Are Voltage Sensors Really Embedded in Muscarinic Receptors?

Unexpectedly, the affinity of the seven-transmembrane muscarinic acetylcholine receptors for their agonists is modulated by membrane depolarization. Recent reports attribute this characteristic to an embedded charge movement in the muscarinic receptor, acting as a voltage sensor. However, this explanation is inconsistent with the results of experiments measuring acetylcholine binding to muscarinic receptors in brain synaptoneurosomes. According to these results, the gating of the voltage-dependent sodium channel (VDSC) acts as the voltage sensor, generating activation of Go-proteins in response to membrane depolarization, and this modulates the affinity of muscarinic receptors for their cholinergic agonists.

A Long-Lasting PARP1-Activation Mediates Signal-Induced Gene Expression.

This overview presents recent evidence for a long-lasting PARP1 activation by a variety of signal transduction mechanisms, mediating signal-induced gene expression and chromatin remodeling. This mode of PARP1 activation has been reported in a variety of cell types, under physiological conditions. In this mechanism, PARP1 is not transiently activated by binding to DNA breaks. Moreover, damaged DNA interfered with this long-lasting PARP1 activation.

Exclusive modifications of NuMA in malignant epithelial cells: A potential therapeutic mechanism.

NuMA (nuclear mitotic apparatus) protein is indispensable in the mitosis of human proliferating cells, both malignant and benign. The progression of mitosis requires stable spindles, which depend on the bipolar clustering of NuMA within the spindles. The phenanthridine PJ34 kills malignant epithelial cells during mitosis and targets NuMA. PJ34 treated healthy cells are not impaired. PJ34 exclusively blocks the post-translational modification of NuMA in a variety of malignant epithelial cells, but not in benign cells. This blockage of the post-translational modification of NuMA affects its protein-binding capacity, causing construction faults in the mitotic spindle poles of PJ34-treated cancer cells, leading to Mitotic Catastrophe cell death. PJ34 is a potent PARP1 inhibitor. Therefore its PARP independent exclusive cytotoxicity in human malignant cells, challenges the currently accepted notion that inhibition of PARP1 halts cancer by preventing DNA repair. Certain molecules that act as PARP1 inhibitors kill cancer cells by targeting other proteins and vital mechanisms.

The Modified Phenanthridine PJ34 Unveils an Exclusive Cell-Death Mechanism in Human Cancer Cells.

This overview summarizes recent data disclosing the efficacy of the PARP inhibitor PJ34 in exclusive eradication of a variety of human cancer cells without impairing healthy proliferating cells. Its cytotoxic activity in cancer cells is attributed to the insertion of specific un-repairable anomalies in the structure of their mitotic spindle, leading to mitotic catastrophe cell death. This mechanism paves the way to a new concept of cancer therapy.

Correction: Exclusive destruction of mitotic spindles in human cancer cells.

[This corrects the article DOI: 10.18632/oncotarget.15343.].

Correction: The phenanthrene derivative PJ34 exclusively eradicates human pancreatic cancer cells in xenografts.

[This corrects the article DOI: 10.18632/oncotarget.27268.].

The phenanthrene derivative PJ34 exclusively eradicates human pancreatic cancer cells in xenografts.

Recent reports demonstrate an exclusive eradication of a variety of human cancer cells by the modified phenanthridine PJ34. Their eradication during mitosis is attributed to PJ34 preventing NuMA clustering in the mitotic spindle poles of human malignant cells, which is crucial for their normal mitosis. Here, the effect of PJ34 is tested in cell cultures and xenografts of human pancreas ductal adenocarcinoma. Evidence is presented for a substantial reduction (80-90%) of PANC1 cancer cells in xenografts, measured 30 days after the treatment with PJ34 has been terminated. Benign cells infiltrated into the PANC1 tumors (stroma) were not affected. Growth, weight gain and behavior of the treated nude mice were not impaired during, and 30 days after the treatment with PJ34. The efficient eradication of malignant cells in human pancreas cancer xenografts presents a new model of pancreas cancer treatment.

Signal-induced PARP1-Erk synergism mediates IEG expression.

A recently disclosed Erk-induced PARP1 activation mechanism mediates the expression of immediate early genes (IEGs) in response to a variety of extra- and intracellular signals implicated in memory acquisition, development and proliferation. Here, we review this mechanism, which is initiated by stimulation-induced binding of PARP1 to phosphorylated Erk translocated into the nucleus. This binding maintains long-lasting synergistic activity of these proteins, which offers a new pattern for targeted therapy.

PARP1-Erk synergism in proliferating cells.

A synergism between PARP1 and phosphorylated Erk mediating IEG (immediate early gene) expression has been recently reported in cerebral neurons and cardiomyocytes. Stimulation induced PARP-Erk synergism was required for IEG expression underlying synaptic plasticity and long-term memory acquisition during learning. It was similarly required for cardiomyocytes development. Here, we identified this mechanism in Erk-induced gene expression promoting proliferation. This mechanism can be targeted in malignant cells.

Exclusive destruction of mitotic spindles in human cancer cells.

We identified target proteins modified by phenanthrenes that cause exclusive eradication of human cancer cells. The cytotoxic activity of the phenanthrenes in a variety of human cancer cells is attributed by these findings to post translational modifications of NuMA and kinesins HSET/kifC1 and kif18A. Their activity prevented the binding of NuMA to α-tubulin and kinesins in human cancer cells, and caused aberrant spindles. The most efficient cytotoxic activity of the phenanthridine PJ34, caused significantly smaller aberrant spindles with disrupted spindle poles and scattered extra-centrosomes and chromosomes. Concomitantly, PJ34 induced tumor growth arrest of human malignant tumors developed in athymic nude mice, indicating the relevance of its activity for cancer therapy.

The effect of poly (ADP-ribose) polymerase inhibition on aminoglycoside-induced acute tubular necrosis in rats.

INTRODUCTION: Aminoglycosides (AG) cause nephrotoxicity in 10 - 20% of patients. One of the mechanisms is by generating reactive oxygen species (ROS), leading to DNA destruction and activation of poly(ADPribose) polymerase (PARP) causing necrotic tubular cell death. PARP inhibition on gentamicin-induced nephrotoxicity was studied. METHODS: 19 female Wistar-Kyoto rats divided into 3 groups: control (3 rats receiving no treatment); gentamicin-treated group (8 rats); and 8 rats treated with gentamicin combined with 3-aminobenzamide (3 AB). Kidney functions, protein, and gentamicin levels as well as urinary trypsin inhibitory activity (TIA) were measured. Tissue microscopic examination and immunohistochemical study for proliferative cell nuclear antigen (PCNA) were determined. The effect of PARP inhibitor on the bactericidal activity of gentamicin was also assessed. RESULTS: The following results were statistically significant: urea (mg/dL) 39.9 ± 5.86, 88.3 ± 50.3, and 48.5 ± 12.7 (p = 0.048); serum creatinine (mg/dL): 0.6 ± 0.26, 1.05 ± 0.7, 0.6 ± 0.06 (p = 0.043); proteinuria (mg/24-hours): 7.27 ± 3.65, 41.2 ± 18.1, and 17.6 ± 13.9 (p = 0.050); the number of tubular macronuclei (per 10 mm2): 18.33 ± 16.07, 218 ± 101.8, 41.7 ± 36.2 (p = 0.012); the number of dilated tubes (per 10 mm2): 61.67 ± 12.58, 276.3 ± 112.7, 140.0 ± 90.9 (p = 0.04); and the number of PCNA positive nuclei (per 10 mm2): 223.3 ± 95.69, 3,585 ± 2,215.3, 626.7 ± 236.9 (p = 0.034) in the control, gentamicin, and gentamicin+3AB-treated groups, respectively. The following biochemical and histologic parameters were also examined, however, they showed no statistically significant difference: TIA (p = 0.055), mitoses (p = 0.14), mononuclear infiltrate (p = 0.188), and intratubular cast formation (p = 0.084). No effect on bactericidal activity was observed. CONCLUSION: This study illustrates that PARP inhibitor significantly attenuates gentamicin-induced nephrotoxicity in rats with no effect on the bactericidal activity.

Cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells.

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.

A phenanthrene derived PARP inhibitor is an extra-centrosomes de-clustering agent exclusively eradicating human cancer cells.

BACKGROUND: 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'. METHODS: We used cell biology and biochemical methods, including flow cytometry, immunocytochemistry and live confocal imaging. RESULTS: 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. CONCLUSION: 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.

Ca2+-induced PARP-1 activation and ANF expression are coupled events in cardiomyocytes.

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.

A selective eradication of human nonhereditary breast cancer cells by phenanthridine-derived polyADP-ribose polymerase inhibitors.

INTRODUCTION: 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. METHODS: In vitro (cell cultures) and in vivo (xenotransplants) experiments were performed. RESULTS: 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. CONCLUSIONS: These results outline a new therapeutic approach for a selective eradication of abundant nonhereditary human breast cancers.

PolyADP-ribosylation is required for long-term memory formation in mammals.

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.

PARP-1 activation in the ERK signaling pathway.

PARP-1 is a highly conserved DNA-binding protein, the most abundant member of the polyADP-ribose polymerases (PARP) family, which catalyzes post-translational modification of proteins by polyADP-ribosylation. This modification affects protein-protein and protein-DNA interactions. Binding of PARP-1 to breakages in damaged DNA causes its activation and auto-polyADP-ribosylation in a process that is pivotal for DNA repair. Our recent findings outlined an alternative mechanism of PARP-1 activation via a direct interaction with phosphorylated ERK2 (externally regulated kinase), which is unrelated to DNA damage and does not involve PARP-1 binding to DNA. Furthermore, ERK2-induced PARP-1 activation dramatically amplifies ERK-signals, enhancing ERK-induced phosphorylation of the transcription factor Elk1 and enhancing core histone acetylation and expression of the Elk1 target gene, c-fos. Thus, PARP-1 activation in the ERK signaling pathway mediates epigenetic mechanisms promoting growth, proliferation and differentiation regulated by the Raf-MEK-ERK phosphorylation cascade.

DNA-independent PARP-1 activation by phosphorylated ERK2 increases Elk1 activity: a link to histone acetylation.

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.

A fatal effect of hornet venom on rat-brain cortical neurons.

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.

Role of poly(ADP-ribosyl)ation during human spermatogenesis.

OBJECTIVE: Genomic stability of cells is known to be linked to their poly(ADP-ribosyl)ation capacity. We aimed to demonstrate, for the first time, the patterns of poly(ADP-ribosyl)ation during human spermatogenesis. DESIGN: Retrospective case-control study. SETTING: Teaching hospital. PATIENT(S): Azoospermic men who underwent testicular biopsy for sperm recovery. INTERVENTION(S): Testicular biopsy evaluation by immunohistochemistry for the expression of poly(ADP-ribose) polymerase-1 (PARP-1) enzyme and of poly(ADP-ribose) (PAR) (an indicator for PARP activity.) MAIN OUTCOME MEASURE(S): The subcellular localization of both markers in testes with full spermatogenesis (obstructive azoospermia), spermatocyte maturation arrest, or Sertoli cell-only syndrome. RESULT(S): Expression of both markers was localized in germ cell nuclei in full spermatogenesis: PAR expression, indicating PARP activity, was exhibited in round and elongating spermatids and in a subpopulation of primary spermatocytes. Strong immunoreactivity for PAR was identified in all of the spermatocytes in maturation arrest at the spermatocyte level. Sertoli cells lacked immunoreactivity for both markers, whereas other somatic testicular cells were rarely immunostained. CONCLUSION(S): The detection of PAR expression in germ-line cells and its subcellular localization in meiotic and postmeiotic prophases demonstrates chromatin modifications occurring during spermatogenesis and establishes a key role for poly(ADP-ribosyl)ation in germ cell differentiation, presumably to safeguard DNA integrity.