Elaine L. Jacobson

Depletion of the 110-Kilodalton Isoform of Poly(ADP-Ribose) Glycohydrolase Increases Sensitivity to Genotoxic and Endotoxic Stress in Mice

ABSTRACT Poly(ADP-ribosylation) is rapidly stimulated in cells following DNA damage. This posttranslational modification is regulated by the synthesizing enzyme poly(ADP-ribose) polymerase 1 (PARP-1) and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG). Although the role of PARP-1 in response to DNA damage has been studied extensively, the function of PARG and the impact of poly(ADP-ribose) homeostasis in various cellular processes are largely unknown. Here we show that by gene targeting in embryonic stem cells and mice, we specifically deleted the 110-kDa PARG protein (PARG110) normally found in the nucleus and that depletion of PARG110 severely compromised the automodification of PARP-1 in vivo. PARG110-deficient mice were viable and fertile, but these mice were hypersensitive to alkylating agents and ionizing radiation. In addition, these mice were susceptible to streptozotocin-induced diabetes and endotoxic shock. These data indicate that PARG110 plays an important role in DNA damage responses and in pathological processes.

DISCOVERING NEW ADP-RIBOSE POLYMER CYCLES: PROTECTING THE GENOME AND MORE

The authors thank Gilbert de Murcia, Valerie Kickhoefer and Susan Smith for very helpful comments on the manuscript. The authors are supported by research grants from the National Institutes of Health (CA43894, CA65579, NS38496) and Niadyne, Inc. (The authors are principals in Niadyne Inc., whose sponsored research is managed in accordance with University of Kentucky conflict-of-interest policies.)

Oral niacin prevents photocarcinogenesis and photoimmunosuppression in mice

Topical nicotinamide (niacinamide) has demonstrable preventive activity against photocarcinogenesis in mice. To better understand how this vitamin prevents ultraviolet (UV) carcinogenesis, we tested systemic administration of another form of the vitamin, niacin, and its capacity to elevate cutaneous nicotinamide-adenine dinucleotide (NAD) content as well as to decrease photoimmunosuppression and photocarcinogenesis. BALB/cAnNTacfBR mice were fed the AIN-76A diet supplemented with 0%, 0.1%, 0.5%, or 1.0% niacin throughout the experiment. UV irradiation consisted of five 30-minute exposures per week to banks of six FS40 Westinghouse sunlamps for 22 weeks in the carcinogenesis experiments, yielding a total cumulative dose of approximately 1.41 x 10(6) Jm-2 of UV-B radiation. Dietary supplementation with 0.1%, 0.5%, or 1.0% niacin reduced the control incidence of skin cancer from 68% to 60%, 48%, and 28%, respectively, at 26.5 weeks after the first UV treatment. Two potential mechanisms by which niacin prevents tumor formation were identified. Photoimmunosuppression, critical for photocarcinogenesis, is measured by a passive transfer assay. Syngeneic, antigenic tumor challenges grew to an average of 91.6 +/- 19.7, 79.8 +/- 11.5, 41.9 +/- 11.7, or 13.2 +/- 4.1 mm2 in naive recipients of splenocytes from UV-irradiated mice treated with 0%, 0.1%, 0.5%, or 1.0% niacin supplementation, respectively, demonstrating niacin prevention of immunosuppression. Niacin supplementation elevated skin NAD content, which is known to modulate the function of DNA strand scission surveillance proteins p53 and poly(ADP-ribose) polymerase, two proteins critical in cellular responses to UV-induced DNA damage. These results clearly demonstrate a dose-dependent preventive effect of oral niacin on photocarcinogenesis and photoimmunosuppression and establish the capacity of oral niacin to elevate skin NAD levels.

Tissue NAD as a biochemical measure of niacin status in humans.

Publisher Summary The known biological roles of niacin are attributable to the function of its active metabolites—nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). In humans, niacin equivalents can be obtained from dietary nicotinate, nicotinamide, and tryptophan. Consequently, niacin status depends on the amount of these in the diet and on factors that influence uptake, distribution, efficiency of conversion to tissue NAD and NADP, and excretion or reutilization of the nicotinamide moiety formed by the turnover of NAD and NADP. Niacin number is chosen as a convenient way to represent niacin status and is defined as NAD/NADP × 100. Expressing the values in this way yields a whole number that is linearly related to intracellular NAD content. Niacin status derived from erythrocytes or whole blood from humans varies over a wide range. The wide range of NAD content is of interest with regard to new questions concerning optimal amounts of dietary niacin raised by the involvement of NAD in ADP-ribose transfer reactions.

Glycation of proteins by ADP-ribose

Numerous metabolic pathways generate free ADP-ribose at many locations within cells. The metabolic fates of this nucleotide are poorly understood and measurement of itin situ is technically difficult at present. Yet considerable evidence has accumulated implicating that protein glycation by ADP-ribose can occur. This evidence is reviewed here along with recent developments in characterizing the chemistry of this reaction and the application of this information to the identification of this posttranslational modification in proteinin situ.

[50] Determination of in Vivo levels of polymeric and monomeric ADP-ribose by fluorescence methods

Publisher Summary This chapter describes the determination of in vivo levels of polymeric and monomeric ADP-ribose by fluorescence methods. Two key features that are common to both methods and are crucial in providing the necessary selectivity and sensitivity include the utilization of immobilized boronate resins to selectively and quantitatively adsorb polymeric or monomeric ADP-ribose from cell or tissue extracts and the conversion of adenine-containing compounds to highly fluorescent 1, N 6 -etheno derivatives which can be quantified at the picomole level. For polymeric ADP-ribose, the adenine-containing compounds are formed by the enzymatic hydrolysis of the polymer to generate unique adenosine derivatives from all internal residues. For monomeric ADP-ribose, the method involves chemical release of intact ADP-ribose residues from protein and quantification following conversion to the 1, N 6 -etheno(ADP-ribose). The assay for measurement of polymeric ADP-ribose is designed for up to 10 8 tissue culture cells or for up to 1.3 g (wet weight) of tissue. A real difficulty with regard to the quantification of monomeric ADP-ribose residues covalently bound to proteins is the limited knowledge of the chemical nature of the linkages that exists in vivo . Enzymes from eukaryotic sources have been purified that can catalyze the covalent attachment of single ADP-ribosyl residues to acceptor proteins via N-glycosylic linkages to the guanidino group of arginine residues.

ADP-Ribose in Glycation and Glycoxidation Reactions

Glycation is initiated by reaction of a reducing sugar with a protein amino group to generate a Schiff base adduct. Following an Amadori rearrangement to form a ketoamine adduct, a complex chemistry involving oxidation often leads to protein glycoxidation products referred to as advanced glycosylation end products (AGE). The AGE include protein carboxymethyllysine (CML) residues and a heterogeneous group of complex modifications characterized by high fluorescence and protein-protein cross links. The sugar sources for the glycoxidation of intracellular proteins are not well defined but pentoses have been implicated because they are efficient precursors for the formation of the fluorescent AGE, pentosidine. ADP-ribose, generated from NAD by ADP-ribose transfer reactions, is a likely intracellular source of a reducing pentose moiety. Incubation of ADP-ribose with histones results in the formation of ketoamine glycation conjugates and also leads to the rapid formation of protein CML residues, histone H1 dimers, and highly fluorescent products with properties similar to the AGE. ADP-ribose is much more efficient than other possible pentose donors for glycation and glycoxidation of protein amino groups. Recently developed methods that differentiate nonenzymic modifications of proteins by ADP-ribose from enzymic modifications now allow investigations to establish whether some protein modifications by monomers of ADP-ribose in vivo represent glycation and glycoxidation.

Molecular heterogeneity and regulation of poly(ADP-ribose) glycohydrolase

We have recently described the isolation and characterization of bovine cDNA encoding poly(ADP-ribose) glycohydrolase (PARG). We describe here the preparation and characterization of antibodies to PARG. These antibodies have been used to demonstrate the presence of multiple forms of PARG in tissue and cell extracts from bovine, rat, mouse, and insects. Our results indicate that multiple forms of PARG previously reported could result from a single gene. Analysis of PARG in cells in which poly(ADP-ribose) polymerase (PARP) has been genetically inactivated indicates that the cellular content of PARG is regulated independently of PARP.

NAD in skin: therapeutic approaches for niacin.

The maintenance and regulation of cellular NAD(P)(H) content and its influence on cell function involves many metabolic pathways, some of which remain poorly understood. Niacin deficiency in humans, which leads to low NAD status, causes sun sensitivity in skin, indicative of deficiencies in responding to UV damage. Animal models of niacin deficiency demonstrate genomic instability and increased cancer development in sensitive tissues including skin. Cell culture models of niacin deficiency have allowed the identification of NAD-dependent signaling events critical in early skin carcinogenesis. Niacin restriction in immortalized keratinocytes leads to an increased expression and activity of NADPH oxidase resulting in an accumulation of ROS, providing a potential survival mechanism as has been shown to occur in cancer cells. Niacin deficient keratinocytes are more sensitive to photodamage, as both poly(ADP-ribose) polymerases and Sirtuins are inhibited by the unavailability of their substrate, NAD+, leading to unrepaired DNA damage upon photodamage and a subsequent increase in cell death. Furthermore, the identification of the nicotinic acid receptor in human skin keratinocytes provides a further link to niacins role as a potential skin cancer prevention agent and suggests the nicotinic acid receptor as a potential target for skin cancer prevention agents. The new roles for niacin as a modulator of differentiation and photo-immune suppression and niacin status as a critical resistance factor for UV damaged skin cells are reviewed here.

Modulation of VP-16 cytotoxicity by carboplatin and 41.8°C hyperthermia

Purpose: To study in vitro the effect of carboplatin and/or hyperthermia in relation to etoposide (VP-16) cytotoxicity in L929 cells. Methodology/results: Cell survival assays demonstrated that the addition of 41.8 °C (×60 min) hyperthermia and carboplatin to VP-16 produced an antagonistic effect relative to VP-16 cytotoxicity in L929 cells; administering carboplatin and hyperthermia 24 h before VP-16 reduced this drug resistance; administering carboplatin and hyperthermia 48 h before VP-16, however, produced a supra-additive cytotoxicity. In order to gain insight into the molecular basis for these observations, we investigated the effect of hyperthermia and/or carboplatin on the stress protein GRP78, which is known to affect VP-16 cytotoxicity. Results obtained were consistent with the hypothesis that carboplatin and hyperthermia perturbation of NAD+ pools results in down-regulation of GRP78 with subsequent modulation of VP-16 cytotoxicity. To further explicate these results we studied G-361 as a control cell line that had significantly higher pretreatment NAD+ levels, which were not affected by carboplatin and/or hyperthermia. This cell line did not exhibit a down-regulation of GRP78 or modulation of VP-16 cytotoxicity as a function of carboplatin and hyperthermia. Conclusions: These data taken collectively, demonstrate a sequence effect (regarding the aforementioned antineoplastic agents), and provide a framework for future studies directed at the therapeutic optimization of the sequential application of carboplatin, hyperthermia, and VP-16.