Transferase-mediated Biotinylated UTP Nick End Labeling Research Articles

AMA0428, A Potent Rock Inhibitor, Attenuates Early and Late Experimental Diabetic Retinopathy

ABSTRACTPurpose: Diabetic retinopathy (DR) is characterized by an early stage of inflammation and vessel leakage, and an advanced vasoproliferative stage. Also, neurodegeneration might play an important role in disease pathogenesis. The aim of this study was to investigate the effect of the Rho kinase (ROCK) inhibitor, AMA0428, on these processes.Methods: The response to ROCK inhibition by AMA0428 (1 µg) was studied in vivo using the murine model for streptozotocin (STZ)-induced diabetes, focusing on early non-proliferative DR features and the oxygen-induced retinopathy (OIR) model to investigate proliferative DR. Intravitreal (IVT) administration of AMA0428 was compared with murine anti-VEGF-R2 antibody (DC101, 6.2 µg) and placebo (H2O/PEG; 1C8). Outcome was assessed by analyzing leukostasis using fluorescein isothiocyanate coupled concanavalin A (FITC-ConA) and vessel leakage (bovine serum albumin conjugated with fluorescein isothiocyanate; FITC-BSA)/neovascularization and neurodegeneration by immunohistological approaches (hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase–mediated biotinylated UTP nick end labeling (TUNEL), Brn3a). ELISA and Western blotting were employed to unravel the consequences of ROCK inhibition (1 µM AMA0428) on myosin phosphatase target protein (MYPT)-1 phosphorylation, endothelial nitric oxide synthase (eNOS) phosphorylation, and vascular endothelial growth factor (VEGF) levels in retinas of diabetic mice, on NF-κβ activity and ICAM-1 expression in endothelial cells (ECs).Results: In vivo, AMA0428 significantly reduced vessel leakage and neovascularization, respectively, in the STZ and OIR model, comparable to DC101 therapy. Additionally, the ROCK inhibitor decreased neurodegeneration in both models and inhibited leukostasis by 30% (p < 0.05) in the STZ model (p < 0.05), while DC101 had no positive effect on the outcome of these latter processes. ROCK activity was upregulated in the diabetic retina and AMA0428 administration resulted in decreased phospho-MYPT-1, enhanced phospho-eNOS, and reduced VEGF levels. In vitro, AMA0428 interfered with NF-κβ activity, thereby inhibiting ICAM-1 expression in ECs.Conclusions: Targeting ROCK with AMA0428 effectively attenuated outcome in an early DR model (STZ) and a late vasoproliferative retinopathy model (OIR). These findings make AMA0428 a promising candidate with an additional anti-inflammatory and neuroprotective benefit for DR patients, as compared with anti-VEGF treatment.

Neuroprotective effect of hydrogen sulfide on acute cauda equina injury in rats

BackgroundHydrogen sulfide (H2S), as a novel gaseous messenger molecule, plays an important role in signal transduction and biological modulation. PurposeIn the present study the effect of H2S after compression injury of cauda equina was studied. Study DesignThe setting of this study is the laboratory investigation. MethodsA total of 162 rats were randomly allocated into three groups: sham group, compression group, and H2S group. Cauda equina compression (CEC) injury in rats was induced by implanting silicone gels (10×1×1 mm) into the epidural spaces L5 and L6; laminectomy was performed at the L4 level of the vertebra in the sham-operated group. The experimental group was treated with sodium hydrosulfide intraperitoneally (20 µmol/kg body weight), whereas the compression and sham groups received equal volumes of physiological saline. Levels of malonaldehyde (MDA) and glutathione (GSH) were determined immediately before CEC surgery, 12 h, 24 h, 48 h, and 72 h after CEC surgery. Furthermore, hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling (TUNEL) assay were performed 48 h after CEC. ResultsHematoxylin and eosin staining showed that myelin sheath and the cauda equina fibers in the compression group were less compact and highly degenerated compared with the sham group, and that H2S treatment could improve the status. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling staining exhibited that decreased number of TUNEL positive cells was found in the H2S group than in the compression group. The level of MDA was increased in the sham and H2S groups compared with the compression group (p<.05, p<.01), whereas the level of GSH was decreased (p<.05, p<.01). ConclusionsWith the above data, we conclude that H2S could reduce the oxidative stress and has neuroprotective effect in acute cauda equina syndrome.

Hyperoxia-induced immature brain injury through the TLR4 signaling pathway in newborn mice

Toll-like receptor 4 (TLR4), a pathogen-associated molecular pattern receptor, is known to initiate an inflammatory cascade in response to certain stimuli within the central nervous system (CNS). Although TLR4 activation is known to be a first-line response of the innate immune system, whether and how hyperoxia influences TLR4 signaling in an immature brain remains unclear. In this study, TLR4 wild-type (W) and TLR4 knock-out(M) mice were exposed to 100% oxygen (the WO2 and MO2 groups, respectively), and control groups were exposed to ambient air (the WA and MA groups, respectively) for 48h after postnatal-day (PND) 3. Next, neuronal apoptosis was quantified, and Morris water maze assays were conducted. The WO2 mice showed increased TLR4 expression compared with the WA mice, additionally, the expression level of Tumor Necrosis Factor-α (TNF-α) in the WO2 mice was significantly increased compared with the levels in the WA, MA and MO2 mice. Electron microscopy and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assays showed a significant increase, compared to the WO2 mice, in neuronal apoptosis within the prefrontal cortex and hippocampal CA1 region in the WO2 mice. In contrast, there were no obvious differences in neuronal apoptosis between the MO2 and MA groups. The results of the Morris water maze tests demonstrated marked deficits in learning and memory in the WO2 mice but much milder deficits in the MO2 mice compared to the WA and MA groups, respectively. Moreover, cultured N9 (TLR4 wild-type, derived from ICR/CD1 mice) microglia exposed to hyperoxia showed an immediate increase in the expression of TLR4 mRNA, followed by an increase in the expression of both TNF-α and reactive oxygen species (ROS), but this increase was abrogated by the loss of TLR4 signaling in TLR4-knockout microglia (primary cells from a C3H/HeJ strain defective in TLR4). Taken together, these data suggest that 1) TLR4 signaling is involved in hyperoxia-induced immune responses in the immature brain and 2) the loss of TLR4 activation may abrogate the neuronal apoptosis and cognitive deficits following hyperoxia exposure in newborn mice.

Protective effects of Zhibai Dihuang Wan on renal tubular cells affected with gentamicin-induced apoptosis

Ethnopharmacological relevanceZhibai Dihuang Wan (ZDW) is an ancient traditional Chinese medicine composed of eight herbal ingredients and has been used to treat chronic kidney inflammation and diabetes for thousands of years. Nonetheless, the influence of ZDW on acute kidney injury is still unknown. We intended to identify the influence of ZDW on cell growth and gentamicin-induced apoptotic injury in renal tubular cells. Materials and methodsWe extracted ZDW with artificial intestinal fluid and treated rat renal tubular cells (NRK-52E) with various concentrations of the ZDW extraction. Cell proliferation and gentamicin-induced apoptosis of NRK-52E cells were evaluated using real-time proliferation monitoring and annexin V staining, respectively. Western blotting was used to evaluate the levels of Bcl-2 and caspase-3 expression. The effect of ZDW on gentamicin-induced kidney injury was also monitored in mice using the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay, and the measurement of serum creatinine and blood urea nitrogen. ResultsWe found that 30μg/ml of ZDW promoted cell proliferation of the rat renal tubular cells. ZDW also expressed a dose-dependent protective effect against gentamicin-induced apoptosis in the cells. Pretreatment with 3μg/ml or 30μg/ml of ZDW maximally increased Bcl-2 and decreased cleaved caspase-3 in the gentamicin-treated NRK-52E cells. Among the herbal ingredients of ZDW, only Phellodendron amurense Rupr., bark (Cortex Phellodendri), and Anemarrhena asphodeloides Bunge, rhizome inhibited both the gentamicin-induced Bcl-2 decrease and cleaved caspase-3 increase. Phellodendron amurense Rupr., bark and Anemarrhena asphodeloides Bunge, rhizome also inhibited gentamicin-induced apoptosis at particular concentrations; however, these two ingredients were less effective than ZDW. In the mouse model of gentamicin-induced nephropathy, the ZDW treatment significantly reduced apoptotic cells in the renal cortex and improved renal function. ConclusionsOur results suggest that ZDW at adequate doses attenuates gentamicin-induced apoptotic injury in renal tubular cells and also protects kidneys from gentamicin-induced injury in mice.

Umbilical Cord Blood-Derived Mesenchymal Stem Cells Inhibit, But Adipose Tissue-Derived Mesenchymal Stem Cells Promote, Glioblastoma Multiforme Proliferation

Mesenchymal stem cells (MSCs) possess self-renewal and multipotential differentiation abilities, and they are thought to be one of the most reliable stem cell sources for a variety of cell therapies. Recently, cell therapy using MSCs has been studied as a novel therapeutic approach for cancers that show refractory progress and poor prognosis. MSCs from different tissues have different properties. However, the effect of different MSC properties on their application in anticancer therapies has not been thoroughly investigated. In this study, to characterize the anticancer therapeutic application of MSCs from different sources, we established two different kinds of human MSCs: umbilical cord blood-derived MSCs (UCB-MSCs) and adipose-tissue-derived MSCs (AT-MSCs). We used these MSCs in a coculture assay with primary glioblastoma multiforme (GBM) cells to analyze how MSCs from different sources can inhibit GBM growth. We found that UCB-MSCs inhibited GBM growth and caused apoptosis, but AT-MSCs promoted GBM growth. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling assay clearly demonstrated that UCB-MSCs promoted apoptosis of GBM via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL was expressed more highly by UCB-MSCs than by AT-MSCs. Higher mRNA expression levels of angiogenic factors (vascular endothelial growth factor, angiopoietin 1, platelet-derived growth factor, and insulin-like growth factor) and stromal-derived factor-1 (SDF-1/CXCL12) were observed in AT-MSCs, and highly vascularized tumors were developed when AT-MSCs and GBM were cotransplanted. Importantly, CXCL12 inhibited TRAIL activation of the apoptotic pathway in GBM, suggesting that AT-MSCs may support GBM development in vivo by at least two distinct mechanisms-promoting angiogenesis and inhibiting apoptosis. The opposite effects of AT-MSCs and UCB-MSCs on GBM clearly demonstrate that differences must be considered when choosing a stem cell source for safety in clinical application.

Neuroprotective effects of breviscapine against apoptosis induced by transient focal cerebral ischaemia in rats

Breviscapine, a flavonoid isolated from the traditional Chinese medicinal herb Erigerin breviscapus, has been proved to be effective in protecting the brain against ischaemic damage, but the mechanisms remain unknown. In this study, we have demonstrated the effects of breviscapine on neuronal apoptosis in a rat model of transient focal cerebral ischaemia. Rats were administered with breviscapine (50 or 100 mg kg(-1)/day) intragastrically for seven successive days, then subjected to 2-h brain ischaemia induced by middle cerebral artery occlusion, followed by 24-h reperfusion. After reperfusion, the rats were killed and the brain samples were collected. Haematoxylin-eosin staining was used to evaluate the histopathological changes. Terminal deoxynucleotidyl transferase-mediated biotiny-lated UTP nick end labeling (TUNEL) and flow cytometry (FCM) analysis were used to detect the level of apoptosis. The expressions of bcl-2 and caspase-3 in the cortex were determined by Western blot. Significant increases in the number of haematoxylin-eosin- and TUNEL-positive staining cells and DNA fragmentation were observed at 24 h after reperfusion, and the increases were inhibited by breviscapine (50 and 100 mg kg(-1)). Breviscapine at both doses markedly inhibited the expression and activation of caspase-3 and up-regulated the expression of bcl-2. These findings suggested that breviscapine attenuated neuroapoptosis and regulated the protein expression related to apoptosis after transient focal cerebral ischaemia, which may have contributed, in part, to the protective effects of breviscapine on cerebral ischaemic damage.

Estradiol protects the cochlea against gentamicin ototoxicity through inhibition of the JNK pathway

Gentamicin induces outer hair cell death through the apoptotic pathway. It has been reported that this death pathway of outer hair cells is mediated by specific apoptotic enzymes including c-jun N-terminal kinase (JNK) and caspases. 17β-Estradiol (E2), the most potent estrogen, is known to function as an antiapoptotic agent to prevent the death of various cell types. The purpose of the present study was to examine the effects of E2 on gentamicin-induced apoptotic cell death in outer hair cells. The basal turn organ of Corti explants from p3 or p4 rats were maintained in a tissue culture and exposed to 100 μM gentamicin for 48 h. The effects of E2 on gentamicin-induced outer hair cell loss, JNK activation, and staining for terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling (TUNEL) were examined. E2 significantly decreased gentamicin-induced outer hair cell loss in a dose-dependent manner. JNK activation and TUNEL staining were observed in organ of Corti explants exposed to gentamicin, and staining levels were significantly decreased by E2 treatment. The results indicate that, through the inhibition of JNK and subsequent apoptotic reactions, E2 decreases outer hair cell loss induced by gentamicin ototoxicity.

Quinacrine protects neuronal cells against heat‐induced injury

The effects of quinacrine (QA) on heat-induced neuronal injury have been explored, with the intention of understanding the mechanisms of QA protection. Primary cultivated striatum neurons from newborn rats were treated with QA 1h before heat treatment at 43 degrees C which lasted for another 1h, and necrosis and apoptosis were detected by Annexin-V-FITC and propidium iodide (PI) double staining. Neuronal apoptosis was determined using terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) techniques. Cell membrane fluidity, activity of cytosolic phospholipase A(2) (cPLA(2)) and the level of arachidonic acid (AA) were also examined. Membrane surface ultrastructure of striatum neurons was investigated by atomic force microscopy (AFM). Results showed that heat treatment induced great striatum neurons death, with many dying neurons undergoing necrosis rather than apoptosis. QA alone had little effect on the survival of striatum neurons, while QA pretreatment before heat treatment decreased necrosis. Heat treatment also resulted in decreased membrane fluidity and increased cPLA(2) activity as well as arachidonic acid level; these effects were reversed by QA pretreatment. QA pretreatment also significantly prevented damage to the membrane surface ultrastructure of heat-treated neurons. These results suggest that QA protects striatum neurons against heat-induced neuronal necrosis, and also demonstrate that inhibition of cPLA(2) activity and stabilization of membranes may contribute to protective effect of quinacrine.

Chronic application of nonylphenol-induced apoptosis via suppression of bcl-2 transcription and up-regulation of active caspase-3 in mouse brain

Nonylphenol (NP) is an endocrine disruptor, which has been reported to have adverse effects on reproductive and immune systems. However, the influence of NP on the central nervous system (CNS) has not been extensively explored. The present study was performed to investigate the effects of chronic administration of NP on the apoptosis-related protein expression in mouse brain by in situ hybridization, RT-PCR and immunoblotting assays. The expression of bcl-2 mRNA was down-regulated by NP at the doses of 100 and 200mg/(kg day) (p<0.05), whereas the expression of bax mRNA was not affected in NP treated mice (p>0.05). Furthermore, as the main executor of apoptosis, the expression of active caspase-3 was up-regulated by 100 and 200mg/(kg day) NP (p<0.01), which is in accord with the results of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) (p<0.05). These results suggest that chronic application of NP could sensitize the mice brain to apoptosis.

Protecting neurons from HIV-1 gp120-induced oxidant stress using both localized intracerebral and generalized intraventricular administration of antioxidant enzymes delivered by SV40-derived vectors

Human immunodeficiency virus-1 (HIV-1) is the most frequent cause of dementia in adults under 40. We sought to use gene delivery to protect from HIV-1-related neuron loss. Because HIV-1 envelope (Env) gp120 elicits oxidant stress and apoptosis in cultured neurons, we established reproducible parameters of Env-mediated neurotoxicity in vivo, then tested neuroprotection using gene delivery of antioxidant enzymes. We injected 100-500 ng mul(-1)gp120 stereotaxically into rat caudate-putamens (CP) and assayed brains for apoptosis by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) 6-h to 14-day post-injection. Peak apoptosis occurred 1 day after injection of 250 and 500 ng microl(-1)gp120. TUNEL-positive cells mostly expressed neuronal markers (NeuroTrace), although some expressed CD68 and so were most likely microglial cells. Finally, we compared neuroprotection from gp120-induced apoptosis provided by localized and generalized intra-central nervous system (CNS) gene delivery. Recombinant SV40 vectors carrying Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) were injected into the CP, where gp120 was administered 4-24 weeks later. Alternatively, we inoculated the vector into the lateral ventricle (LV), with or without prior intraperitoneal (i.p.) administration of mannitol. Intracerebral injection of SV(SOD1) or SV(GPx1) significantly protected neurons from gp120-induced apoptosis throughout the 24-week study. Intraventricular vector administration protected from gp120 neurotoxicity comparably, particularly if preceded by mannitol i.p. Thus, HIV-1 gp120 is neurotoxic in vivo, and intracerebral or intra-ventricular administration of rSV40 vectors carrying antioxidant enzymes is neuroprotective. These findings suggest the potential utility of both localized and widespread gene delivery in treating neuroAIDS and other CNS diseases characterized by excessive oxidative stress.

Cathepsin D Deficiency Induces Persistent Neurodegeneration in the Absence of Bax-Dependent Apoptosis

Neuronal ceroid lipofuscinosces/Batten disease (NCL) is a devastating group of neurodegenerative diseases caused by genetic disruptions in lysosomal function. Cathepsin D (CD) is a major lysosomal protease, and mutations in CD that render it enzymatically defective have been reported recently in subsets of NCL patients. The targeted deletion of CD in mice results in extensive neuropathology, including biochemical and morphological evidence of apoptosis and autophagic stress (aberrant autophagosome accumulation), effects that are similar to those observed in NCL. To determine the contribution of Bax-dependent apoptosis in this mouse model of NCL, combined Bax- and CD-deficient mice were generated. Morphological analysis of CD-deficient mouse brains indicated large numbers of pyknotic neurons and neurons with marked cytoplasmic swellings containing undigested lipofuscin. Cell death and apoptosis were evidenced by increases in terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) reactivity and activation of caspase-3, respectively. DeOlmos silver-positive neurons were abundant in CD-deficient brain and correlated with neuron loss, as indicated by significant decreases in NeuN (neuronal nuclear antigen)-positive neurons. Lysosome dysfunction and autophagic stress were apparent in CD-deficient brain as indicated by the accumulation of autofluorescent storage material and by increased levels of LC3-II (light chain 3-II, a selective autophagosome marker), respectively. Bax deletion significantly inhibited caspase-3 activation and hippocampal TUNEL reactivity but did not prevent the majority of CD deficiency-induced neuropathology, including the persistence of pyknotic neurons, elevated cortical TUNEL reactivity, lysosome dysfunction and autophagic stress, neurodegeneration, and neuron loss. Together, these results suggest that CD deficiency-induced neuropathology does not require Bax-dependent apoptosis and highlights the importance of caspase-independent neuron death and neurodegeneration resulting from the genetic disruption of lysosome function.

Regeneration of Inner Retinal Neurons after Intravitreal Injection of Ouabain in Zebrafish

We examined the regenerative capacity of the adult zebrafish retina by intravitreal injection of a low ouabain concentration to rapidly damage the ganglion cell layer (GCL) and inner nuclear layer (INL) with minimal photoreceptor cell damage. By 24 h after ouabain injection, maximal numbers of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-positive cells were detected in the INL and GCL, with low numbers of TUNEL-positive cells in the outer nuclear layer. Immunolabeling revealed that approximately 85% of the HuC/D-positive amacrine and ganglion cells were lost by 7 d post-ouabain injection (dpi). This ganglion cell loss was consistent with the small, but statistically significant, decrease in the optic nerve diameter. The regeneration response began within 1 dpi with increased proliferating cell nuclear antigen (PCNA) expression in both the INL and GCL. By 3 dpi, PCNA expression is primarily restricted to the Müller glia. By 5 dpi, most of the PCNA expression was localized to neuronal progenitors expressing the olig2:egfp transgene rather than the Müller glia. By 7 dpi, the neuronal progenitors began committing to the ganglion cell fate based on the coexpression of the atoh7:EGFP transgene and the zn5 antigen. The regeneration of ganglion and amacrine cells continued until 60 dpi, when they reached 75% of their uninjected control number. This demonstrates that inner retinal damage, without extensive photoreceptor damage, is sufficient to induce a regeneration response that is marked by the Müller glial cells reentering the cell cycle to produce neuronal progenitor cells that regenerate INL and ganglion cells in the zebrafish retina.

In Vivo Restoration of Physiological Levels of Truncated TrkB.T1 Receptor Rescues Neuronal Cell Death in a Trisomic Mouse Model

Imbalances in neurotrophins or their high-affinity Trk receptors have long been reported in neurodegenerative diseases. However, a molecular link between these gene products and neuronal cell death has not been established. In the trisomy 16 (Ts16) mouse there is increased apoptosis in the cortex, and hippocampal neurons undergo accelerated cell death that cannot be rescued by administration of brain-derived neurotrophic factor (BDNF). Ts16 neurons have normal levels of the TrkB tyrosine kinase receptor but an upregulation of the TrkB.T1 truncated receptor isoform. Here we show that restoration of the physiological level of the TrkB.T1 receptor by gene targeting rescues Ts16 cortical cell and hippocampal neuronal death. Moreover, it corrects resting Ca2+ levels and restores BDNF-induced intracellular signaling mediated by full-length TrkB in Ts16 hippocampal neurons. These data provide a direct link between neuronal cell death and abnormalities in Trk neurotrophin receptor levels.

Loss of Neurons from Laminas I-III of the Spinal Dorsal Horn Is Not Required for Development of Tactile Allodynia in the Spared Nerve Injury Model of Neuropathic Pain

It has been proposed that death of inhibitory interneurons in the dorsal horn contributes to the neuropathic pain that follows partial nerve injury. In this study, we have used two approaches to test whether there is neuronal death in the dorsal horn in the spared nerve injury (SNI) model. We performed a stereological analysis of the packing density of neurons in laminas I-III 4 weeks after operation and found no reduction on the ipsilateral side compared with that seen on the contralateral side or in sham-operated or naive rats. In addition, we used two markers of apoptosis, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining and immunocytochemical detection of cleaved (activated) caspase-3. Neither of these methods demonstrated apoptotic neurons in the dorsal spinal cord 1 week after operation. Although TUNEL-positive cells were present throughout the gray and white matter at this stage, they were virtually all labeled with antibody against ionized calcium-binding adapter molecule 1, a marker for microglia. All animals that underwent SNI showed clear signs of tactile allodynia affecting the ipsilateral hindpaw. These results suggest that a significant loss of neurons from the dorsal horn is not necessary for the development of tactile allodynia in the SNI model.

Modulation of tamoxifen sensitivity by antisense Bcl‐2 and trastuzumab in breast carcinoma cells

Because the overexpression of HER-2 and Bcl-2 is associated with resistance to tamoxifen (TAM), the authors examined the effect of antisense (AS) Bcl-2 on sensitivity to TAM compared with the effect of trastuzumab on sensitivity to TAM in breast carcinoma cell lines. Drug sensitivity was assessed in vitro using a [3-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay with the breast carcinoma cell lines ZR-75-1, MDA-MB-453, and BT-474. AS Bcl-2 18-mer phosphorothioate oligonucleotide was applied. Apoptotic cell death was assessed with the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling method, and gene expression was evaluated with Western blot analysis. The expression of Bcl-2 was identified in ZR-75-1 and BT-474 cells and, to a lesser extent, in MDA-MB-453 cells. Overexpression of HER-2 was identified in BT-474 cells, and moderate expression was identified in MDA-MB-453 and ZR-75-1 cells. Combination treatment with trastuzumab or AS Bcl-2 enhanced TAM sensitivity in ZR-75-1 cells, which showed 50% inhibitory concentration (IC50) values of 0.9 microM (7.2-fold increase) and 0.5 microM (13.0-fold), respectively. Combination treatment with trastuzumab or AS Bcl-2 slightly enhanced TAM sensitivity of BT-474 cells, with IC50 values of 3.0 microM (1.3-fold) and 1.5 microM (2.6-fold), respectively. The sensitivity of MDA-MB-453 cells to TAM was not enhanced by combination with trastuzumab or AS Bcl-2. Modulation of TAM sensitivity by AS Bcl-2 was superior to modulation by trastuzumab in HER-2-expressing and Bcl-2-expressing breast carcinoma cells. Enhanced sensitivity in combination with AS Bcl-2 was associated with down-regulation of Bcl-2 and pAkt, which was correlated with the induction of Bax and caspase-3, leading to apoptosis. AS Bcl-2 appeared to be superior to trastuzumab with respect to regulating the signal-transduction pathways involved in breast carcinoma cells.

Hypoxia-Ischemia Induces DNA Synthesis without Cell Proliferation in Dying Neurons in Adult Rodent Brain

Recent studies suggest that postmitotic neurons can reenter the cell cycle as a prelude to apoptosis after brain injury. However, most dying neurons do not pass the G1/S-phase checkpoint to resume DNA synthesis. The specific factors that trigger abortive DNA synthesis are not characterized. Here we show that the combination of hypoxia and ischemia induces adult rodent neurons to resume DNA synthesis as indicated by incorporation of bromodeoxyuridine (BrdU) and expression of G1/S-phase cell cycle transition markers. After hypoxia-ischemia, the majority of BrdU- and neuronal nuclei (NeuN)-immunoreactive cells are also terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-stained, suggesting that they undergo apoptosis. BrdU+ neurons, labeled shortly after hypoxia-ischemia, persist for >5 d but eventually disappear by 28 d. Before disappearing, these BrdU+/NeuN+/TUNEL+ neurons express the proliferating cell marker Ki67, lose the G1-phase cyclin-dependent kinase (CDK) inhibitors p16INK4 and p27Kip1 and show induction of the late G1/S-phase CDK2 activity and phosphorylation of the retinoblastoma protein. This contrasts to kainic acid excitotoxicity and traumatic brain injury, which produce TUNEL-positive neurons without evidence of DNA synthesis or G1/S-phase cell cycle transition. These findings suggest that hypoxia-ischemia triggers neurons to reenter the cell cycle and resume apoptosis-associated DNA synthesis in brain. Our data also suggest that the demonstration of neurogenesis after brain injury requires not only BrdU uptake and mature neuronal markers but also evidence showing absence of apoptotic markers. Manipulating the aberrant apoptosis-associated DNA synthesis that occurs with hypoxia-ischemia and perhaps neurodegenerative diseases could promote neuronal survival and neurogenesis.

The c-Jun N-terminal protein kinase signaling pathway mediates Bax activation and subsequent neuronal apoptosis through interaction with Bim after transient focal cerebral ischemia.

The c-Jun N-terminal protein kinase (JNK) signaling pathway is implicated in neuronal apoptosis. The mechanism by which activated JNK induces neuronal apoptosis is strongly linked to mitochondrial apoptogenic proteins, although the molecular machinery downstream of JNK has not been precisely elucidated. Our study examined the relevance of proapoptotic Bcl-2 family members in JNK-mediated apoptosis after transient focal cerebral ischemia (tFCI), which, when induced by 60 min of middle cerebral artery (MCA) occlusion, elevated levels of JNK activity and phospho-JNK in the MCA territory. Phospho-JNK was primarily expressed in neurons and colocalized with terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-positive cells. Inhibition of JNK activity by anthra[1,9-cd]pyrazol-6(2H)-one (SP600125), a selective JNK inhibitor, protected neurons from ischemia-induced apoptosis detected by TUNEL staining and an apoptotic-related DNA fragmentation assay. SP600125 blocked translocation of the cell death effector Bax from the cytosol to the mitochondria after tFCI. BimL (Bim long) was induced and phosphorylated parallel to JNK activity. Coimmunoprecipitation studies consistently revealed increased interaction of JNK with BimL, as well as BimL with Bax, after tFCI. SP600125 blocked these interactions at a dose that significantly inhibited JNK-induced neuronal apoptosis. These results suggest that the JNK signaling pathway is involved in ischemia-induced neuronal apoptosis by stimulation, at least in part, of Bax translocation to the mitochondria, in which BimL is likely regulated by JNK as a downstream substrate for transmission of apoptotic signals to Bax.

The toppler mouse: a novel mutant exhibiting loss of Purkinje cells.

We describe the genetic and neurological features of toppler, a spontaneous autosomal mutation that appeared in a colony of FVB/N mice and that manifests as severe ataxia appearing at around 12 days of age, worsening with age. The lifespan of affected mice is 8-12 months, with occasional mice living longer. Both homozygous males and females are fertile, and females are able to nurture litters. Histological examination of brain revealed no striking abnormalities other than the loss of cerebellar Purkinje cells. The toppler mutation was mapped to mouse chromosome 8, and to assess whether it was novel or a recurrence of a previously described chromosome 8 mouse mutant, toppler mice were crossed with the nervous and tottering mouse mutants. These studies demonstrate that toppler is a unique mouse mutation. Purkinje cell abnormalities in toppler mice were obvious around postnatal day (P) 14, i.e., toppler Purkinje cells already exhibited abnormal morphology. Staining for calbindin, a calcium binding protein enriched in Purkinje cells, showed altered dendritic morphology. Between P14 and P30, dramatic Purkinje cell loss occurred, although there were differences in the degree of Purkinje cell loss in each lobule. At P30, the surviving Purkinje cells expressed zebrin II. From P30 through 6 months, many of the remaining Purkinje cells gradually degenerated. Purkinje cell loss was analyzed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL), and Purkinje cells were TUNEL-positive most abundantly at P21. In addition, Bergmann glia were TUNEL positive at P21, and they expressed activated caspase-3 at earlier time points. Interestingly, despite the apparent death of some Bergmann glia, there was up-regulation of glial fibrillary acidic protein, expressed in astrocytes as well as Bergmann glia. Given the changes in both Purkinje cells and glia in toppler cerebellum, this may be a very useful model in which to investigate the developmental interaction of Purkinje cells and Bergmann glia.

Time course of programmed cell death in Ciona intestinalis in relation to mitotic activity and MAPK signaling.

Programmed cell death (PCD) in the ascidian species Ciona intestinalis (Tunicata; Chordata) is investigated from early larvae to juvenile stages, by means of digoxigenin-based terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) technique. At first, PCD in the swimming larva affects trunk mesenchyme and central nervous system (CNS), then it participates extensively to metamorphosis, until it is restricted to developing organs of juveniles. Analysis of patterns of cell death and division in the larval CNS question old models on the genesis of the adult C. intestinalis brain. Upon performing immunochemical and functional assays for mitogen-activated protein kinase (MAPK) kinase kinase-1 (MEKK1), MAPK kinase 1/2 (MEK1/2), c-Jun NH2-terminal kinase (JNK), and dual phosphorylated extracellular regulated kinase 1/2 (dpERK1/2), the neurogenic competence of the larval brain appears to rely on a combinatorial regulation of PCD by the mitogen-activated protein kinase signaling cascade. These results show that, in tunicates, PCD consists of a multistep program implicated in growth and patterning with various roles.

Cumulative labeling of embryonic mouse neural retina with bromodeoxyuridine supplied by an osmotic minipump

I describe a method for providing cumulative label of bromodeoxyuridine (BrdU) to mouse embryos. Commercially available osmotic pumps, which release their contents at a steady rate, were implanted subcutaneously in the interscapular space of pregnant mice on embryonic day (E) 9.5–12.5. Survival times varied from 4 h to 37 days. Tissues (embryonic and neonatal eyes and maternal intestine) were immunochemically labeled for BrdU and examined histologically. The first detectably labeled cells appeared 4–7 h post-implantation (hpi) and all cycling cells were labeled for at least 7 days post-implantation (dpi). Retinal development appeared normal. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) stained retinas that had been exposed to BrdU showed no more apoptotic cells than those unexposed. I conclude that the maternally implanted osmotic pump successfully provides cumulative BrdU labeling in the mouse embryo.