Poly ADP-ribose Polymerase mRNA Research Articles

Cytocidal Antitumor Effects against Human Ovarian Cancer Cells Induced by B-Lactam Steroid Alkylators with Targeted Activity against Poly (ADP-Ribose) Polymerase (PARP) Enzymes in a Cell-Free Assay.

We evaluated three newly synthesized B-lactam hybrid homo-aza-steroidal alkylators (ASA-A, ASA-B and ASA-C) for their PARP1/2 inhibition activity and their DNA damaging effect against human ovarian carcinoma cells. These agents are conjugated with an alkylating component (POPA), which also served as a reference molecule (positive control), and were tested against four human ovarian cell lines in vitro (UWB1.289 + BRCA1, UWB1.289, SKOV-3 and OVCAR-3). The studied compounds were thereafter compared to 3-AB, a known PARP inhibitor, as well as to Olaparib, a standard third-generation PARP inhibitor, on a PARP assay investigating their inhibitory potential. Finally, a PARP1 and PARP2 mRNA expression analysis by qRT-PCR was produced in order to measure the absolute and the relative gene expression (in mRNA transcripts) between treated and untreated cells. All the investigated hybrid steroid alkylators and POPA decreased in vitro cell growth differentially, according to the sensitivity and different gene characteristics of each cell line, while ASA-A and ASA-B presented the most significant anticancer activity. Both these compounds induced PARP1/2 enzyme inhibition, DNA damage (alkylation) and upregulation of PARP mRNA expression, for all tested cell lines. However, ASA-C underperformed on average in the above tasks, while the compound ASA-B induced synthetic lethality effects on the ovarian cancer cells. Nevertheless, the overall outcome, leading to a drug-like potential, provides strong evidence toward further evaluation.

PARP activity and inhibition in fetal and adult oligodendrocyte precursor cells: Effect on cell survival and differentiation

Poly (ADP-ribose) polymerase (PARP) family members are ubiquitously expressed and play a key role in cellular processes, including DNA repair and cell death/survival balance. Accordingly, PARP inhibition is an emerging pharmacological strategy for cancer and neurodegenerative diseases. Consistent evidences support the critical involvement of PARP family members in cell differentiation and phenotype maturation. In this study we used an oligodendrocyte precursor cells (OPCs) enriched system derived from fetal and adult brain to investigate the role of PARP in OPCs proliferation, survival, and differentiation. The PARP inhibitors PJ34, TIQ-A and Olaparib were used as pharmacological tools. The main results of the study are: (i) PARP mRNA expression and PARP activity are much higher in fetal than in adult-derived OPCs; (ii) the culture treatment with PARP inhibitors is cytotoxic for OPCs derived from fetal, but not from adult, brain; (iii) PARP inhibition reduces cell number, according to the inhibitory potency of the compounds; (iv) PARP inhibition effect on fetal OPCs is a slow process; (v) PARP inhibition impairs OPCs maturation into myelinating OL in fetal, but not in adult cultures, according to the inhibitory potency of the compounds. These results have implications for PARP-inhibition therapies for diseases and lesions of the central nervous system, in particular for neonatal hypoxic/ischemic encephalopathy.

Neuropilin-1 modulates vascular endothelial growth factor-induced poly(ADP-ribose)-polymerase leading to reduced cerebrovascular apoptosis

Cerebral ischemia is encompassed by cerebrovascular apoptosis, yet the mechanisms behind apoptosis regulation are not fully understood. We previously demonstrated inhibition of endothelial apoptosis by vascular endothelial growth factor (VEGF) through upregulation of poly(ADP-ribose)-polymerase (PARP) expression. However, PARP overactivation through oxidative stress can lead to necrosis. This study tested the hypothesis that neuropilin-1 (NP-1), an alternative VEGF receptor, regulates the response to cerebral ischemia by modulating PARP expression and, in turn, apoptosis inhibition by VEGF. In endothelial cell culture, NP-1 colocalized with VEGF receptor-2 (VEGFR-2) and acted as its coreceptor. This significantly enhanced VEGF-induced PARP mRNA and protein expression demonstrated by receptor-specific inhibitors and VEGF-A isoforms. NP-1 augmented the inhibitory effect of VEGF/VEGFR-2 interaction on apoptosis induced by adhesion inhibition through the αV-integrin inhibitor cRGDfV. NP-1/VEGFR-2 signal transduction involved JNK and Akt. In rat models of permanent and temporary middle cerebral artery occlusion, the ischemic cerebral hemispheres displayed endothelial and neuronal apoptosis next to increased endothelial NP-1 and VEGFR-2 expression compared to non-ischemic cerebral hemispheres, sham-operated or untreated controls. Increased vascular superoxide dismutase-1 and catalase expression as well as decreased glycogen reserves indicated oxidative stress in the ischemic brain. Of note, protein levels of intact PARP remained stable despite pro-apoptotic conditions through increased PARP mRNA production during cerebral ischemia. In conclusion, NP-1 is upregulated in conditions of imminent cerebrovascular apoptosis to reinforce apoptosis inhibition and modulate VEGF-dependent PARP expression and activation. We propose that NP-1 is a key modulator of VEGF maintaining cerebrovascular integrity during ischemia. Modulating the function of NP-1 to target PARP could help to prevent cellular damage in cerebrovascular disease.

Apoptosis of U937 Cells Induced by Hematoporphyrin Monomethyl Ether-Mediated Sonodynamic Action

The present study aims to investigate apoptosis of U937 cells induced by hematoporphyrin monomethyl ether (HMME)-mediated sonodynamic therapy (SDT). HMME concentration was kept constant at 10 μg/mL. Tumor cells suspended in serum-free RPM1640 were exposed to ultrasound at 1.1 MHz for up to 60 seconds with an intensity of 1 W/cm(2) in the presence and absence of HMME. The viability of cells was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium (MTT) test. Apoptosis was analyzed using a flow cytometer with Annexin V-PE/7-ADD staining as well as fluorescence microscopy with 4'-6-diamidino-2-phenylindole (DAPI) staining. The DNA damage of U937 cells, intracellular reactive oxygen species (ROS), and mitochondria membrane potential (MMP) were also analyzed by a flow cytometer after exposures. Western blotting and reverse transcriptase-polymerase chain reaction were used to analyze the protein and mRNA expression level of caspase-3 and poly(ADP-ribose) polymerase (PARP). Fluorescent imaging revealed that HMME mainly localized in the mitochondria. MTT assay showed 55.6% of cell survival at 4 hours post-SDT. Flow cytometric analysis displayed a significant increase in the early- and late-apoptotic cell populations (35.6%) of U937 cells by HMME-mediated SDT. Compared with the control, ultrasound-alone, and HMME-alone groups, the intracellular ROS and the MMP loss were greatly increased in the combined SDT group. Obvious nuclear condensation was also found with DAPI staining, and the DNA fragment increased to 33.9% at 2 hours post-SDT treatment. Immunofluorescent staining indicated obvious Bax translocation after SDT. Western blot showed visible enhancement of caspase-3 and PARP cleavage. In addition, caspase-3 and PARP mRNA expression of U937 cells increased remarkably after SDT treatment. The findings demonstrated that HMME-mediated sonodynamic action (HMME-SDT) significantly induced apoptosis of U937 cells, suggesting that HMME may be a good sonosensitizer, and HMME-SDT might be a potential therapeutic strategy for cancer treatment.

The Thioredoxin System in Diabetic Nephropathy

32 Hyperhexosemia-Induced Vasoactive Factors and Extracellular Matrix Protein Production in Kidneys are Mediated via NFB. BRADLEY J. DE SOUZA, JANE CHIU, BIAO FENG, BIJU GEORGE AND SUBRATA CHAKRABARTI, Department of Pathology, University of Western Ontario, London, ON Increase in reactive oxygen species production, in diabetes, is critical in the initiation of the cellular responses by activating redox-sensitive transcription factor, nuclear factor kappa B (NFB). NFB activation may lead to cellular structural and functional alteration via altered expression of cytokines. We investigated the role of NFB in the development of early changes in diabetic nephropathy We used a hyperhexosemic, normoinsulinimic model of chronic diabetic complication to avoid potential problems associated with systemic insulin therapy needed for longterm diabetic animals. Hence, NFB(p50) knockout mice and their wild-type controls were fed with a 30% galactose diet for 2 months. Renal tissues were analyzed. Galactose-fed animals showed increased in serum reducing sugars, without any alterations of body weight. Wild type galactose-fed animals demonstrated increase in the mRNA expression of ET-1 and TGF1. Furthermore ECM proteins, regulated by ET-1 and TGF 1, such as fibronectin and collagen1 4 were upregulated. Interestingly, poly ADP ribose polymerase mRNA expression was not altered. Such alterations were prevented in the kidneys of galactose-fed knockout animals. These results demonstrate that early changes in diabetic nephropathy may be mediated through NFB-dependent signaling pathways. Understanding these mechanisms is important in identifying potential novel treatment targets. Supported by CDA

ERCC1 and XPF mediated upregulation of Fibronectin (FN) in Diabetes.

ERCC1 and XPF mediated upregulation of Fibronectin (FN) in Diabetes. BIJU GEORGE*, SHALI CHEN, BIAO FENG, JANE CHIU AND SUBRATA CHAKRABARTI. Dept. of Pathology, Univ. of Western Ontario, London, ON. Excision Repair Cross Complimenting1 (ERCC-1) and Xeroderma Pigmentosum group F (XPF) are important DNA repair enzymes. They play important roles in aging, glucose metabolism and oxidative stress and demonstrate interactive relationships with Poly (ADP-Ribose) polymerase (PARP), another DNA repair protein, upregulated in diabetes. We studied the role of ERCC1 and XPF in glucose induced FN production. Human Umbilical Vein endothelial cells were grown in low (5mM) and high glucose (25mM) containing media. They were also exposed to siRNA against ERCC1 and XPF, histone deacetylase blocker TSA, PARP blocker ABA and p300 blocker Curcumin. Quantitative RT-PCR were performed for ERCC1, XPF, FN, PARP and p300 mRNA. High glucose led to upregulation of ERCC1, XPF, FN, PAPR, p300 mRNA levels compared to the cells in low glucose. ERCC1 and XPF silencing reduced such upregulation. ABA and curcumin also reduced both basal and glucose induced ERCC1 and XPF expression. TSA however, only prevented glucose induced ERCC1 and XPF upregulation. Western blots of ERCC1 and XPF from the nuclear proteins showed similar changes. Data from this study indicated that glucose induced upregulation of ERCC1 and XPF may lead to increased FN production via PARP, P300 pathway. These novel mechanisms may play important roles in diabetic complications. These studies were supported in part by CDA and CIHR 116

Glucose-induced Alteration of Endothelial microRNA

ERCC1 and XPF mediated upregulation of Fibronectin (FN) in Diabetes. BIJU GEORGE*, SHALI CHEN, BIAO FENG, JANE CHIU AND SUBRATA CHAKRABARTI. Dept. of Pathology, Univ. of Western Ontario, London, ON. Excision Repair Cross Complimenting1 (ERCC-1) and Xeroderma Pigmentosum group F (XPF) are important DNA repair enzymes. They play important roles in aging, glucose metabolism and oxidative stress and demonstrate interactive relationships with Poly (ADP-Ribose) polymerase (PARP), another DNA repair protein, upregulated in diabetes. We studied the role of ERCC1 and XPF in glucose induced FN production. Human Umbilical Vein endothelial cells were grown in low (5mM) and high glucose (25mM) containing media. They were also exposed to siRNA against ERCC1 and XPF, histone deacetylase blocker TSA, PARP blocker ABA and p300 blocker Curcumin. Quantitative RT-PCR were performed for ERCC1, XPF, FN, PARP and p300 mRNA. High glucose led to upregulation of ERCC1, XPF, FN, PAPR, p300 mRNA levels compared to the cells in low glucose. ERCC1 and XPF silencing reduced such upregulation. ABA and curcumin also reduced both basal and glucose induced ERCC1 and XPF expression. TSA however, only prevented glucose induced ERCC1 and XPF upregulation. Western blots of ERCC1 and XPF from the nuclear proteins showed similar changes. Data from this study indicated that glucose induced upregulation of ERCC1 and XPF may lead to increased FN production via PARP, P300 pathway. These novel mechanisms may play important roles in diabetic complications. These studies were supported in part by CDA and CIHR 116

Oxidative DNA damage and augmentation of poly(ADP-ribose) polymerase/nuclear factor-kappa B signaling in patients with Type 2 diabetes and microangiopathy

Although oxidative stress and the subsequent DNA damage is one of the obligatory signals for poly(ADP-ribose) polymerase (PARP) activation and nuclear factor-kappa B (NFkappaB) alterations, these molecular aspects have not been collectively examined in epidemiological and clinical settings. Therefore, this study attempts to assess the oxidative DNA damage and its downstream effector signals in peripheral blood lymphocytes from Type 2 diabetes subjects without and with microangiopathy along with age-matched non-diabetic subjects. The basal DNA damage, lipid peroxidation and protein carbonyl content were significantly (p<0.05) higher in patients with and without microangiopathy compared to control subjects. Formamido Pyrimidine Glycosylase (FPG)-sensitive DNA strand breaks which represents reliable indicator of oxidative DNA damage were also significantly (p<0.001) higher in diabetic patients with (19.41+/-2.5) and without microangiopathy (16.53+/-2.0) compared to control subjects (1.38+/-0.85). Oxidative DNA damage was significantly correlated to poor glycemic control. PARP mRNA expression and PARP activity were significantly (p<0.05) increased in cells from diabetic patients with (0.31+/-0.03 densitometry units; 0.22+/-0.02PARPunits/mgprotein, respectively) and without (0.35+/-0.02; 0.42+/-0.05) microangiopathy compared to control (0.19+/-0.02; 0.11+/-0.02) subjects. Diabetic subjects with and without microangiopathy exhibited a significantly (p<0.05) higher (80%) NFkappaB binding activity compared to control subjects. In diabetic patients, FPG-sensitive DNA strand breaks correlated positively with PARP gene expression, PARP activity and NFkappaB binding activity. This study provides a comprehensive molecular evidence for increased oxidative stress and genomic instability in Type 2 diabetic subjects even prior to vascular pathology and hence reveals a window of opportunity for early therapeutic intervention.

Protection of hepatocytes from apoptosis by a novel substance from actinomycetes culture medium

A novel substance, #675, found from an Streptomyces sp. SM675 culture medium, dose-dependently stimulates the proliferation of human functional liver cell 4 (FLC4). When FLC4 cells were incubated under conditions without fetal bovine serum (FBS), typical features of apoptotic cell death such as shrinkage and nuclear condensation appeared; high molecular weight (HMW) DNA fragments were found; and caspase-3 and poly (ADP-ribose) polymerase (PARP) proteins were cleaved. When FLC4 cells were incubated with #675 and without FBS, the cells grew healthy, no HMW DNA fragments were found, and caspase-3 and PARP cleavage weakened, suggesting that #675 protects FLC4 cells from apoptosis induced by FBS-deprivation. The quantitative reverse-transcribed polymerase chain reaction did not show differences in PARP or Bcl-2 mRNA expression in FLC4 cells incubated with or without #675, indicating other genes may be involved in this anti-apoptosis effect. These results show that #675 enhances FLC4 proliferation via an apoptosis-inhibition pathway, implying potential pharmacological and clinical applications.

Developmental changes of poly(ADP-ribose) polymerase expression in &lt;i&gt;Sarcophaga peregrina&lt;/i&gt;

To elucidate the physiological role of poly(ADP-ribose) polymerase (PARP), we studied the levels of PARP mRNA and protein during the developmental stages of Sarcophaga peregrina. PARP mRNA expression changed remarkably throughout the developmental stages. The level of PARP mRNA (the molecular ratio of PARP mRNA to the total RNA) was highest in unfertilized eggs and that of PARP protein (the molecular ratio of PARP protein to the total protein of the crude extract) was high in unfertilized and fertilized eggs and in 1st instar larvae. During the embryogenesis period, the levels of PARP mRNA and protein gradually decreased. The levels of PARP mRNA during larval and pupal periods became less than about 5% of that in unfertilized eggs. After the emergence of adult flies, the levels of PARP mRNA and protein increased both in female and male flies. PARP activity normalized with the total amount of protein in the crude extract changed in parallel to the level of PARP protein throughout the developmental stages. The biological significance of the drastic change of mRNA and protein levels of PARP still remains to be clarified. (Contributed by Takashi SUGIMURA, M.J.A.)

Overexpression of Poly(ADP-ribose) Polymerase Disrupts Organization of Cytoskeletal F-actin and Tissue Polarity inDrosophila

Poly(ADP-ribose) polymerase (PARP) may play important roles in nuclear events such as cell cycle, cell proliferation, and maintenance of chromosomal stability. However, the exact biological role played by PARP or how PARP is involved in these cellular functions is still unclear. To elucidate the biological functions of PARP in vivo, we have constructed transgenic flies that overexpress Drosophila PARP in the developing eye primordia. These flies showed mild roughening of the normally smooth ommatidial lattice and tissue polarity disruption caused by improper rotation and chirality of the ommatidia. To clarify how this phenotypical change was induced, here we analyzed transgenic flies overexpressing PARP in the developing eye, embryo, and adult in detail. PARP mRNA level and the phenotype were enhanced in flies carrying more copies of the transgene. Developing eyes from third instar larvae were analyzed by using the neural cell marker to examine the involvement of PARP in cell fate. Morphological disorder of non-neuronal accessory cells was observed in PARP transgenic flies. Interestingly, overexpression of PARP did not interfere with the cell cycle or apoptosis, but it did disrupt the organization of cytoskeletal F-actin, resulting in aberrant cell and tissue morphology. Furthermore, heat-induced PARP expression disrupted organization of cytoskeletal F-actin in embryos and tissue polarity in adult flies. Because these phenotypes closely resembled mutants or transgenic flies of the tissue polarity genes, genetic interaction of PARP with known tissue polarity genes was examined. Transgenic flies expressing either PARP or RhoA GTPase in the eye were crossed, and co-expression of PARP suppressed the effect of RhoA GTPase. Our results indicate that PARP may play a role in cytoskeletal or cytoplasmic events in developmental processes of Drosophila.

Genetic and functional analysis of PARP, a DNA strand break-binding enzyme

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by binding to a single- or double-strand break of DNA and is one of the death substrates for caspase-3 in apoptosis. The nuclear function of PARP is well studied and recent PARP-knockout studies indicate that PARP takes part in chromosomal stability. To analyze the effect of PARP overexpression, or loss of function, we have cloned PARP cDNA and the gene from Drosophila melanogaster and studied its function in developmental stages. Organization of exons corresponds to the functional domains of PARP. An alternatively spliced form of PARP lacking exon 5, which encodes the auto-modification domain, is found in Drosophila. Expression of the PARP gene is at high levels in embryos at 0-6h after egg laying and gradually decreased. In situ mRNA hybridization indicates localization of PARP mRNA in cells along the central nervous system at a late stage of embryogenesis. Overexpression of the gene in the developing eye primordia of D. melanogaster is an excellent experimental model to analyze the cell cycle and programmed cell death. We introduced PARP expression vector overexpresses PARP in the eye discs of Drosophila, and established the PARP transgenic flies by P element-mediated germ line transformation. These flies showed mild roughening of the normally smooth ommatidial lattice involving tissue polarity disruption characterized by missrotation and incorrect chirality of ommatidia. Possible mechanisms of involvement of PARP in the development are discussed.

Effects of transient global ischemia and kainate on poly(ADP-ribose) polymerase (PARP) gene expression and proteolytic cleavage in gerbil and rat brains

Poly (ADP-ribose) polymerase (PARP) is involved in various cellular functions, including DNA repair, the cell cycle and cell death. While PARP activation could play a critical role in repairing ischemic brain damage, PARP inactivation caused by caspase 3-cleavage may also be important for apoptotic execution. In this study we investigated the effects of transient global ischemia and kainic acid (KA) neurotoxicity, in gerbil and rat brains, respectively, on PARP gene expression and protein cleavage. PARP mRNA increased in the dentate gyrus of gerbil brains 4 h after 10 min of global ischemia, which returned to basal levels 8 h after ischemia. KA injection (10 mg/kg) also induced a marked elevation in PARP mRNA level selectively in the dentate gyrus of rat brains 1 h following the injection, which returned to basal levels 4 h after the injection. These observations provide the first evidence of altered PARP gene expression in brains subjected to ischemic and excitotoxic insults. Using both monoclonal and polyclonal antibodies to PARP cleavage products, little evidence of significant PARP cleavage was found in gerbil brains within the first 3 days after 10 min of global ischemia. In addition, there was little evidence of significant PARP cleavage in rat brains within 2 days after kainate (KA) injection. Though these findings show that caspase induced PARP cleavage is not substantially activated by global ischemia and excitotoxicity in whole brain, the PARP mRNA induction could suggest a role for PARP in repairing DNA following brain injury.

Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia.

We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP-ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37-fold at 1 h (p < 0.05 vs. sham) and 1.73-fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly(ADP-ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3-aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single-strand breaks assessed as DNA polymerase I-mediated biotin-dATP nick-translation (PANT)-positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3-aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain.

Correlation of poly(ADP-ribose)polymerase and p53 expression levels in high-grade lymphomas.

The steady-state levels of mRNA for the poly(ADP-ribose)polymerase (PARP), c-myc, p53, and histone H3 genes were investigated in 31 high-grade B-cell lymphomas by northern blot analysis. The panel included 15 nodal large B-cell lymphomas, nine mediastinal large B-cell lymphomas, and seven sporadic Burkitt's lymphomas. The PARP mRNA level was significantly higher in lymphomas than in control tissues and corresponded with the amount of PARP protein, as assessed by immunoblot analysis in six samples. The level of PARP mRNA was positively correlated with that of p53 mRNA. No correlation was found between the mRNA expression levels of PARP and histone H3, suggesting that PARP expression levels are independent of the proliferation rate of neoplastic cells. In this setting, the strong correlation between PARP and p53 suggests that the high expression of PARP may be associated with ongoing DNA damage in high-grade lymphomas.

Engineered Human Skin Model Using Poly(ADP-Ribose) Polymerase Antisense Expression Shows a Reduced Response to DNA Damage

Poly(ADP-ribose) polymerase (PADPRP) modifies nuclear proteins in response to DNA-damaging agents. The principal organ subject to exposure to many of these agents is the skin. To understand the role of PADPRP in the maintenance of the epidermis, a model system has been developed in which we have selectively lowered the levels of this enzyme by the use of induced expression of antisense RNA. Human keratinocyte lines were stably transfected with the cDNA for human PADPRP in the antisense orientation under an inducible promoter. Induction of this antisense RNA in cultured cells selectively lowers the levels of PADPRP mRNA, protein, and enzyme activity. Induction of antisense RNA also led to a reduction in the levels of PADPRP in individual cell nuclei, as well as the loss of the ability of cells to synthesize and modify proteins by poly(ADP-ribose) polymer in response to DNA damage. When keratinocyte clones containing the antisense construct or empty vector alone were grafted onto nude mice, they formed histologically normal human skin. The PADPRP antisense construct was also inducible in vivo by the topical application of dexamethasone to the reconstituted epidermis. In addition, poly(ADP-ribose) polymer could be induced and detected in vivo following the topical application of a DNA alkylating agent to the grafted transfected skin layers. Accordingly, a model system has been developed in which the levels of PADPRP can be selectively manipulated in human keratinocytes in cell culture, and potentially in reconstituted epidermis as well. This system will be a useful tool to study the role of PADPRP and DNA repair in general in essential biologic processes in the epidermis.

Model systems for the study of the role of PADPRP in essential biological processes

The nuclear enzyme poly(ADP-ribose) polymerase (PADPRP) is implicated in a number of cellular processes, including DNA repair, replication, and differentiation. We have been using several model systems to examine these potential roles of PADPRP. A human keratinocyte model system has been developed in which stable lines of epidermal cells contain an inducible construct harboring the antisense cDNA to PADPRP. When PADPRP antisense RNA is induced in culture, endogenous PADPRP mRNA, protein, and enzymatic activity is lowered, and the pattern of poly(ADP) ribosylation in response to alkylating agents is altered. When keratinocyte clones containing the antisense construct or empty vector alone were grafted onto nude mice, they formed histologically normal human skin. The PADPRP antisense construct was also inducible in vivo by the topical application of dexamethasone to the reconstituted epidermis, as determined by in situ hybridization. In addition, poly(ADP-ribose) polymer could be induced and detected in vivo following the topical application of a DNA alkylating agent to the grafted transfected skin layers. Thus, a model system has been developed in which the levels of PADPRP can be selectively manipulated in human keratinocytes in cell culture, and potentially in reconstituted epidermis as these keratinocyte lines can be grafted to nude mice, whereupon they form a histologically and immunocytochemically normal human epidermis. Another system that we have been utilizing to examine the role of PADPRP in proliferation and differentiation is stable lines of mouse preadipocytes that contain an inducible antisense PADPRP RNA. Similar to the keratinocyte system, these cells can inducibly express antisense PADPRP RNA, and subsequently lower levels of endogenous PADPRP.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of transfection of human poly(ADP-ribose) polymerase in Chinese hamster cells on mutagen resistance

Poly(ADP-ribose)polymerase (PARP) is a DNA-binding protein that is activated upon induction of DNA breaks and supposed to play a role in DNA repair. To elucidate the effect of overexpression of PARP on the resistance of cells to mutagens, Chinese hamster ovary cells (both the line CHO-9 and the mutagen-hypersensitive derivative 27-1) were transfected with the human PARP cDNA along with pSV2neo. Treatment of the transfected cell population with a high dose of MNNG and selection with G418 gave rise to a significant increase of neo+ clones, as compared to the control transfection with pSV2neo + salmon sperm DNA. The frequency of survivors in these mass culture experiments was lower, however, than after transfection with the bacterial ada gene encoding the DNA repair protein O6-alkylguanine-DNA alkyltransferase. Thus transfection of PARP cDNA in CHO cells is only weakly effective in inducing alkylation resistance. This was confirmed by analyzing the mutagen resistance of individual PARP transfectant clones derived from CHO-9 and 27-1 cells that expressed increased levels of PARP mRNA, protein and PARP activity. These strains were slightly more resistant to the toxic effect of MMS and showed a reduced frequency of MMS-induced chromosomal aberrations. CHO-9-PARP transfectants also gained resistance to UV. From these data we conclude that, in CHO cells, PARP is limiting in handling critical lesions during the repair process and that increase of the amount of PARP protein can elicit some protection against genotoxic effects of mutagens.

Expression of recombinant placental prolactin-like protein B in mammalian cells

Poly(ADP-ribose)polymerase (PARP) is a DNA-binding protein that is activated upon induction of DNA breaks and supposed to play a role in DNA repair. To elucidate the effect of overexpression of PARP on the resistance of cells to mutagens, Chinese hamster ovary cells (both the line CHO-9 and the mutagen-hypersensitive derivative 27-1) were transfected with the human PARP cDNA along with pSV2neo. Treatment of the transfected cell population with a high dose of MNNG and selection with G418 gave rise to a significant increase of neo+ clones, as compared to the control transfection with pSV2neo + salmon sperm DNA. The frequency of survivors in these mass culture experiments was lower, however, than after transfection with the bacterial ada gene encoding the DNA repair protein O6-alkylguanine-DNA alkyltransferase. Thus transfection of PARP cDNA in CHO cells is only weakly effective in inducing alkylation resistance. This was confirmed by analyzing the mutagen resistance of individual PARP transfectant clones derived from CHO-9 and 27-1 cells that expressed increased levels of PARP mRNA, protein and PARP activity. These strains were slightly more resistant to the toxic effect of MMS and showed a reduced frequency of MMS-induced chromosomal aberrations. CHO-9-PARP transfectants also gained resistance to UV. From these data we conclude that, in CHO cells, PARP is limiting in handling critical lesions during the repair process and that increase of the amount of PARP protein can elicit some protection against genetoxic effects of mutagens.

Isolation of cDNAs Encoding the Catalytic Domain of Poly(ADP-Ribose) Polymerase from Xenopus laevis and Cherry Salmon Using Heterologous Oligonucleotide Consensus Sequences

We have isolated and sequenced cDNAs encoding the catalytic domain of poly(ADP-ribose) polymerase (PARP) from Xenopus laevis and Oncorhyncus masou (cherry salmon). The cDNAs were amplified by polymerase chain reaction using heterologous oligonucleotides corresponding to the conserved sequences of mammalian cDNAs as primers. The deduced amino acid sequences of Xenopus laevis and cherry salmon cDNA showed 84.4% and 75.6% similarities to that of human PARP, respectively. In both species, mRNA for PARP was identified as a single band of 4 kb, and PARP mRNA was abundant in ovary and brain. Thus, mixed oligonucleotide-primed amplification is a useful method in the cloning of cDNAs from different species, and the catalytic domain of PARP is conserved structurally among phylogenetically different species, suggesting an importance of poly(ADP-ribosyl)ation.