Expression Levels Of Poly ADP-ribose Polymerase Research Articles

Pravastatin Administration Alleviates Kanamycin-Induced Cochlear Injury and Hearing Loss.

The effect of statins on aminoglycoside-induced ototoxicity is controversial. This study aimed to explore the role of pravastatin (PV) in kanamycin-induced hearing loss in rats. Adult rats were intraperitoneally treated with 20 mg/kg/day of kanamycin (KM) for 10 days. In the PV- and PV + KM-treated rats, 25 mg/kg/day of PV was intraperitoneally administered for 5 days. The auditory brainstem response (ABR) thresholds were measured before and after drug treatment using a smartEP system at 4, 8, 16, and 32 kHz. Cochlear changes in poly ADP-ribose (PAR) polymerase (PARP), PAR, and caspase 3 were estimated using Western blotting. PV administration did not increase the ABR thresholds. The KM-treated rats showed elevated ABR thresholds at 4, 8, 16, and 32 kHz. The PV + KM-treated rats demonstrated lower ABR thresholds than the KM-treated rats at 4, 8, and 16 kHz. The cochlear outer hair cells and spiral ganglion cells were relatively preserved in the PV + KM-treated rats when compared with that in the KM-treated rats. The cochlear expression levels of PARP, PAR, and caspase 3 were higher in the KM-treated rats. The PV + KM-treated rats showed lower levels of PARP, PAR, and caspase 3 than the KM-treated rats. PV protected cochleae from KM-induced hearing loss in rats. The regulation of autophagy and apoptosis mediated the otoprotective effects of PV.

Chlorogenic acid alleviates neurobehavioral disorders and brain damage in focal ischemia animal models

Cerebral ischemia leads to neuronal cell death, causes neurological disorder and permanent disability. Chlorogenic acid has antioxidant, anti-inflammatory, and anti-apoptotic properties. This study investigated the neuroprotective effects of chlorogenic acid against cerebral ischemia. Focal cerebral ischemia was induced in male adult rats via middle cerebral artery occlusion (MCAO). Chlorogenic acid (30 mg/kg) or vehicle was injected in the intraperitoneal cavity 2 h after MCAO operation. Neurological behavior tests were performed 24 h after MCAO, brain edema and infarction were measured. Oxidative stress was assessed by investigating the levels of reactive oxygen species (ROS) and lipid peroxidation (LPO) levels. MCAO damage leaded to severe neurobehavioral deficits, increased ROS and LPO levels, and induced brain edema and infarction. MCAO damage caused histopathological damages and increased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the cerebral cortex. However, chlorogenic acid treatment improved neurological behavioral deficits caused by MCAO and attenuated the increase in ROS and LPO levels. It also alleviated MCAO-induced brain edema, infarction, and histopathological lesion. Chlorogenic acid treatment attenuated the increase in the number of TUNEL-positive cells in the cerebral cortex of MCAO animals. We also investigated caspase proteins expression to elucidate the neuroprotective mechanism of chlorogenic acid. Caspase-3, caspase-7, and poly ADP-ribose polymerase expression levels were increased in the MCAO damaged cortex, while chlorogenic acid mitigated these increases. These results showed that MCAO injury leads to severe neurological damages and chlorogenic acid exerts neuroprotective effects by regulating oxidative stress and caspase proteins expressions. Thus, our findings suggest that chlorogenic acid acts as a potent neuroprotective agent by modulating the apoptotic-related proteins.

Abstract 291: Functionalizing novel modulators in lung cancer

Abstract Lung cancer is the leading cause of cancer mortality globally, with lung adenocarcinoma being the most prevalent subtype. The discovery of driver mutations has paved the way for the use of targeted therapy, notably EGFR inhibitors, in the management of advanced disease. However, the effectiveness of targeted therapy is limited firstly by the eventual development of treatment resistance, and secondly by the sizeable percentage of cases in which the driver mutations remain unknown. Furthermore, lung adenocarcinoma (LUAD) in Asians is known to exhibit a molecular profile distinct from that in Caucasians, yet the majority of existing cohort studies have been focused on the latter. To address this gap, we and our collaborators have spearheaded the largest Asian LUAD cohort study, comprising genomic and transcriptomic data from 305 patients. The MutSigCV and 20/20+ driver prediction algorithms were applied to whole exome sequencing data from our cohort and yielded seven novel candidate drivers that had not been previously implicated in LUAD. We proceeded to functionally validate these drivers by overexpressing or knocking them down in patient-derived transformed lung cell lines and observing their effects on tumorigenicity via a number of in vitro and in vivo tumorigenicity assays. We have identified a poly (ADP-ribose) polymerase (PARP) gene, which was mutated at a relatively high frequency within our cohort, and show that PARP is crucial for modulating the tumorigenicity of lung cancer cells. In LUAD patients, reduced PARP expression levels were correlated with poorer prognosis, suggesting a putative tumor suppressor role. We further observed a loss of PARP copy number both within our cohort as well as the Caucasian The Cancer Genome Atlas (TCGA) LUAD cohort. We are now working to dissect the underlying mechanisms and explore PARP's clinical utility. This work illustrates the use of a platform to systematically model genetics in patient-derived models to study novel driver functions. Citation Format: Yi Fei Lee, Ju Yuan, Edwin Lim, Jianbin Chen, Weiwei Zhai, Nguan Soon Tan, Wai Leong Tam. Functionalizing novel modulators in lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 291.

Epigallocatechin gallate alleviates neuronal cell damage against focal cerebral ischemia in rats.

Cerebral ischemia is a neurological disorder that causes permanent disability and is sometimes fatal. Epigallocatechin gallate (EGCG) is a natural polyphenol that exerts beneficial antioxidant and anti-inflammatory effects. The aim of this study was to investigate the neuroprotective effects of EGCG against cerebral ischemia. Middle cerebral artery occlusion was surgically initiated to induce focal cerebral ischemia in adult male rats. EGCG (50 mg/kg) or vehicle was intraperitoneally injected just prior to middle cerebral artery occlusion (MCAO) induction. Neuronal behavior tests were performed 24 hr after MCAO. Brain tissues were isolated to evaluate infarct volume, histological changes, apoptotic cell death, and caspase-3 and poly ADP-ribose polymerase (PARP) levels. MCAO injury led to serious functional neurological deficits and increased infarct volume. Moreover, it induced histopathological lesions and increased the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the cerebral cortex. However, EGCG improved MCAO-induced neurological deficits and reduced infarct volume, alleviated histopathological changes, and decreased TUNEL-positive cells in the cerebral cortex of MCAO rats. Western blot analysis showed increases of caspase-3 and PARP expression levels in MCAO rats with vehicle, whereas EGCG administration alleviated these increases after MCAO injury. These results demonstrate that EGCG exerts a neuroprotective effect by regulating caspase-3 and PARP proteins during cerebral ischemia. In conclusion, we suggest that EGCG acts as a potent neuroprotective agent by modulating the apoptotic signaling pathway.

Breviscapine Induces Breast Cancer Cell Cycle Arrest and Apoptosis by Modulating the Jak-Stat Pathway

The aim of this study was to investigate the effects and possible mechanisms of breviscapine arrest in the development of breast cancer cells. Cell viability, cycle arrest, and apoptosis were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoliumbromide assay and flow cytometry in MCF-7 cells treated with increasing concentrations of breviscapine. In addition, the expression level of cyclin D1, p21, cleaved caspase-3, and poly ADP-ribose polymerase, p-Janus kinase 2 and p-signal transducers and activators of transcription 3 was analyzed using western blot assay. The results showed that breviscapine suppressed cell viability and induced apoptosis in MCF-7 cells in a dose-dependent fashion. Breviscapine also increased MCF-7 cell number in the G1 phase and decreased that in the S phase. Moreover, cyclin D1, p-JAK2/JAK2, and p-STAT3/STAT3 expression levels were down-regulated, while p21, cleaved caspase-3, and poly ADP-ribose polymerase expression levels were up-regulated in breviscapine-treated MCF-7 cells. In conclusion, breviscapine regulates breast cancer viability, cell cycle arrest, and apoptosis in a concentration-dependent manner by modulating the JAK-STAT pathway.

Tanshinone IIA inhibits proliferation and induces apoptosis of human nasopharyngeal carcinoma cells via p53‑cyclin B1/CDC2

Tanshinone IIA exhibits natural antioxidative and antineoplastic activity. However, to the best of our knowledge, the effects of tanshinone IIA on human nasopharyngeal carcinoma cells remains unknown. The present study aimed to investigate whether tanshinone IIA inhibits proliferation and induces apoptosis of human nasopharyngeal carcinoma cells via p53-cyclin B1/cell division cycle gene 2 (CDC2). Cell proliferation, cytotoxicity and apoptosis of 13-9B cells were evaluated by an MTT assay, lactate dehydrogenase assay and flow cytometry, respectively. ELISA and western blot analysis were used to analyze caspase-3 activity and poly (ADP-ribose) polymerase (PARP), p53, cyclin B1 and CDC2 protein expression in 13-9B cells. Treatment of 13-9B cells with tanshinone IIA significantly suppressed cell proliferation and significantly induced cytotoxicity and apoptosis of 13-9B cells. Furthermore, tanshinone IIA significantly increased caspase-3 activity, and significantly increased the protein expression levels of PARP, p53, cyclin B1 and CDC2 in 13-9B cells. In summary, the current results indicate that tanshinone IIA inhibits proliferation and induces apoptosis of human nasopharyngeal carcinoma cells via PARP, p53, cyclin B1/CDC2 and caspase-3-mediated signaling.

Abstract 3816: Corilagin could help to overcome PARP inhibitor resistance by inhibiting ERK signaling pathways

Abstract We have identified that Corilagin is a major anti-tumor active component extracted from a well-known hepatoprotective and antiviral medicinal herb, Phyllanthus niruri L. Our previous work found that Corilagin inhibited the growth of ovarian cancer cells via TGF-β/AKT/ERK signaling pathways. Corilagin also sensitizes Paclitaxel and Carboplatin by primarily inhibiting Snail-glycolysis pathways. Recently, we have investigated if Corilagin could give a help to overcome poly ADP ribose polymerase (PARP) inhibitors (PARPi) resistance in ovarian cancer cells. As we knew, PARP inhibitors are DNA repair drugs, have been approved for ovarian cancer therapy, but drug resistance is a faced problem, combination therapy may give helps. In this study, two pairs of PARPi sensitive/resistant cells: A2780CP/A2780CP_R and UMB1.289/UMB1.289_R (Sci Transl Med. 2017; 9:392) were used. Cells were cloned again and confirmed their sensitivities to PARPi. The IC50 of PARPi-BMN673 is 0.02μM to A2780CP, &gt 10μM to A2780CP_R, 0.12μM to UMB1.289 and 60μM to UMB1.289_R. The combination assays showed Corilagin had almost equally cytotoxic activities to these two pairs and had synergistic effects with BMN673 to A2780CP_R and UMB1.289_R . Using CulcuSyn, the combination indexes (CI) presented at IC50 are 0.17365 and 0.62787 in A2780_R and UMB1.289_R, respectively. Signaling analysis found that PARPi did down-regulate the expression levels of PARP and up-regulate of pH2AX, PARPi only inhibited pERK activity in sensitive cells, but not in resistant cells. In contrast, Corilagin did not have DNA repair function, but it inhibited pERK activity in both sensitive and resistant cells. In summary, we demonstrate that combined treatment with Corilagin and PARPi evokes synergistic cytotoxic effects in two pairs of ovarian cancer cell models. Corilagin is an herb medicine with lower toxic effects to normal cells, especially hepatoprotective, which could be an ideal complimentary medicine when combinating with PARPi in ovarian cancer therapy. Citation Format: Baoxin Luan, Hongbo Zhao, Robert C. Bast, Jr., Zhen Lu, Yinhua Yu. Corilagin could help to overcome PARP inhibitor resistance by inhibiting ERK signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3816.

Lycium barbarum polysaccharides exert an antioxidative effect on rat chondrocytes by activating the nuclear factor (erythroid-derived 2)-like 2 signaling pathway.

IntroductionOxidative stress is the main cause of osteoarthritis (OA). Lycium barbarum polysaccharides (LBP) have antioxidant properties. Thus, the potential effect of LBP on H2O2-stimulated chondrocytes was examined.Material and methodsThe cell viability was detected by CCK-8. The reactive oxygen species (ROS) production and apoptosis rates were determined by flow cytometric analysis. The DNA damage was detected by comet assay. Real-time polymerase chain reaction (qPCR) and Western blot assays were performed to examine the expression of histone 2A family member X (γH2AX), checkpoint kinase 1 (Chk1), poly ADP-ribose polymerase (PARP), cysteinyl aspartate specific proteinase (caspase)-3/8/9, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its antioxidant-response element (ARE) dependent factors including heme oxygenase-1 (HO-1) and quinine oxidoreductase-1 (NQO-1).ResultsCompared to the H2O2 group, LBP inhibited the ROS production and DNA damage caused by H2O2 (p < 0.05), respectively. LBP inhibited the mRNA and protein expressions of γH2AX and Chk1 (p < 0.05). Meanwhile, LBP significantly decreased apoptosis (p < 0.05). And LBP inhibited the expression levels of PARP and Caspase-3/8/9 (p < 0.05). Moreover, LBP increased the expression of Nrf2, HO-1and NQO-1 (p < 0.05). Furthermore, the depletion of Nrf2 that mediated by RNA interference reversed the apoptosis and DNA damage inhibition effect of LBP (p < 0.05).ConclusionsLBP protected chondrocytes through inhibiting DNA damage and apoptosis caused by H2O2, in which the Nrf2/ARE signaling pathway played a positive role. It provided an inspiration for clinical application – developing LBP as a therapeutic agent and Nrf2 as a promising candidate.

Curcumin regulates intracellular calcium release and inhibits oxidative stress parameters, VEGF, and caspase-3/-9 levels in human retinal pigment epithelium cells.

In this study, we aimed to observe whether curcumin (cur), a polyphenolic compound derived from the dietary spice turmeric, a yellow substance obtained from the root of the plant Curcuma longa Linn, has any protective effect against blue light irradiation in human retinal pigment epithelium (ARPE-19) cells. For this purpose, we evaluated the intracellular calcium release mechanism, poly ADP ribose polymerase (PARP), procaspase-3/-9 protein expression levels, caspase activation, and reactive oxygen species levels. ARPE-19 cells were divided into four main groups, such as control, cur, blue light, and cur + blue light. Results were evaluated by Kruskal-Wallis and Mann-Whitney U tests as post hoc tests. The cells in cur and cur + blue light samples were incubated with 20μM cur. Blue light exposure was performed for 24h in an incubator. Lipid peroxidation and cytosolic-free Ca2+ [Ca2+]i concentrations were higher in the blue light exposure samples than in the control samples; however, their levels were determined as significantly lower in the cur and cur + blue light exposure samples than in the blue light samples alone. PARP and procaspase-3 levels were significantly higher in blue light samples. Cur administration significantly decreased PARP and procaspase-3 expression levels. Reduced glutathione and glutathione peroxidase values were lower in the blue light exposure samples, although they were higher in the cur and cur + blue light exposure samples. Caspase-3 and -9 activities were lower in the cur samples than in the blue light samples. Moreover, vascular endothelial growth factor (VEGF) levels were significantly higher in the blue light exposure samples. In conclusion, cur strongly induced regulatory effects on oxidative stress, intracellular Ca2+ levels, VEGF levels, PARP expression levels, and caspase-3 and -9 values in an experimental oxidative stress model in ARPE-19 cells.

Anti-tumor effects of bakuchiol on human gastric carcinoma cell lines are mediated through PI3K/AKT and MAPK signaling pathways

Bakuchiol is extracted from Psoraleacorylifolia, a member of the Leguminosae family, has been used in Indian Ayurvedic and Chinese traditional medicine, and it possesses an anticancer effect. The primary aim of the present study was to identify the molecular mechanisms underlying the anticancer effect of bakuchiol monoterpenes. Bakuchiol treatment significantly inhibited NUGC3 human gastric cancer cell viability in a concentration dependent manner. In addition, bakuchiol significantly increased the apoptotic cell population in the sub‑G1 phase, and Annexin‑V‑fluorescein isothiocyanate/propidium iodide double staining confirmed the increase in apoptosis. Nuclear fragmentation and the formation of apoptotic organelles were promoted in bakuchiol‑treated NUGC3 cells. Western blotting results indicated that bakuchiol treatment significantly decreased procaspase‑3,6,8,9 and poly (ADP‑ribose) polymerase (PARP) expression levels, increased cleaved caspase‑3 and cleaved PARP expression levels, and increased the B cell lymphoma‑2 associated X, apoptosis regulator:B cell lymphoma‑extra large ratio. Bakuchiol‑treated NUGC3 cells demonstrated significantly reduced phosphorylated (p‑) protein kinase B (AKT) protein expression levels and elevated p‑extracellular signal related kinase 1/2 (ERK1/2), p‑c‑Jun N‑terminal kinase (JNK) and p‑p38. Bakuchiol‑induced cell death was mitochondrial dependent, through modulation of phosphoinositide 3‑kinase/AKT and mitogen‑activated protein kinase signaling pathways. These findings demonstrated that bakuchiol possesses potential for treating human gastric cancer.

Expression of Caspase Signaling Components in the Outer Membranes of Chronic Subdural Hematomas

Chronic subdural hematoma (CSDH) is fundamentally treatable through surgery, although CSDH recurs in some cases. We have observed several cases of spontaneous resolution of CSDH outer membranes, including in trabecular CSDH, after trepanation surgery. In this study, we examined the expression of molecules involved in caspase signaling in CSDH outer membranes. Eight patients whose outer membranes were obtained successfully during trepanation surgery were included in this study. The expression of Fas; Fas-associated death domain (FADD); tumor necrosis factor receptor type 1-associated death domain (TRADD); receptor-interacting protein (RIP); caspases 3, 7, 8, and 9; poly-(ADP-ribose) polymerase (PARP); DNA fragmentation factor 45 (DFF45) and β-actin was examined by Western blot analysis. The expression levels of PARP, caspase-3, and cleaved caspase-3 were also examined by immunohistochemistry. Fas; FADD; TRADD; RIP; caspases 3, 7, 8, and 9; PARP, and DFF45 were detected in nearly all samples. Caspase-3 and PARP were localized in the endothelial cells of vessels and in fibroblasts in CSDH outer membranes. In addition, cleaved caspase-3 was detected in fibroblasts. We detected molecules of the caspase signaling pathway in CSDH outer membranes. In particular, cleaved caspase-3 was detected, which suggests that apoptosis may occur within these membranes. Thus, during the growth of CSDH outer membranes, the caspase signaling pathway may be restrained. Once the pathway is activated, gradual resolution of CSDH outer membranes may occur. Therefore, these molecules may be novel therapeutic targets for intractable CSDH.

The efficiency of Poly(ADP-Ribose) Polymerase (PARP) cleavage on detection of apoptosis in an experimental model of testicular torsion.

The aim of this study was to evaluate the histopathological and apoptotic changes occurring in the rat ipsilateral and contralateral testes, after experimental spermatic cord torsion, and to explore and the role of poly(ADP-ribose) polymerase (PARP) cleavage in testicular torsion-detorsion injury. A total of 37 Wistar albino rats were subjected to 720° unilateral spermatic cord torsion for 1, 2 and 4 h, followed by 4-h reperfusion, or else to a sham operation (control group). Histology of the testicle was evaluated using haematoxylin-eosin (H&E) staining and Johnsen's scoring system. Germ cell apoptosis was evaluated via active caspase-3 immunostaining, and PARP expression levels were evaluated via Western blotting. The mean Johnsen's tubular biopsy scores (JTBS) of the ipsilateral testicles were lower for all torsion groups than for the controls (P < 0.05), but the JTBS of the contralateral testicles were only lower in the 4-h torsion group (P < 0.05). The mean apoptosis score (AS) of the ipsilateral and contralateral testicles was significantly higher in the torsion groups than in the sham group. AS increased correlatively with torsion time, in both testicles. The effect of testicular torsion on PARP cleavage was time dependent, with the highest effect observed after 4 h of testicular torsion (P < 0.05). Testicular torsion caused time-dependent histological changes, apoptosis and increases in PARP cleavage. Our results suggest that testicular torsion-detorsion injury caused cell damage and germ cell apoptosis that apparently involved cleavage of PARP. Increased PARP cleavage could, in turn, lead to enhanced apoptosis.

Poly (ADP‑ribose) polymerase‑ and cytochromec‑mediated apoptosis induces hepatocyte injury in a rat model of hyperammonia‑induced hepatic failure.

Hepatic failure (HF) is caused by several factors, which induce liver cell damage and dysfunction. However, the specific mechanism of HF remains to be fully elucidated. The present study aimed to investigate the underlying cause of hepatocyte injury and liver dysfunction. Liver cells were isolated from healthy female Sprague-Dawley rats, aged between 6 and 8 weeks, weighing ~230 g. The liver cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. An MTT assay was used to examine the inhibitory rate of liver growth in each group. Flow cytometric analysis was performed to detect liver cells undergoing apoptosis. The protein expression levels of poly (ADP-ribose) polymerase (PARP) and cytochrome c (Cyt C) were detected by western blotting. The level of calmodulin-dependent kinase (CaMK) was assessed using an ELISA. The results indicated that the growth inhibitory rate of rat liver cells was significantly increased following treatment with increasing concentrations of NH4Cl. The results of flow cytometric analysis demonstrated that the apoptotic rate in the BAPTA-acetoxymethyl ester group was significantly lower compared with the NH4Cl group (P<0.05). Treatment with NH4Cl increased the protein expression levels of PARP and Cyt C in the liver cells. The mRNA expression of CaMK decreased gradually following treatment with increasing concentrations of NH4Cl for 6, 12 and 24 h. The results suggested that hepatocyte injury and liver dysfunction may be caused by inducing apoptosis via the PARP and Cyt C pathways. Additionally, downregulation of CaMK may be associated with the apoptosis observed in hepatocyte injury.

Effect of celecoxib, a specific cyclooxygenase 2 inhibitor, on the apoptotic and mitotic indexes of breast cancer in patients with early stage disease

14132 Background: To assess whether the administration of celecoxib, a specific cyclooxegenease-2 (COX-2) inhibitor, to patients with breast cancer alters the proliferative and apoptotic indexes of their tumors. Methods: Women newly diagnosed with non metastatic breast cancer were enrolled into the study after undergoing a diagnostic core needle biopsy. Patients received celecoxib treatment at 400 mg orally twice a day for 14 days, and then underwent surgical excision of their tumor. Core biopsies obtained at the time of initial diagnostic procedure and surgical excision specimens were stained for Ki-67, as well as COX-2 and cleaved poly (ADP-ribose) polymerase (PARP) expression (as an apoptosis marker). Appropriate negative and positive controls were included. We assessed the difference in Ki- 67, COX-2 and cleaved PARP expression levels, before and after treatment using the Wilcoxon’s matched-pair ranks test and the McNemar’s test. Results: 16 patients were enrolled. The median age was 54.6 years. ER and/or PR expression was present in 81% of tumors; Her-2 neu overexpression was present in 25%. No significant change in COX-2 or cleaved PARP expression was noticed in the post intervention specimen compared to the core biopsies. Surprisingly, there was a significant increase in the Ki-67 expression (p &lt; 0.009). Conclusions: we have conducted a short term prospective study to assess the effects of celecoxib, on the proliferative and apoptotic indexes in patients with early stage breast cancer. We have found an increase in the Ki-67 activity, with no significant down regulation of COX-2 or increase in cleaved PARP expression with 14 days of therapy. This could be partly due to the small sample size. [Table: see text]

Effects of Chronic Alcohol Consumption on Hepatic Poly‐ADP‐Ribosylation in the Rat

Background: Poly‐adenosine diphosphate (ADP)‐ribosylation is involved in a variety of biological processes, which include DNA repair, malignant transformation, and apoptosis. It is of interest how this reaction is altered after long‐term alcohol intake. Therefore, we determined long‐term alcohol effects on hepatic poly‐ADP‐ribosylation in the rat.Methods: Male Sprague Dawley® rats (four pairs) were pair‐fed a nutritionally adequate liquid diet that contained ethanol as 36% of total energy and an isocaloric control diets for 4 weeks. Liver tissue homogenates and nuclear fractions were subjected to ADP‐ribosylation with [32P]nicotinamide adenine dinucleotide. The ADP‐ribosylated proteins were separated by SDS‐PAGE, followed by autoradiography. Expression of poly‐ADP‐ribose polymerase (PARP) also was evaluated by Western blotting.Results: Incubation of rat liver homogenates in ADP‐ribosylation reaction mixture resulted in a radiolabeling of a 116 kDa protein, most likely auto‐ribosylation of PARP. This poly‐ADP‐ribosylation was increased significantly (p &lt; 0.025) after long‐term alcohol intake. This alcohol effect was reproducible in nuclear fractions as well. Expression levels of PARP, however, were comparable between alcohol‐fed rats and their pair‐fed controls.Conclusion: Poly‐ADP‐ribosylation, an important posttranslational modification of nuclear proteins, was increased significantly after chronic alcohol consumption in the rat.

Effects of chronic alcohol consumption on hepatic poly-ADP-ribosylation in the rat.

Poly-adenosine diphosphate (ADP)-ribosylation is involved in a variety of biological processes, which include DNA repair, malignant transformation, and apoptosis. It is of interest how this reaction is altered after long-term alcohol intake. Therefore, we determined long-term alcohol effects on hepatic poly-ADP-ribosylation in the rat. Male Sprague Dawley(R) rats (four pairs) were pair-fed a nutritionally adequate liquid diet that contained ethanol as 36% of total energy and an isocaloric control diets for 4 weeks. Liver tissue homogenates and nuclear fractions were subjected to ADP-ribosylation with [32P]nicotinamide adenine dinucleotide. The ADP-ribosylated proteins were separated by SDS-PAGE, followed by autoradiography. Expression of poly-ADP-ribose polymerase (PARP) also was evaluated by Western blotting. Incubation of rat liver homogenates in ADP-ribosylation reaction mixture resulted in a radiolabeling of a 116 kDa protein, most likely auto-ribosylation of PARP. This poly-ADP-ribosylation was increased significantly (p < 0.025) after long-term alcohol intake. This alcohol effect was reproducible in nuclear fractions as well. Expression levels of PARP, however, were comparable between alcohol-fed rats and their pair-fed controls. Poly-ADP-ribosylation, an important posttranslational modification of nuclear proteins, was increased significantly after chronic alcohol consumption in the rat.

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.

Regulation of the human poly(ADP-ribose) polymerase promoter by the ETS transcription factor.

Ewing's sarcoma (EWS) cells accumulate elevated steady-state levels of poly (ADP-ribose) polymerase (PARP) mRNA and protein. To understand the molecular mechanisms underlying PARP upregulation, we cloned and analysed the 5'-flanking region of the PARP gene from EWS cells. Nucleotide sequence analysis demonstrated no variations in the PARP promoter region in EWS cells. The PARP promoter encompasses multiple binding motifs for the ETS transcription factor. We have also observed that there is a coordinated up-regulation of the expression of both PARP and ETS1, relative to cells of other human tumor types expressing lower levels of PARP. Transient co-expression of ETS1 in EWS cells resulted in a strong enhancement of PARP-promoter activity. The participation of ETS in the regulation of PARP gene expression was further demonstrated in EWS cells stably transfected with Ets1 antisense cDNA constructs. Antisense-mediated down-regulation of endogenous ETS1 resulted in the inhibition of PARP expression in EWS cells, and sensitized these cells to ionizing radiation. These data provide support for ETS regulation of PARP expression levels, and implicate ETS transcription factors in the radiation response of EWS cells.