Antiretroviral drugs induce oxidative stress and neuronal damage in the central nervous system.

HIV-associated neurocognitive disorder (HAND), characterized by a wide spectrum of behavioral, cognitive, and motor dysfunctions, continues to affect approximately 50 % of HIV(+) patients despite the success of combination antiretroviral drug therapy (cART) in the periphery. Of note, potential toxicity of antiretroviral drugs in the central nervous system (CNS) remains remarkably underexplored and may contribute to the persistence of HAND in the cART era. Previous studies have shown antiretrovirals (ARVs) to be neurotoxic in the peripheral nervous system in vivo and in peripheral neurons in vitro. Alterations in lipid and protein metabolism, mitochondrial damage, and oxidative stress all play a role in peripheral ARV neurotoxicity. We hypothesized that ARVs also induce cellular stresses in the CNS, ultimately leading to neuronal damage and contributing to the changing clinical and pathological picture seen in HIV-positive patients in the cART era. In this report, we show that ARVs are neurotoxic in the CNS in both pigtail macaques and rats in vivo. Furthermore, in vitro, ARVs lead to accumulation of reactive oxygen species (ROS), and ultimately induction of neuronal damage and death. Whereas ARVs alone caused some activation of the endogenous antioxidant response in vitro, augmentation of this response by a fumaric acid ester, monomethyl fumarate (MMF), blocked ARV-induced ROS generation, and neuronal damage/death. These findings implicate oxidative stress as a contributor to the underlying mechanisms of ARV-induced neurotoxicity and will provide an access point for adjunctive therapies to complement ARV therapy and reduce neurotoxicity in this patient population.

Calpain-mediated degradation of MDMx/MDM4 contributes to HIV-induced neuronal damage.

Neuronal damage in HIV-associated Neurocognitive Disorders (HAND) has been linked to inflammation induced by soluble factors released by HIV-infected, and non-infected, activated macrophages/microglia (HIV M/M) in the brain. It has been suggested that aberrant neuronal cell cycle activation determines cell fate in response to these toxic factors. We have previously shown increased expression of cell cycle proteins such as E2F1 and phosphorylated pRb in HAND midfrontal cortex in vivo and in primary neurons exposed to HIV M/M supernatants in vitro. In addition, we have previously shown that MDMx (also referred to as MDM4), a negative regulator of E2F1, was decreased in the brain in a primate model of HIV-induced CNS neurodegeneration. Thus, we hypothesized that MDMx provides indirect neuroprotection from HIV-induced neurodegeneration in our in vitro model. In this report, we found significant reductions in MDMx protein levels in the mid-frontal cortex of patients with HAND. In addition, treatment of primary rat neuroglial cultures with HIV M/M led to NMDA receptor- and calpain-dependent degradation of MDMx and decreased neuronal survival, while overexpression of MDMx conferred partial protection from HIV M/M toxicity in vitro. Further, our results demonstrate that MDMx is a novel and direct calpain substrate. Finally, blocking MDMx activity led to neuronal death in vitro in the absence of toxic stimulus, which was reversed by calpain inhibition. Overall, our results indicate that MDMx plays a pro-survival role in neurons, and that strategies to stabilize and/or induce MDMx can provide neuroprotection in HAND and in other neurodegenerative diseases where calpain activation contributes to neuropathogenesis.

Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration.

Neurocognitive deficits seen in HIV-associated neurocognitive disorders (HANDs) are attributed to the release of soluble factors from CNS-resident, HIV-infected and/or activated macrophages and microglia. To study HIV-associated neurotoxicity, we used our in vitro model in which primary rat neuronal/glial cultures are treated with supernatants from cultured human monocyte-derived macrophages, infected with a CNS-isolated HIV-1 strain (HIV-MDM). We found that neuronal damage, detected as a loss of microtubule-associated protein-2 (MAP2), begins as early as 2h and is preceded by a loss of mitochondrial membrane potential (Δψ(m)). Interestingly, inhibitors of calpains, but not inhibitors of caspases, blocked MAP2 loss, however neither type of inhibitor prevented the loss of Δψ(m). To facilitate throughput for these studies, we refined a MAP2 cell-based-ELISA whose data closely compare with our standardized method of hand counting neurons. In addition, we developed a tetramethyl rhodamine methyl ester (TMRM)-based multi-well fluorescent plate assay for the evaluation of whole culture Δψ(m). Together, these findings indicate that calpain activation and loss of Δψ(m) may be parallel pathways to death in HIV-MDM-treated neurons and also demonstrate the validity of plate assays for assessing multiple experimental parameters as is useful for screening neurotherapeutics for neuronal damage and death.

E2F1 localizes predominantly to neuronal cytoplasm and fails to induce expression of its transcriptional targets in human immunodeficiency virus-induced neuronal damage.

As human immunodeficiency virus (HIV) does not induce neuronal damage by direct infection, the mechanisms of neuronal damage or loss in HIV-associated dementia (HAD) remain unclear. We have shown previously that immunoreactivity of transcription factor, E2F1, increases in neurons, localizing predominantly to the cytoplasm, in HIV-associated pathologies. Here we confirm that E2F1 localization is predominantly cytoplasmic in primary postmitotic neurons in vitro and cortical neurons in vivo. To determine whether E2F1 contributes to neuronal death in HAD via transactivation of target promoters, we assessed the mRNA and protein levels of several classical E2F1 transcriptional targets implicated in cell cycle progression and apoptosis in an in vitro model of HIV-induced neurotoxicity and in cortical autopsy tissue from patients infected with HIV. By Q-PCR, we show that mRNA levels of E2F1 transcriptional targets implicated in cell cycle progression (E2F1, Cyclin A, proliferating cell nuclear antigen (PCNA), and dyhydrofolate reductase (DHFR)) and apoptosis (caspases 3, 8, 9 and p19(ARF)) remain unchanged in an in vitro model of HIV-induced neurotoxicity. Further, we show that protein levels of p19(ARF), Cyclin A, and PCNA are not altered in vitro or in the cortex of patients with HAD. We propose that the predominantly cytoplasmic localization of E2F1 in neurons may account for the lack of E2F1 target transactivation in neurons responding to HIV-induced neurotoxicity.

Improved mobilization of peripheral blood CD34+ cells and dendritic cells by AMD3100 plus granulocyte-colony-stimulating factor in non-Hodgkin's lymphoma patients.

AMD3100 is a drug capable of mobilizing peripheral blood stem cells (PBSCs) in donors and in cancer patients as a single agent or in combination with granulocyte-colony-stimulating factor (G-CSF). We initiated a phase II study of 11 refractory or relapsed non-Hodgkin's lymphoma (NHL) patients, receiving 16 microg/kg daily of G-CSF for 4 days followed by 240 microg/kg of AMD3100 given subcutaneously on a new schedule of 9-10 h before apheresis collection on day 5. Our aims were to assess the effect of AMD3100 on the mobilization of CD34+ cells, dendritic cells (DCs) and lymphoma cells. Administration of G-CSF and AMD3100 were continued daily until >or=2 x 10(6) CD34+ cells/kg were collected. Adequate collection of the target of CD34+ cells was achieved in all but 1 patient within 2 days, and 10/11 patients were transplanted within 2 months. All transplanted patients engrafted with a mean of 10 and 12 days for neutrophils and platelets, respectively. Addition of AMD3100 to G-CSF resulted with >2.5-fold increase in CD34+ cells/microl (p = 0.0001) and in a >2-fold increase in pDC1 and pDC2 cells/microl (p = 0.003). Adverse events related to AMD3100 were minimal. AMD3100 was generally safe and improved PBSC and DC cell mobilization with no apparent contamination of lymphoma cells.

Activation of cyclin-dependent kinase 5 by calpains contributes to human immunodeficiency virus-induced neurotoxicity.

Although the specific mechanism of neuronal damage in human immunodeficiency virus (HIV) -associated dementia is not known, a prominent role for NMDA receptor (NMDAR)-induced excitotoxicity has been demonstrated in neurons exposed to HIV-infected/activated macrophages. We hypothesized NMDAR-mediated activation of the calcium-dependent protease, calpain, would contribute to cell death by induction of cyclin-dependent kinase 5 (CDK5) activity. Using an in vitro model of HIV neurotoxicity, in which primary rat cortical cultures are exposed to supernatants from primary human HIV-infected macrophages, we have observed increased calpain-dependent cleavage of the CDK5 regulatory subunit, p35, to the constitutively active isoform, p25. Formation of p25 is dependent upon NMDAR activation and calpain activity and is coincident with increased CDK5 activity in this model. Further, inhibition of CDK5 by roscovitine provided neuroprotection in our in vitro model. Consistent with our observations in vitro, we have observed a significant increase in calpain activity and p25 levels in midfrontal cortex of patients infected with HIV, particularly those with HIV-associated cognitive impairment. Taken together, our data suggest calpain activation of CDK5, a pathway activated in HIV-infected individuals, can mediate neuronal damage and death in a model of HIV-induced neurotoxicity.

Arsenic trioxide induces p53-dependent apoptotic signals in myeloma cells with SiRNA-silenced p53: MAP kinase pathway is preferentially activated in cells expressing inactivated p53.

Mutations in p53 are the most common genetic abnormality in cancers. Arsenic trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia (APL) and is being tested in phase II studies in various types of cancers. We have shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging primarily the intrinsic apoptotic pathway in cells expressing w.t. p53 and the extrinsic apoptotic pathway in cells expressing mutant p53. To further establish the differential apoptotic signals of ATO in relation to p53 functional status we studied the activation of the intrinsic and the extrinsic apoptotic pathways in IM9 myeloma cells expressing w.t. p53 following silencing of p53 and p21 with the corresponding SiRNAs-GFP constructs. In untransfected cells or in cells transfected with GFP-empty vector construct we observed weak apoptosis concomitant with mild depolarization of mitochondrial membrane, depletion of reduced glutathione and release of cytochrome c. Following silencing of p53 or p21 we observed extensive apoptosis concomitant with extensive depolarization of mitochondrial membrane and depletion of reduced glutathione. We also observed in these cells activation of the extrinsic apoptotic pathway through upregulation of APO2/TRAIL and APO2/TRAIL-R2, activation of caspase 8, degradation of FLIP-L and release of apoptosis inducing factors from mitochondria, instead of cytochrome c. In addition, we observed marked activation of the MAP kinase pathway and dephosphorylation of Akt in p53 or p21 silenced cells. Hence, silencing of p53 or p21 in IM9 myeloma cells results in diversion of apoptosis to the extrinsic pathway and sensitization of myeloma cells to ATO.

Granulocyte colony-stimulating factor mobilizes more dendritic cell subsets than granulocyte-macrophage colony-stimulating factor with no polarization of dendritic cell subsets in normal donors.

Dendritic cells (DCs) are effective antigen-presenting cells. We hypothesized that increasing the DC populations in donor lymphocyte infusions (DLIs) may augment the graft versus malignancy effect, particularly if granulocyte-macrophage colony-stimulating factor (GM-CSF) mobilization resulted in increased precursor dendritic cell (pDC) 1 cells. Mature DCs, pDC1 cells, pDC2 cells, and CD34(+) cells from the same donor were compared after granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cell collections and GM-CSF mobilized DLI collections. Mobilization with G-CSF resulted in up to a 10-fold larger number of CD34(+) cells per kg and a 3-5-fold larger number of mature DCs, pDC1 cells, and pDC2 cells within the same donor compared with GM-CSF. The ratio of pDC1 to pDC2 in each donor remained constant with either cytokine. In this small sample of normal donors, it appears that G-CSF mobilizes more CD34(+) cells, mature DCs, pDC1 cells, and pDC2 cells within the same donor than does GM-CSF, with no significant polarization by G-CSF or GM-CSF for either pDC1 or pDC2 cells.

No polarization of type 1 or type 2 precursor dendritic cells in peripheral blood stem cell collections of non-hodgkin's lymphoma patients mobilized with cyclophosphamide plus G-CSF, GM-CSF, or GM-CSF followed by G-CSF.

Dendritic cells (DCs) are the most efficient antigen-presenting cells and play a role in immune reconstitution after autologous transplantation. Recent reports suggest that mobilization with granulocyte colony-stimulating factor (G-CSF) containing regimens polarizes DCs into pDC2, which could potentially result with increased Th2 response and decreased graft-versus-host disease (GVHD) in allogeneic transplantation and with decreased cytotoxic Th1 response and graft versus tumor effect, which in autologous transplantation could translate into increased relapse rate. Previously, we have shown that non-Hodgkin's lymphoma (NHL) patients receiving cyclophosphamide (CTX) plus granulocyte- macrophage (GM)-CSF, G-CSF or GM-CSF followed by G-CSF for stem cell collection, mobilize up to five-fold more mature CD80(+) DCs compared to CTX plus G-CSF mobilized patients. Here, we analyzed samples from the same study for the number of pDC1 and pDC2 subsets in blood and apheresis products obtained from these patients. Samples from 29 patients were collected. Patients mobilized with CTX plus G-CSF collected a mean of 1.2 +/- 0.4 x 10(6) pDC1/kg per day and 2.2 +/- 1 x 10(6) pDC2/kg per day, whereas patients mobilized with CTX plus GM-CSF collected a mean of 1.1 +/- 0.5 x 10(6) pDC1 and 1.5 +/- 0.9 x 10(6) pDC2/kg per day. Patients mobilized with CTX plus GM-CSF followed by G-CSF collected 2.5 +/- 1.1 x 10(6) pDC1 and 2 +/- 0.5 x 106 pDC2/kg per day, with significantly higher levels of pDC1 +/- pDC2 cells. No significant difference was observed in pDC1/pDC2 ratio between the three mobilization arms. Patients mobilized with the GM-CSFcontaining regimen had a higher probability for survival compared to patients receiving G-CSF alone (median of 55 months vs. 15 months; p = 0.02). These results support the hypothesis that higher levels of DCs in the graft might be associated with prolonged survival of autotransplanted NHL patients. Further similar studies are merited in a larger population of NHL patients.

Arsenic trioxide: an anti cancer missile with multiple warheads.

The proven efficacy of ATO in the treatment of APL and the emerging importance of ATO in other diseases prompted extensive studies of the mechanisms of action of ATO in APL and in other types of cancers. In this review we will focus on downstream events in ATO-induced intrinsic and extrinsic apoptotic pathways with an emphasis on the role of pro-apoptotic and anti-apoptotic proteins and the role of p53 in ATO-induced apoptosis including its effect on cell cycle, its anti-mitotic effect and the role of apoptosis inducing factors (AIF) in ATO-induced apoptosis, chromatin condensation and nuclear fragmentation in myeloma cells as a model.

Arsenic trioxide and paclitaxel induce apoptosis by different mechanisms.

Arsenic trioxide (ATO) and paclitaxel (TAXOL) are effective in the treatment of various types of cancers. Both drugs induce G2/M arrest. We have previously shown that ATO is a potent inducer of apoptosis in myeloma cells expressing mutant p53 engaging both the intrinsic and extrinsic apoptotic pathways. Here we compared the effect of ATO and TAXOL on myeloma cells expressing mutant p53 and varying levels of Bcl-2. ATO rapidly induced Apo2/TRAIL, activation of caspase 8, cleavage of BID, depolarization of mitochondrial membrane (MM) and release of AIF from mitochondria in a Bcl-2 independent fashion. Apoptosis was associated with early formation of ring-like perinuclear condensed chromatin colocalized with AIF. In contrast, paclitaxel-induced apoptosis MM depolarization, cytochrome C release and activation of caspase 9 were all blocked by Bcl-2. Apoptosis was associated with a random chromatin condensation and nuclear fragmentation with no early involvement of AIF.

Mobilization of myeloma cells involves SDF-1/CXCR4 signaling and downregulation of VLA-4.

Adhesion molecules and stromal cell-derived factor-1 (SDF-1)/CXCR4 signaling play key roles in homing and mobilization of hematopoietic stem cells (HSC). Active signaling through SDF-1/CXCR4 and upregulation of adhesion molecules are required for homing, whereas downregulation of adhesion molecules and disruption of SDF-1/CXCR4 signaling are required for mobilization of HSC. We studied the surface expression of CXCR4 very late activation antigen (VLA)-4 and VLA-5 on myeloma cells mobilized with cyclophosphamide and GM-CSF in 12 multiple myeloma patients undergoing HSC mobilization for autologous transplantation. We also studied the plasma levels of SDF-1 in apheresis collection of these patients. We observed a statistically significant decrease in the levels of SDF-1 and surface expression of CXCR4 on myeloma cells in four consecutive apheresis collections compared with premobilization bone marrow specimens. We also observed a statistically significant decrease in surface expression of VLA-4 in myeloma cells in the apheresis collections compared with premobilization bone marrow samples. Furthermore, myeloma cells derived from apheresis collections had decreased adhesion and trans-stromal migration in response to SDF-1, which could be reversed by short incubation with interleukin-6. Hence, mobilization of myeloma cells involves SDF-1/CXCR4 signaling and downregulation of VLA-4.

Arsenic trioxide selectively induces early and extensive apoptosis via the APO2/caspase-8 pathway engaging the mitochondrial pathway in myeloma cells with mutant p53.

Arsenic trioxide (ATO) is effective in the treatment of acute promyelocytic leukemia (APL) and induces apoptosis in APL cells and in a great variety of other cancer cells. We have previously shown that ATO induces apoptosis in myeloma cells in two different modes depending on p53 status in the cells. In cells expressing mutated p53, ATO induced, G2/M arrest and activation caspase 8 and 3 and rapid and extensive apoptosis. Myeloma cells expressing w.t. p53, ATO induced G1 arrest and delayed apoptosis with activation of caspase 9 and 3. APO2/TRAIL receptor expression was induced in both cell types and APO2/TRAIL synergized with ATO in the induction of apoptosis. Here we tested the effect of ATO on mitochondrial membrane potential (MMP) in myeloma cells expressing mutated or w.t. p53. In myeloma cells expressing mutated p53, depolarization of MMP occurred early, concomitant with induction of APO2/TRAIL, activation of BID and release of AIF, preceding apoptosis. However, in cells expressing w.t. p53, APO2/TRAIL is not induced, BID is not cleaved and depolarization of MMP occurs concurrently with cytochrome c release and apoptosis. These results explain the greater sensitivity to ATO of cells with mutated p53 and suggest perhaps a general mechanism for ATO-induced apoptosis.