Diana Casper

ATM protein expression correlates with radioresistance in primary glioblastoma cells in culture

PURPOSE Glioblastoma multiforme (GBM) is one of the malignancies most resistant to radiation therapy. In contrast, cells derived from individuals with ataxia telangiectasia (AT), possessing mutations in the ATM gene, demonstrate increased sensitivity to ionizing radiation. Using a collection of glioma specimens adapted to tissue culture and several established GBM cell lines, we investigated the relationship between ATM protein expression and radiosensitivity. The three aims of our study were to: (1) quantify ATM protein levels in cultured glioma cells; (2) measure the correlation between ATM protein levels and radiation sensitivity; and (3) examine the dependence of ATM on p53 status. METHODS AND MATERIALS Glioma specimens were collected, catalogued, and adapted to grow in culture. Levels of ATM, p53, and p21 proteins were determined by Western blot. Radiation sensitivities were determined by clonogenic assays. p53 mutation status was determined by DNA sequencing. Correlations were identified by linear regression analysis. RESULTS ATM protein levels were variable in the primary gliomas. Glioma cell lines demonstrated significantly lower levels of ATM protein. Clonogenic assays of cell strains and cell lines yielded survival fractions (SF2s) consistent with the radioresistant behavior of GBM tumors in vivo. Regression analysis revealed a high correlation between ATM protein levels and SF2 for primary glioma cell strains, but not for established GBM cell lines. p53 status failed to predict radiosensitivity. CONCLUSION We have demonstrated that while our collection of low passage cell cultures depends on ATM for their resistance to IR, established cell lines may acquire adaptive characteristics which downplay the role of the ATM gene product in vitro. Therefore, attenuating ATM gene expression may be a successful strategy in the treatment of GBM tumors.

PGE2 receptor EP1 renders dopaminergic neurons selectively vulnerable to low-level oxidative stress and direct PGE2 neurotoxicity

Oxidative stress and increased cyclooxygenase‐2 (COX‐2) activity are both implicated in the loss of dopaminergic neurons from the substantia nigra (SN) in idiopathic Parkinsons disease (PD). Prostaglandin E2 (PGE2) is one of the key products of COX‐2 activity and PGE2 production is increased in PD. However, little is known about its role in the selective death of dopaminergic neurons. Previously, we showed that oxidative stress evoked by low concentrations of 6‐hydroxydopamine (6‐OHDA) was selective for dopaminergic neurons in culture and fully dependent on COX‐2 activity. We postulated that this loss was mediated by PGE2 acting through its receptors, EP1, EP2, EP3, and EP4. Using double‐label immunohistochemistry for specific EP receptors and tyrosine hydroxylase (TH), we identified EP1 and EP2 receptors on dopaminergic neurons in rat SN. EP2 receptors were also found in non‐dopaminergic neurons of this nucleus, as were EP3 receptors, whereas the EP4 receptor was absent. PGE2, 16‐phenyl tetranor PGE2 (a stable synthetic analogue), and 17‐phenyl trinor PGE2 (an EP1 receptor–selective agonist) were significantly toxic to dopaminergic cells at nanomolar concentrations; EP2‐ and EP3‐selective agonists were not. We challenged dopaminergic neurons in embryonic rat mesencephalic primary neuronal cultures and tested whether these receptors mediate selective 6‐OHDA toxicity. The nonselective EP1–3 receptor antagonist AH‐6809 and two selective EP1 antagonists, SC‐19220 and SC‐51089, completely prevented the 40%–50% loss of dopaminergic neurons caused by exposure to 5 μM 6‐OHDA. Together, these results strongly implicate PGE2 activation of EP1 receptors as a mediator of selective toxicity in this model of dopaminergic cell loss.

Electromagnetic fields as first messenger in biological signaling: Application to calmodulin-dependent signaling in tissue repair

BACKGROUND The transduction mechanism for non-thermal electromagnetic field (EMF) bioeffects has not been fully elucidated. This study proposes that an EMF can act as a first messenger in the calmodulin-dependent signaling pathways that orchestrate the release of cytokines and growth factors in normal cellular responses to physical and/or chemical insults. METHODS Given knowledge of Ca(2+) binding kinetics to calmodulin (CaM), an EMF signal having pulse duration or carrier period shorter than bound Ca(2+) lifetime may be configured to accelerate binding, and be detectable above thermal noise. New EMF signals were configured to modulate calmodulin-dependent signaling and assessed for efficacy in cellular studies. RESULTS Configured EMF signals modulated CaM-dependent enzyme kinetics, produced several-fold increases in key second messengers to include nitric oxide and cyclic guanosine monophosphate in chondrocyte and endothelial cultures and cyclic adenosine monophosphate in neuronal cultures. Calmodulin antagonists and downstream blockers annihilated these effects, providing strong support for the proposed mechanism. CONCLUSIONS Knowledge of the kinetics of Ca(2+) binding to CaM, or for any ion binding specific to any signaling cascade, allows the use of an electrochemical model by which the ability of any EMF signal to modulate CaM-dependent signaling can be assessed a priori or a posteriori. Results are consistent with the proposed mechanism, and strongly support the Ca/CaM/NO pathway as a primary EMF transduction pathway. GENERAL SIGNIFICANCE The predictions of the proposed model open a host of significant possibilities for configuration of non-thermal EMF signals for clinical and wellness applications that can reach far beyond fracture repair and wound healing.

Dopaminergic neurotoxicity by 6-OHDA and MPP+: differential requirement for neuronal cyclooxygenase activity.

Cyclooxygenase (COX), a key enzymatic mediator of inflammation, is present in microglia and surviving dopaminergic neurons in Parkinsons disease (PD), but its role and place in the chain of neurodegenerative events is unclear. Epidemiologic evidence showed that regular use of nonsteroidal antiinflammatory drugs (NSAIDs), specifically non‐aspirin COX inhibitors like ibuprofen, lowers the risk for PD; however, the putative cause‐and‐effect relationship between COX activity in activated microglia and neuronal loss was challenged recently. We examined whether neuronal COX activity is involved directly in dopaminergic cell death after neurotoxic insult. Using low concentrations of 6‐hydroxydopamine (6‐OHDA) and 1‐methyl‐4‐phenylpyridium ion (MPP+), neurotoxicants used to model selective dopaminergic cell loss in PD, and cultures of embryonic rat mesencephalic neurons essentially devoid of glia, we tested whether the nonselective COX inhibitor ibuprofen attenuated 6‐OHDA and MPP+ neurotoxicity. At levels close to its IC50 for both COX isoforms, ibuprofen protected dopaminergic neurons against 6‐OHDA but not MPP+ toxicity. Experiments with selective inhibitors of COX‐1 (SC‐560) and COX‐2 (NS‐398 and Cayman 10404), indicated that COX‐2, but not COX‐1, was involved in 6‐OHDA toxicity. Accordingly, 6‐OHDA, but not MPP+, increased prostaglandin (PG) levels twofold and this increase was blocked by ibuprofen. At concentrations well above its IC50 for COX, ibuprofen also prevented MPP+ toxicity, but had only limited efficacy against loss of structural complexity. Taken together, our data suggest that selective 6‐OHDA toxicity to dopaminergic neurons is associated with neuronal COX‐2, whereas MPP+ toxicity is COX independent. This difference may be important for understanding and manipulating mechanisms of dopaminergic cell death.

Sustained high levels of neuregulin‐1 in the longest‐lived rodents; a key determinant of rodent longevity

Naked mole‐rats (Heterocephalus glaber), the longest‐lived rodents, live 7–10 times longer than similarly sized mice and exhibit normal activities for approximately 75% of their lives. Little is known about the mechanisms that allow them to delay the aging process and live so long. Neuregulin‐1 (NRG‐1) signaling is critical for normal brain function during both development and adulthood. We hypothesized that long‐lived species will maintain higher levels of NRG‐1 and that this contributes to their sustained brain function and concomitant maintenance of normal activity. We monitored the levels of NRG‐1 and its receptor ErbB4 in H. glaber at different ages ranging from 1 day to 26 years and found that levels of NRG‐1 and ErbB4 were sustained throughout development and adulthood. In addition, we compared seven rodent species with widely divergent (4–32 year) maximum lifespan potential (MLSP) and found that at a physiologically equivalent age, the longer‐lived rodents had higher levels of NRG‐1 and ErbB4. Moreover, phylogenetic independent contrast analyses revealed that this significant strong correlation between MLSP and NRG‐1 levels was independent of phylogeny. These results suggest that NRG‐1 is an important factor contributing to divergent species MLSP through its role in maintaining neuronal integrity.

Enhanced vascularization and survival of neural transplants with ex vivo angiogenic gene transfer

Restoration of brain function by neural transplants is largely dependent upon the survival of donor neurons. Unfortunately, in both rodent models and human patients with Parkinsons disease the survival rate of transplanted neurons has been poor. We have employed a strategy to increase the availability of nutrients to the transplant by increasing the rate at which blood vessels are formed. Replication-deficient HSV-1 vectors containing the cDNA for human vascular endothelial growth factor (HSVhvegf) and the bacterial β-galacto-sidase gene (HSVlac) have been transduced in parallel into nonadherent neuronal aggregate cultures made of cells from embryonic day 15 rat mesencephalon. Gene expression from HSVlac was confirmed in fixed preparations by staining with X-gal. VEGF expression as determined by sandwich ELISA assay of culture supernatant was up to 322-fold higher in HSVhvegf-infected than HSVlac-infected sister cultures. This peptide was also biologically active, inducing endothelial cell proliferation in vitro. Adult Sprague-Dawley rats received bilateral transplants into the striatum, with HSVlac on one side and HSVhvegf on the other. At defined intervals up to 8 weeks, animals were sacrificed and vibratome sections of the striatum were assessed for various parameters of cell survival and vascularization. Results demonstrate dose-dependent increases in blood vessel density within transplants transduced with HSVhvegf. These transplants were vascularized at a faster rate up to 4 weeks after transplantation. After 8 weeks, the average size of the HSVhvegf-infected transplants was twice that of controls. In particular, the survival of transplanted dopaminergic neurons increased 3.9-fold. Taken together these experiments provide convincing evidence that the rate of vascularization may be a major determinant of neuronal survival that can be manipulated by VEGF gene transduction.

Prostaglandin receptor EP2 protects dopaminergic neurons against 6-OHDA-mediated low oxidative stress.

Dopaminergic neurons in the substantia nigra (SN) selectively die in Parkinsons disease (PD), but it is unclear how and why this occurs. Recent findings implicate prostaglandin E(2) (PGE(2)) and two of its four receptors, namely EP1 and EP2, as mediators of degenerative and protective events in situations of acute and chronic neuronal death. EP1 activation can exacerbate excitotoxic damage in stroke models and our recent study showed that EP1 activation may explain the selective sensitivity of dopaminergic neurons to oxidative stress. Conversely, EP2 activation may be neuroprotective, although toxic effects have also been demonstrated. Here we investigated if and how EP2 activation might alter the survival of dopaminergic neurons following selective low-level oxidative injury evoked by the neurotoxin 6-hydroxydopamine (6-OHDA) in primary neuronal cultures prepared from embryonic rat midbrain. We found that cultured dopaminergic neurons displayed EP2 receptors. Butaprost, a selective EP2 agonist, significantly reduced 6-OHDA neurotoxicity. EP2 receptors are coupled to stimulatory G-proteins (Gs), which activate adenylate cyclase, increasing cAMP synthesis, which then activates protein kinase A (PKA). Both dibutyryl cAMP and forskolin reduced dopaminergic cell loss after 6-OHDA exposure. Conversely, KT5720 and H-89, two structurally distinct high-affinity PKA inhibitors, abolished the protective effect of butaprost, implicating cAMP-dependent PKA activity in the neuroprotection by EP2 activation. Finally, we show that melanized dopaminergic neurons in the human SN express EP2. This pathway warrants consideration as a neuroprotective strategy for PD.

Stimulation of choline acetyltransferase activity by retinoic acid and sodium butyrate in a cultured human neuroblastoma

Choline acetyltransferase (Acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6, abbreviated ChAT), the biosynthetic enzyme for acetylcholine and acetylcholinesterase (EC 3.1.1.7, abbreviated AChE) are expressed in a human cholinergic neuroblastoma cell line, MC-IXC. We have shown that ChAT activity can be regulated in culture by retinoic acid, an active metabolite of vitamin A, and by sodium butyrate, an organic fatty acid. Optimal concentrations of these agents produce 4.3-fold and 1.6-fold increases in ChAT activity, respectively. The effects of retinoic acid are statistically significant after 24 h, whereas for sodium butyrate significant differences are seen only after 48 h. Since retinoic acid stimulation of ChAT activity was reversed only by trypsin treatment and not by removal of retinoic acid from the medium, this suggests that this agent may be acting at the level of the cell surface. Other differentiating conditions, such as culture in serum-free medium or addition of 1-2% dimethylsulfoxide did not increase ChAT activity. Acetylcholinesterase activity was shown to increase only in the presence of sodium butyrate, suggesting that retinoic acid and sodium butyrate may be acting via different pathways. Retinoic acid and sodium butyrate both seem to be permissive rather than instructive in regulating ChAT activity in that they are unable to induce ChAT expression de novo in cell lines which do not already express ChAT activity.

β-DICARBONYL ENOLATES: A NEW CLASS OF NEUROPROTECTANTS

J. Neurochem. (2011) 116, 132–143.

Aberrant expression of interleukin-1β and inflammasome activation in human malignant gliomas.

Objective Glioblastoma is the most frequent and malignant form of primary brain tumor with grave prognosis. Mounting evidence supports that chronic inflammation (such as chronic overactivation of IL-1 system) is a crucial event in carcinogenesis and tumor progression. IL-1 also is an important cytokine with species-dependent regulations and roles in CNS cell activation. While much attention is paid to specific anti-tumor immunity, little is known about the role of chronic inflammation/innate immunity in glioma pathogenesis. In this study, we examined whether human astrocytic cells (including malignant gliomas) can produce IL-1 and its role in glioma progression. Methods We used a combination of cell culture, real-time PCR, ELISA, western blot, immunocytochemistry, siRNA and plasmid transfection, micro-RNA analysis, angiogenesis (tube formation) assay, and neurotoxicity assay. Results Glioblastoma cells produced large quantities of IL-1 when activated, resembling macrophages/microglia. The activation signal was provided by IL-1 but not the pathogenic components LPS or poly IC. Glioblastoma cells were highly sensitive to IL-1 stimulation, suggesting its relevance in vivo. In human astrocytes, IL-1β mRNA was not translated to protein. Plasmid transfection also failed to produce IL-1 protein, suggesting active repression. Suppression of microRNAs that can target IL-1α/β did not induce IL-1 protein. Glioblastoma IL-1β processing occurred by the NLRP3 inflammasome, and ATP and nigericin increased IL-1β processing by upregulating NLRP3 expression, similar to macrophages. RNAi of annexin A2, a protein strongly implicated in glioma progression, prevented IL-1 induction, demonstrating its new role in innate immune activation. IL-1 also activated Stat3, a transcription factor crucial in glioma progression. IL-1 activated glioblastoma-conditioned media enhanced angiogenesis and neurotoxicity. Conclusions Our results demonstrate unique, species-dependent immune activation mechanisms involving human astrocytes and astrogliomas. Specifically, the ability to produce IL-1 by glioblastoma cells may confer them a mesenchymal phenotype including increased migratory capacity, unique gene signature and proinflammatory signaling.