase in NAD kinase activity. This is the first report showing induction of these three enzymes as part of the mitogenic response in purified human T-lymphocytes. Maximum increases in activity of each of these three enzymes required the combined presence of TPA and monoclonal antibody to human T-cell T3 antigen anti-T3. Analysis of the relative enzyme levels indicates that NMN pyrophosphorylase is the rate-limiting enzyme for NAD synthesis and NAD kinase is the rate-limiting enzyme for NADP synthesis.
Antibodies, Monoclonal↗
Pyridine nucleotide analog interference with metabolic processes in mitogen-stimulated human T lymphocytes.
The differential metabolic effects of three nicotinamide analogs, 6-aminonicotinamide, 3-aminobenzamide, and 5-methylnicotinamide, were analyzed in mitogen-stimulated preparations of human T lymphocytes. Mitogen stimulation with the phorbol ester TPA and a monoclonal antibody to the T3 cell surface antigen caused an increase in cellular NAD and ATP levels and a marked increase in glucose metabolism as demonstrated by an increase in cellular levels of glucose 6-phosphate and a sevenfold increase in radioactive CO2 formation from [l-14C]glucose. 6-Aminonicotinamide had drastic inhibitory effects on the mitogen-stimulated increases in NAD and ATP levels as well as on the metabolism of glucose. Treatment of the mitogen-stimulated cells with 6-aminonicotinamide also caused a marked increase in cellular levels of 6-phosphogluconate, suggesting inhibition of the hexose monophosphate shunt at 6-phosphogluconate dehydrogenase. Radioactive CO2 formation from [6-14C]glucose showed that metabolism through the tricarboxylic acid cycle was not used to compensate for the inhibition of the hexose monophosphate shunt pathway. Treatment of cells with 3-aminobenzamide had the opposite effect of 6-aminonicotinamide in that cellular NAD levels increased, presumable due to inhibition of poly(ADP-ribose) polymerase. 3-Aminobenzamide did not interfere with ATP or glucose 6-phosphate levels and did not cause significant elevations of 6-phosphogluconate. Thus, 6-aminonicotinamide appears to have direct inhibitory effects on the synthesis of both pyridine nucleotides and poly(ADP-ribose), whereas 3-aminobenzamide has its major inhibitory effect on poly(ADP-ribose) synthesis. 5-Methylnicotinamide also interferes with the mitogen-stimulated increase in NAD levels but not as effectively as 6-aminonicotinamide. The alterations in pyridine nucleotide metabolism resulting from treatment with these nicotinamide analogs can produce drastic and diverse alterations in pathways of glucose utilization and energy generation.
6-Aminonicotinamide↗
Strategy for selection of cell variants deficient in poly(ADP-ribose) polymerase.
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.
Adenosine Triphosphate↗
Role of nicotinamide adenine dinucleotide and adenosine triphosphate in glucocorticoid-induced cytotoxicity in susceptible lymphoid cells.
The possibility that corticosteroid cytotoxicity could be mediated by activation of poly(ADP-ribose) polymerase and consequent depletion of NAD and ATP was evaluated in steroid-sensitive S49.1 and steroid-resistant S49.143R mouse lymphoma cells and in lymphocytes from a patient with chronic lymphocytic leukemia. All cell types were shown to have the enzyme poly(ADP-ribose) polymerase and to increase activity in response to DNA strand breaks. Incubation of susceptible cells with 1 microM dexamethasone resulted in DNA strand breaks. Susceptible cells also showed a dose-dependent decrease in NAD and ATP that preceded loss of cell viability. These studies suggest that steroid-induced cytotoxicity in susceptible lymphocytes is due to the presence of DNA strand breaks that activate poly(ADP-ribose) polymerase to a sufficient degree to consume cellular pools of NAD with a consequent depletion of ATP and loss of cell viability.
Adenosine Triphosphate↗
DNA repair, ADP-ribosylation and pyridine nucleotide metabolism as targets for cancer chemotherapy.
DNA repair mechanisms serve as useful targets for modulating the cytotoxic and chemotherapeutic effects of many agents whose mechanism of action involves the induction of DNA damage. For example, the modified base O6-methylguanine can inactivate the repair protein O6-alkylguanine alkyltransferase, thereby sensitizing cells to the cytotoxic effects of clinically useful nitrosoureas such as BCNU. Some of the cytotoxic DNA adducts induced by BCNU are repaired by O6-alkylguanine alkyltransferase; thus, inactivation of the protein by O6-methylguanine converts cells that are relatively resistant to BCNU into sensitive cells. Another cellular enzyme, poly(ADP-ribose) polymerase, responds to DNA strand breaks by cleaving its substrate, NAD+, and using the resultant ADP-ribose moieties to synthesize homopolymers of ADP-ribose. The use of agents such as benzamide derivatives to inhibit enzyme function results in the accumulation of DNA strand breaks and potentiates the tumoricidal effects of some DNA strand-breaking agents such as bleomycin. Poly(ADP-ribose) polymerase can also affect pyridine nucleotide metabolism in a manner that initiates biochemical alterations leading directly to cell death. Thus, the amount of NAD used in the synthesis of poly(ADP-ribose) is dependent on the number of DNA strand breaks present in the cells. DNA damage can sufficiently activate the enzyme to rapidly consume NAD and consequently deplete ATP levels, resulting in the cessation of all energy-dependent functions and cell death. Understanding this biochemical pathway that leads to cell death provides a new basis for modulating chemotherapy. For example, agents such as Tiazofurin and/or 6-aminonicotinamide can each be used to alter pyridine nucleotide metabolism, lower NAD pools and potentiate the cytotoxic effects of other chemotherapeutic agents whose primary target is the induction of DNA damage.
Antineoplastic Agents↗
Metabolic consequences of DNA damage: DNA damage induces alterations in glucose metabolism by activation of poly (ADP-ribose) polymerase.
In this communication we show that activation of poly(ADP-ribose) polymerase by DNA damage can produce drastic alterations in carbohydrate metabolism. We examined alterations in NAD+, NADP+, ATP and glucose-6-phosphate in L1210 murine leukemia cells, following exposure to different concentrations of N-methyl-N'-nitro-N-nitrosoguanidine. Treatment of cells with 20 micrograms/ml MNNG produced rapid depletion of NAD+ and ATP. The G-6-P pool showed a biphasic change: first the pool size decreased, then increased to a level greater than that present in control cells. Nicotinamide treatment prevented the total depletion of NAD+ and this in turn helped preserve the ATP pools and prevented the biphasic alteration in G-6-P pool sizes.
Adenosine Triphosphate↗
Metabolic consequences of DNA damage: the role of poly (ADP-ribose) polymerase as mediator of the suicide response.
Explore the source record for details and available documents.
Adenosine Triphosphate↗
An enzymatic method for the measurement of nicotinamide mononucleotide pyrophosphorylase in cells and tissues.
A simple enzymatic method is described for the measurement of NMN pyrophosphorylase in tissue homogenates at levels as low as 10(-12) to 10(-9) mol. The product, nicotinamide mononucleotide, is converted to NAD using NAD pyrophosphorylase and the NAD is quantified in an enzymatic cycling assay. The enzyme described here is stimulated more at low concentrations of Mn2+ than Mg2+. ATP is not required for NMN pyrophosphorylase activity; the reaction is neither stimulated nor inhibited by ATP concentrations as high as 3 mM. The enzyme is totally dependent on phosphoribosylpyrophosphate. The method is highly reproducible in all tissues examined. Various cell lines and tissues from mouse were analyzed for NMN pyrophosphorylase.
Adenosine Triphosphate↗
Digital intravenous angiography for the study of hemodialysis vascular access.
Digital intravenous angiography is a simple and safe procedure and can be performed on an outpatient basis. Poor function of a hemodialysis vascular access due to increased graft resistance, reduced arterial inflow or shunt steal can be evaluated by this technique. Complications of direct graft puncture or conventional arteriography can be avoided using digital angiography.
Angiography↗
Poly(ADP-ribose) polymerase activity in proliferating and quiescent murine mammary carcinoma cells.
Previous studies have shown that the well-oxygenated but nutrient-deprived quiescent (QI) cells of the 67 murine mammary carcinoma line are significantly more sensitive to radiation-induced cell killing than the well-oxygenated proliferating (P) cells. These QI cells also sustain more initial strand breaks per radiation dose and repair them more slowly than do P cells. We now report that NAD+ levels and rates of poly(ADP-ribosylation) show a trend similar in that both these metabolic parameters are lower in QI cells by a factor of two or more. NAD+ levels were measured in acid extracts of intact monolayers using an enzymatic cycling assay, while poly(ADP-ribosylation) was followed through the incorporation of radioactive NAD+ into polymer by permeabilized cells. The major proteins labeled by [32P]NAD+ were the same in P and QI cells. However, qualitative differences exist among minor poly(ADP-ribosylated) proteins, with some bands labeled in P cells but not detectably labeled in QI cells. These studies suggest similarities between the physiological state of QI cells and that of mature resting lymphocytes.
Animals↗