Esther Priel

PolyADP-Ribosylation Is Involved in Neurotrophic Activity

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 Alzheimers disease neurotoxin β-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.

HU-331, a novel cannabinoid-based anticancer topoisomerase II inhibitor

Anthracyclines, a large group of quinonoid compounds, are used to treat some forms of cancer. Although highly effective in cancer therapy, the mechanism of action of these compounds is not specific; they act on cancer and other cells by numerous mechanisms. A new anticancer quinone (HU-331) was synthesized from cannabidiol. It shows significant high efficacy against human cancer cell lines in vitro and against in vivo tumor grafts in nude mice. In this study, we investigated its mode of action and present evidence on its unique mechanism. HU-331 does not cause cancer cell cycle arrest, cell apoptosis, or caspase activation. HU-331–caused cell death of human cancer cell lines is not mediated by reactive oxygen intermediates/species, as exposure to HU-331 failed to elicit the generation of reactive oxygen species. HU-331 inhibits DNA topoisomerase II even at nanomolar concentrations but has only a slight nonsignificant effect on DNA topoisomerase I action. The cannabinoid quinone HU-331 is a highly specific inhibitor of topoisomerase II, compared with most known anticancer quinones. It might represent a new potent anticancer drug. [Mol Cancer Ther 2007;6(1):173–83]

Topoisomerase I activity associated with human immunodeficiency virus (HIV) particles and equine infectious anemia virus core.

In the present study, we found a topoisomerase I (topo I) activity in two strains of human immunodeficiency virus type 1 (HIV‐1) and equine infectious anemia virus (EIAV) particles. The topo I activity was located in the EIAV cores and differed from the cellular topo I in its ionic requirements and response to ATP, indicating that these were two distinct forms of this enzyme. Topo I activity was removed from the viral lysates and viral cores by anti‐topo I antiserum. The only protein recognized by this antiserum was an 11.5 kd protein in HIV lysate and 11 kd in EIAV lysate. We showed that the 11 kd protein recognized by the anti‐topo I antiserum is the EIAV p11 nucleocapsid protein. Furthermore, purified topo I protein blocked the binding of the antibodies to the p11 protein and vice versa, purified p11 protein blocked the binding of these antibodies to the cellular topo I. These results suggest that the EIAV p11 nucleocapsid protein and the cellular topo I share similar epitopes.

Novel telomerase‐increasing compound in mouse brain delays the onset of amyotrophic lateral sclerosis

Telomerase is expressed in the neonatal brain, in distinct regions of adult brain, and was shown to protect developing neurons from apoptosis. Telomerase reactivation by gene manipulation reverses neurodegeneration in aged telomerase‐deficient mice. Hence, we and others hypothesized that increasing telomerase expression by pharmaceutical compounds may protect brain cells from death caused by damaging agents. In this study, we demonstrate for the first time that the novel compound AGS‐499 increases telomerase activity and expression in the mouse brain and spinal cord (SC). It exerts neuroprotective effects in NMDA‐injected CD‐1 mice, delays the onset and progression of the amyotrophic lateral sclerosis (ALS) disease in SOD1 transgenic mice, and, after the onset of ALS, it increases the survival of motor neurons in the SC by 60%. The survival of telomerase‐expressing cells (i.e. motor neurons), but not telomerase‐deficient cells, exposed to oxidative stress was increased by AGS‐499 treatment, suggesting that the AGS‐499 effects are telomerase‐mediated. Therefore, a controlled and transient increase in telomerase expression and activity in the brain by AGS‐499 may exert neuroprotective effects.

Critical Role for Telomerase in the Mechanism of Flow Mediated Dilation in the Human Microcirculation

Supplemental Digital Content is available in the text.

DNA topoisomerase I in the mouse central nervous system: Age and sex dependence

Topoisomerase I (topo I) is a nuclear enzyme responsible for the topological state of DNA and therefore participates in most DNA transactions, particularly in transcription. Topo I, a ubiquitous enzyme, was identified and characterized in various cell types and tissues; however, the characterization of topo I in the intact central nervous system was not performed. Here we investigated, for the first time, the activity, level, and distribution pattern of topo I in the various selected brain regions in the mouse. In the visual cortex, cerebellum, and striatum the activity of topo I was 3–4‐fold higher compared to that found in the hippocampus and hypothalamus. Immunohistochemical and immunofluorescence analyses revealed specific distribution patterns of topo I protein in neurons of each of the areas examined. The highest topo I levels were observed in inhibitory neurons. In addition to the expected nuclear localization of this protein, some neurons exhibited significant cytoplasmic content as well. The activity and level of topo I is age‐ and gender‐dependent. It increases from birth to maturity and decreases, more significantly in males, with senescence. These results point to a possible importance and involvement of topo I activity and regulation in various brain functions. J. Comp. Neurol. 493:357–369, 2005.

Moxifloxacin enhances antiproliferative and apoptotic effects of etoposide but inhibits its proinflammatory effects in THP-1 and Jurkat cells.

Etoposide (VP-16) is a topoisomerase II (topo II) inhibitor chemotherapeutic agent. Studies indicate that VP-16 enhances proinflammatory cytokines secretion from tumour cells, including IL-8, a chemokine associated with proangiogenic effects. Fluoroquinolones inhibit topo II activity in eukaryotic cells by a mechanism different from that of VP-16. The fluoroquinolone moxifloxacin (MXF) has pronounced anti-inflammatory effects in vitro and in vivo. We studied the effects of MXF and VP-16 on purified human topo II activity and further analysed their combined activity on proliferation, apoptosis and caspase-3 activity in THP-1 and Jurkat cells. Moxifloxacin alone slightly inhibited the activity of human topo II; however, in combination with VP-16 it led to a 73% reduction in enzyme activity. VP-16 inhibited cell proliferation in a time and dose-dependent manner. The addition of moxifloxacin for 72 h to low-dose VP-16 doubled its cytotoxic effect in THP-1 and Jurkat cells (1.8- and 2.6-fold decrease in cell proliferation, respectively) (P<0.004). Moxifloxacin given alone did not induce apoptosis but enhanced VP-16-induced apoptosis in THP-1 and Jurkat cells (1.8- and two-fold increase in annexin V positive cells and caspase-3 activity, respectively) (P<0.04). VP-16 induced the release of IL-8 in a time and dose-dependent manner from THP-1 cells. Moxifloxacin completely blocked the enhanced release of IL-8 induced by 0.5 and 1 μg ml−1 VP-16, and decreased IL-8 release from cells incubated for 72 h with 3 μg ml−1 VP-16 (P<0.001). VP-16 enhanced the release of IL-1β and TNF-α from THP-1 cells, whereas the addition of MXF prevented the enhanced cytokine secretion (P<0.001). We conclude that MXF significantly enhances VP-16 cytotoxicity in tumour-derived cells while preventing VP-16-induced proinflammatory cytokine release. This unique combination may have clinical benefits and cytotoxic drug ‘sparing effect’ and should be further studied in vivo.

Topoisomerase-II activity in human leukemic and lymphoblastoid cells

Topoisomerase-II activity was analyzed in various human leukemic and lymphoblastoid cell-lines with comparison to normal human peripheral blood lymphocytes. All of the examined tumor cells contained this enzyme in both the nuclear and cytoplasmic fractions, whereas no appreciable activity of the enzyme was detected in either fraction of the resting normal lymphocytes. Using pBR322 plasmid as a substrate, undialyzed extracts of the tumor cells exhibited the typical ATP-dependent relaxation of supercoiled circles and formation of linear and catenated structures, as well as the ATP-independent knotting activity. On the other hand, dialyzed extracts exerted only the ATP-dependent supercoil relaxation. Novobiocin inhibited the linearization and catenation but not the supercoil relaxing or knotting activities. This study provides indications for an excessive level of a structurally abnormal topoisomerase-II in these tumor cell-lines.

Oxidative stress protection by novel telomerase activators in mesenchymal stem cells derived from healthy and diseased individuals.

Human Bone Marrow Mesenchymal Stem cells (hMSC) are a promising candidate for cytotherapy. However, the therapeutic potential is limited since the therapy requires ex-vivo cell culturing in which deterioration in cellular viability and aging is observed with time.Telomerase ribonucleoprotein complex re-elongates telomeres and therefore promotes genomic integrity, proliferation and lifespan. Recently we showed that increasing telomerase reverse transcriptase (TERT) expression by novel compound confers resistance from apoptosis induced by oxidative stress. Here we investigated the possibility that a controlled induction of human TERT (hTERT) levels by chemical compounds (AGS-499 and AGS-500) might improve the functionality of hMSC derived from healthy and neurodegenerative diseased individuals. We demonstrate that AGS treatments of hMSC increased telomerase activity and hTERT levels in a time and dose dependent manner. Prolonged treatments with the compounds increased the average telomeres length, without altering population doublings (PD) or inducing chromosomal aberrations. AGS treatments of hMSC protected the cells from apoptosis and DNA damages induced by H2O2, and from the toxicity induced by long term exposure to DMSO. These AGS effects were shown to be mediated by telomerase since they were not observed when TERT was depleted from hMSC or in mouse embryonic stem cells derived from TERT knockout mice. Furthermore, AGS compounds did not alter the functionality of hMSC as examined by their ability to differentiate into various lineages in the presence of the compounds. These results suggest that pharmaceutical increase of telomerase may confer a beneficial therapeutic advantage in regenerative medicine when hMSC therapy is applied.

DNA binding properties of the zinc‐bound and zinc‐free HIV nucleocapsid protein: supercoiled DNA unwinding and DNA‐protein cleavable complex formation

The HIV nucleocapsid (NC) protein contains, as those of other retroviruses, two Cys‐His arrays which function as zinc finger binding domains. The nucleic acid binding properties of retroviral NC have been previously demonstrated. In this study, we characterized the DNA binding ability of the zinc‐bound and zinc‐free forms of HIV NC. We found that in addition to binding single‐stranded DNA, both forms bind and unwind supercoiled plasmid DNA. The binding ability of the zinc‐bound form was higher than the zinc‐free form. In addition we showed the formation of NC protein‐DNA cleavable complex which is the result of a presumably covalent bond formed between the protein and the phosphate moiety of the DNA backbone. The NC unwinding activity and the protein‐DNA cleavable complex formation resembles the first step of the relaxing mechanism of DNA topoisomerase. Our results shed light on the possibility of a novel physiological function for the HIV NC protein in the viral life cycle.