Search

Inhibitors of PARP-1(Poly(ADP-ribose) polymerase-1) act by competing with NAD(+), the enzyme physiological substrate, which play a protective role in many pathological conditions characterized by PARP-1 overactivation. It has been shown that PARP-1 also promotes tumor growth and progression through its DNA repair activity. Since angiogenesis is an essential requirement for these activities, we sought to determine whether PARP inhibition might affect rat brain microvascular endothelial cells (GP8.3) migration, stimulated by C6-glioma conditioned medium (CM). Through wound-healing experiments and MTT analysis, we demonstrated that PARP-1 inhibitor PJ-34 [N-(6-Oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide] abolishes the migratory response of GP8.3 cells and reduces their viability. PARP-1 also acts in a DNA independent way within the Extracellular-Regulated-Kinase (ERK) signaling cascade, which regulates cell proliferation and differentiation. By western analysis and confocal laser scanning microscopy (LSM), we analyzed the effects of PJ-34 on PARP-1 expression, phospho-ERK and phospho-Elk-1 activation. The effect of MEK (mitogen-activated-protein-kinase-kinase) inhibitor PD98059 (2-(2-Amino-3-methoxyphenyl)-4 H-1-benzopyran-4-one) on PARP-1 expression in unstimulated and in CM-stimulated GP8.3 cells was analyzed by RT-PCR. PARP-1 expression and phospho-ERK activation were significantly reduced by treatment of GP8.3 cells with PJ-34 or PD98059. By LSM, we further demonstrated that PARP-1 and phospho-ERK are coexpressed and share the same subcellular localization in GP8.3 cells, in the cytoplasm as well as in nucleoplasm. Based on these data, we propose that PARP-1 and phospho-ERK interact in the cytosol and then translocate to the nucleus, where they trigger a proliferative response. We also propose that PARP-1 inhibition blocks CM-induced endothelial migration by interfering with ERK signal-transduction pathway., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Poly(ADP-ribose) polymerases (PARPs) play a crucial role in DNA damage surveillance through their nick sensor functions. Since PARPs' over activation leads to an excessive consumption of NAD(+) and ATP depletion, these enzymes also are involved in the early events of programmed cell death as well as in necrosis. In order to verify the protective action of L: -carnosine and trehalose against NO induced cell death, in the present study we examined their effects on the expression of PARP-1, PARP-2 and iNOS in primary rat astrocyte and oligodendrocyte cells, treated with lipopolysaccharide (LPS) and interferon gamma (INFγ), through semi-quantitative PCR and western analysis. To further characterize the molecular mechanisms underlying L-carnosine and trehalose action, we measured cell viability, nitrite production and LDH release. The data obtained clearly demonstrate that in the stress model employed L-carnosine and trehalose down regulate PARP-1 and PARP-2 expression in both cell phenotypes, thus suggesting their possible application in clinical trials.

The enzyme poly(ADP-ribose)polymerase (PARP) has a leader role in the DNA damage survey mechanisms by its nick-sensor function, but it is also involved in the early events of the programmed cell death, particularly during inflammatory injury, as a coactivator of NF-kB. In the present study, we evaluated the PARP involvement in the mechanisms of protection and/or cell death in rat astroglial cell cultures during the early phase of proinflammatory commitment after lipopolysaccharide and interferon gamma treatment. According with the recent findings that PARP-1 phosphorylation by MAPK/ERK-2 pathway seems to modulate PARP activation, in time course experiments we demonstrated that a very early PARP activation and expression is able to trigger a cell death pathway, DNA damage independent, during strong proinflammatory insults, maintaining its role of guardian of the genome stability only during the normal cell cycling.

Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I) and insulin (INS) are powerful mitogens and may regulate gene expression in cultured astrocytes by ADP-ribosylation process. Nuclear poly-ADP ribose polymerase (PARP) and mitochondrial monoADP-ribosyltransferase (ADPRT) are the key enzymes involved in poly-ADP-ribosylation and mono ADP-ribosylation, respectively. In this investigation the effect of EGF, bFGF, IGF-I or INS on nuclear PARP and mitochondrial ADPRT activities were assessed in nuclei and mitochondria purified from developing (30 DIV) or aging (90 and 190 DIV) primary rat astrocyte cultures. A marked increase of PARP activity in bFGF or IGF-I treated astroglial cell cultures at 30 DIV was found. Nuclear PARP and mitochondrial ADPRT activities were greatly stimulated by treatment with EGF or INS alone or together in astrocyte cultures at 30 DIV. Nuclear PARP and mitochondrial ADPRT activities showed a more remarkable increase in control untreated astrocyte cultures at 190 DIV than at 90 DIV. These findings suggest that ADP-ribosylation process is involved in DNA damage and repair during cell differentiation and aging in culture. Twelve hours treatment with EGF, INS or bFGF significantly stimulated nuclear PARP and mitochondrial ADPRT activities in 190 DIV aging astrocyte cultures. The above results indicate that EGF, INS and bFGF may play a crucial role in the post-translational modification of chromosomal proteins including ADP-ribosylation process in in vitro models. This suggests that growth factors regulate genomic stability in glial cells during development and maturation, stimulating nuclear and mitochondrial ADP-ribosylation processes in developing or aging astrocyte cultures.