Mary B. Sevigny

Poly(ADP-ribose) glycohydrolase mediates oxidative and excitotoxic neuronal death

Excessive activation of poly(ADP-ribose) polymerase 1 (PARP1) leads to NAD+ depletion and cell death during ischemia and other conditions that generate extensive DNA damage. When activated by DNA strand breaks, PARP1 uses NAD+ as substrate to form ADP-ribose polymers on specific acceptor proteins. These polymers are in turn rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG), a ubiquitously expressed exo- and endoglycohydrolase. In this study, we examined the role of PARG in the PARP1-mediated cell death pathway. Mouse neuron and astrocyte cultures were exposed to hydrogen peroxide, N-methyl-d-aspartate (NMDA), or the DNA alkylating agent, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Cell death in each condition was markedly reduced by the PARP1 inhibitor benzamide and equally reduced by the PARG inhibitors gallotannin and nobotanin B. The PARP1 inhibitor benzamide and the PARG inhibitor gallotannin both prevented the NAD+ depletion that otherwise results from PARP1 activation by MNNG or H2O2. However, these agents had opposite effects on protein poly(ADP-ribosyl)ation. Immunostaining for poly(ADP-ribose) on Western blots and neuron cultures showed benzamide to decrease and gallotannin to increase poly(ADP-ribose) accumulation during MNNG exposure. These results suggest that PARG inhibitors do not inhibit PARP1 directly, but instead prevent PARP1-mediated cell death by slowing the turnover of poly(ADP-ribose) and thus slowing NAD+ consumption. PARG appears to be a necessary component of the PARP-mediated cell death pathway, and PARG inhibitors may have promise as neuroprotective agents.

The Role of Cadmium and Nickel in Estrogen Receptor Signaling and Breast Cancer: Metalloestrogens or Not?

During the past half-century, incidences of breast cancer have increased globally. Various factors—genetic and environmental—have been implicated in the initiation and progression of this disease. One potential environmental risk factor that has not received a lot of attention is the exposure to heavy metals. While several mechanisms have been put forth describing how high concentrations of heavy metals play a role in carcinogenesis, it is unclear whether chronic, low-level exposure to certain heavy metals (i.e., cadmium and nickel) can directly result in the development and progression of cancer. Cadmium and nickel have been hypothesized to play a role in breast cancer development by acting as metalloestrogens—metals that bind to estrogen receptors and mimic the actions of estrogen. Since the lifetime exposure to estrogen is a well-established risk factor for breast cancer, anything that mimics its activity will likely contribute to the etiology of the disease. However, heavy metals, depending on their concentration, are capable of binding to a variety of proteins and may exert their toxicities by disrupting multiple cellular functions, complicating the analysis of whether heavy metal-induced carcinogenesis is mediated by the estrogen receptor. The purpose of this review is to discuss the various epidemiological, in vivo, and in vitro studies that show a link between the heavy metals, cadmium and nickel, and breast cancer development. We will particularly focus on the studies that test whether these two metals act as metalloestrogens in order to assess the strength of the data supporting this hypothesis.

Glycosylation regulates turnover of cyclooxygenase-2

Cyclooxygenase‐2 (COX‐2) catalyzes the rate‐limiting step in the prostanoid biosynthesis pathway, converting arachidonic acid into prostaglandin H2. COX‐2 exists as 72 and 74 kDa glycoforms, the latter resulting from an additional oligosaccharide chain at residue Asn580. In this study, Asn580 was mutated to determine the biological significance of this variable glycosylation. COS‐1 cells transfected with the mutant gene were unable to express the 74 kDa glycoform and were found to accumulate more COX‐2 protein and have five times greater COX‐2 activity than cells expressing both glycoforms. Thus, COX‐2 turnover appears to depend upon glycosylation of the 72 kDa glycoform.

Expression and activity of poly(ADP-ribose) glycohydrolase in cultured astrocytes, neurons, and C6 glioma cells.

Poly(ADP-ribose) metabolism plays a major role in DNA repair, transcription, replication, and recombination. Poly(ADP-ribose) polymerases are localized primarily to the nucleus, whereas significant levels of poly(ADP-ribose) glycohydrolase (PARG) are believed to be located in the cytoplasm. Only one PARG gene has been identified, but prior studies have reported multiple products of this gene. Here we studied PARG activity and PARG gene expression in several CNS cell types that span the cell growth spectrum: rapidly dividing C6 glioma tumor cells, dividing astrocytes, non-dividing astrocytes (due to contact inhibition), and post-mitotic neurons. Activity assays showed no overall differences between these cell types, but the nuclear to cytoplasmic ratio of PARG activity was highest in C6 glioma cells and lowest in neurons. Western blotting revealed full-length PARG as well as lower molecular weight PARG species in all four cell types.

Acute and chronic cadmium exposure promotes E-cadherin degradation in MCF7 breast cancer cells.

Cadmium is an environmental carcinogen that usually enters the body at minute concentrations through diet or cigarette smoke and bioaccumulates in soft tissues. In past studies, cadmium has been shown to contribute to the development of more aggressive cancer phenotypes including increased cell migration and invasion. This study aims to determine if cadmium exposure—both acute and chronic—contributes to breast cancer progression by interfering with the normal functional relationship between E‐cadherin and β‐catenin. An MCF7 breast cancer cell line (MCF7‐Cd) chronically exposed to 10−7 M CdCl2 was previously developed and used as a model system to study chronic exposures, whereas parental MCF7 cells exposed to 10−6 M CdCl2 for short periods of time were used to study acute exposures. Cadmium exposure of MCF7 cells led to the degradation of the E‐cadherin protein via the ubiquitination pathway. This resulted in fewer E‐cadherin/β‐catenin complexes and the relocation of active β‐catenin to the nucleus, where it interacted with transcription factor TCF‐4 to modulate gene expression. Interestingly, only cells chronically exposed to cadmium showed a significant decrease in the localization of β‐catenin to the plasma membrane and an increased distance between cells. Our data suggest that cadmium exposure promotes breast cancer progression by (1) down‐regulating E‐cadherin, thus decreasing the number of E‐cadherin/β‐catenin adhesion complexes, and (2) enhancing the nuclear translocation of β‐catenin to increase expression of cancer‐promoting proteins (i.e., c‐Jun and cyclin D1).

Glycosylation of human cyclooxygenase-2 (COX-2) decreases the efficacy of certain COX-2 inhibitors

Prostanoids play an important role in a variety of physiological and pathophysiological processes including inflammation and cancer. The rate-limiting step in the prostanoid biosynthesis pathway is catalyzed by cyclooxygenase-2 (COX-2). COX-2 exists as two glycoforms, 72 and 74 kDa, the latter resulting from an additional glycosylation at Asn(580). In this study, Asn(580) was mutated, and the mutant and wild-type COX-2 genes were expressed in COS-1 cells to determine how glycosylation affects the inhibition of COX-2 activity by aspirin, flurbiprofen, ibuprofen, celecoxib, and etoricoxib. Results indicate that certain inhibitors were 2-5 times more effective at inhibiting COX-2 activity when the glycosylation site was eliminated, indicating that glycosylation of COX-2 at Asn(580) decreases the efficacy of some inhibitors.

Heat Shock - Induced Hsp70 Expression in Murine Astrocytes Does not Require Poly(ADP-ribose) Polymerase Activity

Poly(ADP-ribose) polymerase (PARP) activity is involved in DNA repair, replication, recombination, and transcription. Extensive activation of the most abundant PARP, PARP-1, during ischemia or inflammation can promote cell death. PARP inhibitors reduce this cell death and are currently under investigation for use as therapeutic agents. A recent study found that PARP activation was required for Hsp70 upregulation in heat-exposed Drosophila larvae. Here we sought to determine whether PARP activity is likewise required for Hsp70 upregulation in mammalian cells, since many of the settings in which PARP inhibitors are candidate therapeutic agents are also settings in which Hsp70 expression is an important component of the stress response. We examined this issue using murine astrocyte cultures, a mammalian preparation in which the Hsp70 response has been well characterized, and found that PARP inhibitors had no effect on heat shock-induced Hsp70 protein expression. PARP-1-/- astrocytes gave similar results. The present findings indicate that PARP activity, and specifically PARP-1, is not required for upregulation of Hsp70 expression in mammalian cells.Basel