[show abstract][hide abstract] ABSTRACT: Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent γ-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered γ-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.
[show abstract][hide abstract] ABSTRACT: Cells of most human cancers have supernumerary centrosomes. To enable an accurate chromosome segregation and cell division, these cells developed a yet unresolved molecular mechanism, clustering their extra centrosomes at two poles, thereby mimicking mitosis in normal cells. Failure of this bipolar centrosome clustering causes multipolar spindle structures and aberrant chromosomes segregation that prevent normal cell division and lead to 'mitotic catastrophe cell death'.
We used cell biology and biochemical methods, including flow cytometry, immunocytochemistry and live confocal imaging.
We identified a phenanthrene derived PARP inhibitor, known for its activity in neuroprotection under stress conditions, which exclusively eradicated multi-centrosomal human cancer cells (mammary, colon, lung, pancreas, ovarian) while acting as extra-centrosomes de-clustering agent in mitosis. Normal human proliferating cells (endothelial, epithelial and mesenchymal cells) were not impaired. Despite acting as PARP inhibitor, the cytotoxic activity of this molecule in cancer cells was not attributed to PARP inhibition alone.
We identified a water soluble phenanthridine that exclusively targets the unique dependence of most human cancer cells on their supernumerary centrosomes bi-polar clustering for their survival. This paves the way for a new selective cancer-targeting therapy, efficient in a wide range of human cancers.
[show abstract][hide abstract] ABSTRACT: The nuclear protein PARP-1 [poly(ADP-ribose) polymerase-1] is activated in cardiomyocytes exposed to hypoxia causing DNA breaks. Unlike this stress-induced PARP-1 activation, our results provide evidence for Ca(2+)-induced PARP-1 activation in contracting newborn cardiomyocytes treated with growth factors and hormones that increased their contraction rate, induced intracellular Ca(2+) mobilization and its rhythmical and transient translocation into the nucleus. Furthermore, activated PARP-1 up-regulated the activity of phosphorylated ERK (extracellular-signal-regulated kinase) in the nucleus, promoting expression of the Elk1 target gene c-fos. Up-regulation of the transcription factor c-Fos/GATA-4 promoted ANF (atrial natriuretic factor) expression. Given that expression of ANF is known to be implicated in morphological changes, growth and development of cardiomyocytes, these results outline a PARP-1-dependent signal transduction mechanism that links contraction rate and Ca(2+) mobilization with the expression of genes underlying morphological changes in cardiomyocytes.
[show abstract][hide abstract] ABSTRACT: PARP-1 (polyADP-ribose polymerase-1) is known to be activated in response to DNA damage, and activated PARP-1 promotes DNA repair. However, a recently disclosed alternative mechanism of PARP-1 activation by phosphorylated externally regulated kinase (ERK) implicates PARP-1 in a vast number of signal-transduction networks in the cell. Here, PARP-1 activation was examined for its possible effects on cell proliferation in both normal and malignant cells.
In vitro (cell cultures) and in vivo (xenotransplants) experiments were performed.
Phenanthridine-derived PARP inhibitors interfered with cell proliferation by causing G2/M arrest in both normal (human epithelial cells MCF10A and mouse embryonic fibroblasts) and human breast cancer cells MCF-7 and MDA231. However, whereas the normal cells were only transiently arrested, G2/M arrest in the malignant breast cancer cells was permanent and was accompanied by a massive cell death. In accordance, treatment with a phenanthridine-derived PARP inhibitor prevented the development of MCF-7 and MDA231 xenotransplants in female nude mice. Quiescent cells (neurons and cardiomyocytes) are not impaired by these PARP inhibitors.
These results outline a new therapeutic approach for a selective eradication of abundant nonhereditary human breast cancers.
Breast cancer research: BCR 11/2009; 11(6):R78. · 5.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: PolyADP-ribosylation is a post-translational modification of nuclear proteins, catalyzed by polyADP-ribose polymerases (PARPs). In the nucleus, polyADP-ribosylation catalyzed by PARP-1 alters protein–protein and protein–DNA interactions, and is implicated in chromatin remodeling, DNA transcription, and repair. Previous results linked the activation of PARP-1 with long-term memory formation during learning in the marine mollusk Aplysia ( Science 2004, 304:1820–1822). Furthermore, PARP-1 was highly activated in mammalian cerebral neurons treated with neurotrophins and neurotrophic peptides promoting neurite outgrowth and synaptic plasticity. Here, we examine the possibility that PARP-1 activation is required for memory formation during learning in mammals. Mice were tested in two learning paradigms, object recognition and fear conditioning. PolyADP-ribosylation of PARP-1 and histone H1 were detected in their cerebral cortex and hippocampus immediately after their training session. Moreover, in both behavioral paradigms, suppression of PARP activity in the CNS during learning impaired their long-term memory formation, without damaging their short-term memory. These findings implicate PARP-1 activation in molecular processes underlying long-term memory formation during learning.
[show abstract][hide abstract] ABSTRACT: PolyADP-ribose polymerases (PARPs) catalyze a posttranslational modification of nuclear proteins by polyADP-ribosylation. The catalytic activity of the abundant nuclear protein PARP-1 is stimulated by DNA strand breaks, and PARP-1 activation is required for initiation of DNA repair. Here we show that PARP-1 also acts within extracellular signal-regulated kinase (ERK) signaling cascade that mediates growth and differentiation. The findings reveal an alternative mode of PARP-1 activation, which does not involve binding to DNA or DNA damage. In a cell-free system, recombinant PARP-1 was intensively activated and thereby polyADP-ribosylated by a direct interaction with phosphorylated ERK2, and the activated PARP-1 dramatically increased ERK2-catalyzed phosphorylation of the transcription factor Elk1. In cortical neurons treated with nerve growth factors and in stimulated cardiomyocytes, PARP-1 activation enhanced ERK-induced Elk1-phosphorylation, core histone acetylation, and transcription of the Elk1-target gene c-fos. These findings constitute evidence for PARP-1 activity within the ERK signal-transduction pathway.
[show abstract][hide abstract] ABSTRACT: In a previous study, it was shown that the hornet venom or, more specifically, its venom sac extract (VSE) possesses deoxyribonuclease activity that exerts an effect both on insects as well as on mammals. We have now examined the effect of hornet VSE on primary culture of rat cortical neurons. Judging on the basis of our results, VSE induces a rapid cell death by a) permeabilizing the cell membrane, b) inducing DNA breaks, and c) cleaving the nuclear protein poly-ADP-ribose polymerase (PARP-1), thereby preventing DNA repair.
[show abstract][hide abstract] ABSTRACT: PolyADP-ribosylation is a transient posttranslational modification of proteins, mainly catalyzed by poly(ADP-ribose)polymerase-1 (PARP-1). This highly conserved nuclear protein is activated rapidly in response to DNA nick formation and promotes a fast DNA repair. Here, we examine a possible association between polyADP-ribosylation and the activity of neurotrophins and neuroprotective peptides taking part in life-or-death decisions in mammalian neurons. The presented results indicate an alternative mode of PARP-1 activation in the absence of DNA damage by neurotrophin-induced signaling mechanisms. PARP-1 was activated in rat cerebral cortical neurons briefly exposed to NGF-related nerve growth factors and to the neuroprotective peptides NAP (the peptide NAPVSIPQ, derived from the activity-dependent neuroprotective protein ADNP) and ADNF-9 (the peptide SALLRSIPA, derived from the activity-dependent neurotrophic factor ADNF) In addition, polyADP-ribosylation was involved in the neurotrophic activity of NGF-induced and NAP-induced neurite outgrowth in differentiating pheochromocytoma 12 cells as well as in the neuroprotective activity of NAP in neurons treated with the Alzheimer's disease neurotoxin beta-amyloid. A fast loosening of the highly condensed chromatin structure by polyADP-ribosylation of histone H1, which renders DNA accessible to transcription and repair, may underlie the role of polyADP-ribosylation in neurotrophic activity.
Journal of Neuroscience 09/2005; 25(32):7420-8. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: PolyADP-ribose-polymerase 1 is activated in neurons that mediate several forms of long-term memory in Aplysia. Because polyADP-ribosylation of nuclear proteins is a response to DNA damage in virtually all eukaryotic cells, it is surprising that activation of the polymerase occurs during learning and is required for long-term memory. We suggest that fast and transient decondensation of chromatin structure by polyADP-ribosylation enables the transcription needed to form long-term memory without strand breaks in DNA.
[show abstract][hide abstract] ABSTRACT: Synaptoneurosomes (1-3 microm in diameter), prepared from rat brain stem or brain cortex, were fused with liposomes, producing a high yield of giant synaptosomes (10-60 microm in diameter). Single channel currents were measured by using the cell-attach patch-clamp technique. The membrane of the majority of these giant synaptosomes retained the cell membrane selective permeability. However, nonpermeating molecules, such as guanine nucleotides and antibodies directed against GTP-binding region in the alpha-subunit of trimeric GTP-binding proteins, were trapped in the giant synaptosomes during their preparation. Activation of Go proteins was assayed in high [K(+)]-depolarized giant synaptosomes, indicating the advantage of this preparation for tracing signal-transduction mechanisms in stimulated synaptic membranes. Stimulation-induced interactions between membrane proteins, either native or reconstituted, can be studied in the giant synaptosomes.
Journal of Molecular Neuroscience 03/2003; 20(1):73-80. · 2.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: We present the first evidence for a fast activation of the nuclear protein poly(ADP-ribose) polymerase (PARP) by signals evoked in the cell membrane, constituting a novel mode of signaling to the cell nucleus. PARP, an abundant, highly conserved, chromatin-bound protein found only in eukaryotes, exclusively catalyzes polyADP-ribosylation of DNA-binding proteins, thereby modulating their activity. Activation of PARP, reportedly induced by formation of DNA breaks, is involved in DNA transcription, replication, and repair. Our findings demonstrate an alternative mechanism: a fast activation of PARP, evoked by inositol 1,4,5,-trisphosphate-Ca(2+) mobilization, that does not involve DNA breaks. These findings identify PARP as a novel downstream target of phospholipase C, and unveil a novel fast signal-induced modification of DNA-binding proteins by polyADP-ribosylation.
The Journal of Cell Biology 08/2000; 150(2):293-307. · 10.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Evidence for depolarization-induced activation of G-proteins in membranes of rat brain synaptoneurosomes has been previously reported (Cohen-Armon, M., and Sokolovsky, M. (1991) J. Biol. Chem. 266, 2595-2605; Cohen-Armon, M., and Sokolovsky, M. (1993) J. Biol. Chem. 268, 9824-9838). In the present work we identify the activated G-proteins as Go-proteins by tracing their depolarization-induced in situ photoaffinity labeling with [alpha32P]GTP-azidoanilide (GTPAA). Labeled GTPAA was introduced into transiently permeabilized rat brain-stem synaptoneurosomes. The resealed synaptoneurosomes, while being UV-irradiated, were depolarized. Relative to synaptoneurosomes at resting potential, the covalent binding of [alpha32P]GTPAA to Galphao1- and Galphao3-proteins, but not to Galphao2- isoforms, was enhanced by 5- to 7-fold in depolarized synaptoneurosomes, thereby implying an accelerated exchange of GDP for [alpha32P]GTPAA. Their depolarization-induced photoaffinity labeling was independent of stimulation of Go-protein-coupled receptors and could be reversed by membrane repolarization, thus excluding induction by transmitters release. It was, however, dependent on depolarization-induced activation of the voltage-gated sodium channels (VGSC), regardless of Na+ current. The alpha subunit of VGSC was cross-linked and co-immunoprecipitated with Galphao-proteins in depolarized brain-stem and cortical synaptoneurosomes. VGSC alpha subunit most efficiently cross-linked with guanosine 5'-O-2-thiodiphosphate-bound rather than to guanosine 5'-O-(3-thiotriphosphate)-bound Galphao-proteins in isolated synaptoneurosomal membranes. These findings support a possible involvement of VGSC in depolarization-induced activation of Go-proteins.
Journal of Biological Chemistry 04/1999; 274(11):7431-40. · 4.65 Impact Factor