TdT-mediated dUTP Nick-end Labeling Staining Research Articles

Phenylephrine-Induced Cardiomyocyte Injury Is Triggered by Superoxide Generation through Uncoupled Endothelial Nitric-Oxide Synthase and Ameliorated by 3-[2-[4-(3-Chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyindazole (DY-9836), a Novel Calmodulin Antagonist

The pathophysiological relevance of endothelial nitric-oxide synthase (eNOS)-induced superoxide production in cardiomyocyte injury after prolonged phenylephrine (PE) exposure remains unclear. The aims of this study were to define the mechanism of O2(*) production by uncoupled eNOS and evaluate the therapeutic potential of a novel calmodulin antagonist 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyindazole (DY-9836) to rescue hypertrophied cardiomyocytes from PE-induced injury. In cultured rat cardiomyocytes, prolonged exposure for 96 h to PE led to translocation from membrane to cytosol of eNOS and breakdown of caveolin-3 and dystrophin. When NO and O2(*) production were monitored in PE-treated cells by 4-amino-5-methylamino-2',7'-difluorofluorescein and dihydroethidium, respectively, Ca(2+)-induced NO production elevated by 5.7-fold (p < 0.01) after 48-h PE treatment, and the basal NO concentration markedly elevated (16-fold; p < 0.01) after 96-h PE treatment. On the other hand, the O2(*) generation at 96 h was closely associated with an increased uncoupled eNOS level. Coincubation with DY-9836 (3 microM) during the last 48 h inhibited the aberrant O2(*) generation nearly completely and NO production by 72% (p < 0.01) after 96 h of PE treatment and inhibited the breakdown of caveolin-3/dystrophin in cardiomyocytes. PE-induced apoptosis assessed by TdT-mediated dUTP nick-end labeling staining was also attenuated by DY-9836 treatment. These results suggest that O2(*) generation by uncoupled eNOS probably triggers PE-induced cardiomyocyte injury. Inhibition of abnormal O2(*) and NO generation by DY-9836 treatment represents an attractive therapeutic strategy for PE/hypertrophy-induced cardiomyocyte injury.

N-Acetylcysteine-Mediated Antioxidation Prevents Hyperglycemia-Induced Apoptosis and Collagen Synthesis in Rat Mesangial Cells

Background/Aims: High-glucose (HG)-induced mesangial apoptosis and fibrogenesis possibly involves reactive oxygen species (ROS) formation and activated mitochondrial stress. We investigated the therapeutic effect of the antioxidant N-acetylcysteine (NAC) on cellular apoptosis and matrix accumulation in HG-treated rat mesangial cells (RMCs). Methods: RMCs were cultured in media containing 5 (control) or 35 mM (HG) glucose. Cellular apoptosis was assayed by TdT-mediated dUTP nick-end labeling staining. Collagen and transforming growth factor-1 gene expression were measured by reverse transcriptase-polymerase chain reaction or Northern blotting. Mitochondrial capacity and intracellular ROS generation was assayed by fluorescence microscopy and flow cytometry, respectively. Cellular ATP production and malondialdehyde (MDA) formation were determined by a luciferin-luciferase reaction and high-performance liquid chromatography, respectively. Cytochrome c release, caspase activation and poly(ADP)ribose polymerase cleavage were assayed by Western blotting. Results: HG-treated RMCs displayed enhanced cellular apoptosis (65%) and collagen gene expression (1.8-fold increase); these reactions could be significantly suppressed by 1 mM NAC (p < 0.05). Intracellular ROS generation, production of ATP and MDA, and caspase-3, -8 and -9 activities were significantly increased in HG-treated RMCs, and were effectively attenuated by addition of NAC. Conclusion: It is concluded that NAC prevents HG-induced mesangial apoptosis and fibrogenesis pathways by the reduction of oxidative stress.

Mode of Growth Hormone Action in Osteoblasts

Growth hormone (GH) affects bone size and mass in part through stimulating insulin-like growth factor type 1 (IGF-1) production in liver and bone. Whether GH acts independent of IGF-1 in bone remains unclear. To define the mode of GH action in bone, we have used a Cre/loxP system in which the type 1 IGF-1 receptor (Igf1r) has been disrupted specifically in osteoblasts in vitro and in vivo. Calvarial osteoblasts from mice homozygous for the floxed IGF-1R allele (IGF-1R(flox/flox)) were infected with adenoviral vectors expressing Cre. Disruption of IGF-1R mRNA (>90%) was accompanied by near elimination of IGF-1R protein but retention of GHR protein. GH-induced STAT5 activation was consistently greater in osteoblasts with an intact IGF-1R. Osteoblasts lacking IGF-1R retained GH-induced ERK and Akt phosphorylation and GH-stimulated IGF-1 and IGFBP-3 mRNA expression. GH-induced osteoblast proliferation was abolished by Cre-mediated disruption of the IGF-1R or co-incubation of cells with an IGF-1-neutralizing antibody. By contrast, GH inhibited apoptosis in osteoblasts lacking the IGF-1R. To examine the effects of GH on osteoblasts in vivo, mice wild type for the IGF-1R treated with GH subcutaneously for 7 days showed a doubling in the number of osteoblasts lining trabecular bone, whereas osteoblast numbers in similarly treated mice lacking the IGF-1R in osteoblasts were not significantly affected. These results indicate that although direct IGF-1R-independent actions of GH on osteoblast apoptosis can be demonstrated in vitro, IGF-1R is required for anabolic effects of GH in osteoblasts in vivo.

A Critical Role for IκB Kinase β in Metallothionein-1 Expression and Protection against Arsenic Toxicity

Arsenic is a widespread environmental toxic agent that has been shown to cause diverse tissue and cell damage and at the same time to be an effective anti-cancer therapeutic agent. The objective of this study is to explore the signaling mechanisms involved in arsenic toxicity. We show that the IkappaB kinase beta (IKKbeta) plays a crucial role in protecting cells from arsenic toxicity. Ikkbeta(-)(/)(-) mouse 3T3 fibroblasts have decreased expression of antioxidant genes, such as metallothionein 1 (Mt1). In contrast to wild type and IKKbeta-reconstituted Ikkbeta(-)(/)(-) cells, IKKbeta-null cells display a marked increase in arsenic-induced reactive oxygen species (ROS) accumulation, which leads to activation of the MKK4-c-Jun NH(2)-terminal kinase (JNK) pathway, c-Jun phosphorylation, and apoptosis. Pretreatment with the antioxidant N-acetylcysteine (NAC) and expression of MT1 in the Ikkbeta(-)(/)(-) cells prevented JNK activation; moreover, NAC pretreatment, MT1 expression, MKK4 ablation, and JNK inhibition all protected cells from death induced by arsenic. Our data show that two signaling pathways appear to be important for modulating arsenic toxicity. First, the IKK-NF-kappaB pathway is crucial for maintaining cellular metallothionein-1 levels to counteract ROS accumulation, and second, when this pathway fails, excessive ROS leads to activation of the MKK4-JNK pathway, resulting in apoptosis.

16 Myocardial Expression of Mutant P53 Protects Against Doxorubicin-Induced Cardiomyocyte Apoptosis and Preserves Heart Function.

Doxorubicin, a topoisomerase II inhibitor that is commonly used to treat childhood cancer, induces p53-mediated cardiomyocyte apoptosis which can ultimately lead to heart failure. We hypothesized that cardiac-restricted expression of a dominant interfering p53 mutant (CB7) would protect cardiomyocytes from doxorubicin-induced apoptosis in vivo. Age-matched CB7 transgenic mice and control non-transgenic (NTG) mice were treated with a total of 20mg/kg of doxorubicin (2 intraperitoneal injections of 10mg/kg at 3-day interval). The prevalence of cardiomyocyte apoptosis was assessed via anti-activated caspase-3 immune reactivity and also via terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining. Cleavage of poly (ADP-ribose) polymerase (PARP, a substrate of caspase-3) and the level of the anti-apoptotic protein Bcl-xL were also monitored. Cardiac function was assessed using the two dimensional echocardiography. Our results show that the prevalence of cardiomyocyte apoptosis was reduced in CB7 mice as compared to NTG mice following doxorubicin injection (activated caspase 3 immune reactivity: 0.85± 0.14 positive cardiomyocytes/mm2 vs. 1.98±0.24 positive cardiomyocytes/mm2, p<0.05, n=6; TUNEL staining: 0.045±0.006% of total cardiomyocyte nuclei vs. 0.146±0.009%, p<0.01, n=5). In agreement with this, caspase-dependent PARP cleavage was not detected in the doxorubicin-treated CB7 hearts but was detected in the doxorubicin-treated NTG. Bcl-xL expression was induced to a greater extent in CB7 mice as compared to NTG mice. Finally, fractional shortening was better preserved in the CB7 mice as compared to the NTG mice (7.79±1.35% reduction post-injection vs. 28.87±5.48%, p<0.05, n=6). Interestingly, the extent of functional preservation observed in DOX treated CB7 mice appeared to exceed that which would be expected based on the reduced levels of cardiomyocyte apoptosis. In conclusion, cardiac expression of CB7 protects against doxorubicin-induced cardiomyocyte apoptosis and preserves heart function. The anti-apoptotic effects of CB7 might result from the increased level of Bcl-xL. The disproportionate preservation of cardiac function suggests that antagonization of p53 may engender additional cardioprotective activity.

Cytochrome c‐d regulates developmental apoptosis in the Drosophila retina

The role of cytochrome c (Cyt c) in caspase activation has largely been established from mammalian cell-culture studies, but much remains to be learned about its physiological relevance in situ. The role of Cyt c in invertebrates has been subject to considerable controversy. The Drosophila genome contains distinct cyt c genes: cyt c-p and cyt c-d. Loss of cyt c-p function causes embryonic lethality owing to a requirement of the gene for mitochondrial respiration. By contrast, cyt c-d mutants are viable but male sterile. Here, we show that cyt c-d regulates developmental apoptosis in the pupal eye. cyt c-d mutant retinas show a profound delay in the apoptosis of superfluous interommatidial cells and perimeter ommatidial cells. Furthermore, there is no apoptosis in mutant retinal tissues for the Drosophila homologues of apoptotic protease-activating factor 1 (Ark) and caspase 9 (Dronc). In addition, we found that cyt c-d--as with ark and dronc-regulates scutellar bristle number, which is known to depend on caspase activity. Collectively, our results indicate a role of Cyt c in caspase regulation of Drosophila somatic cells.

Nrf2 Negatively Regulates Osteoblast Differentiation via Interfering with Runx2-dependent Transcriptional Activation

Nrf2 (nuclear factor E2 p45-related factor 2) is believed to be a transcription factor essential for the regulation of many detoxifying and antioxidative genes in different tissues. In the present study, we investigated the role of Nrf2 in the regulation of osteoblastic differentiation. nrf2 mRNA expression was significantly up-regulated in femur isolated from ovariectomized mice, whereas in situ hybridization analysis revealed that up-regulation of nrf2 mRNA was mainly found in osteoblasts attached on cancellous bone in femur of ovariectomized mice. Expression of Nrf2 protein was also seen in osteoblasts in neonatal mouse tibia and calvaria. In osteoblastic MC3T3-E1 cells stably transfected with nrf2 expression vector, significant inhibition was seen in the maturation-dependent increase in alkaline phosphatase activity as well as the mineralized matrix formation. Stable overexpression of nrf2 significantly impaired Runx2 (runt-related transcription factor 2)-dependent stimulation of osteocalcin promoter activity and recruitment of Runx2 on osteocalcin promoter without affecting the expression of runx2 mRNA. Coimmunoprecipitation and mammalian two-hybrid assay revealed a physical interaction between Runx2 and Nrf2, whereas cellular distribution of endogenous Runx2 was not apparently changed by nrf2 overexpression in MC3T3-E1 cells. Alternatively, Nrf2 bound to antioxidant-responsive element-like-2 sequence of osteocalcin promoter. The inhibition by nrf2 on runx2-dependent osteocalcin promoter activity was partially prevented by the introduction of reporter of deletion mutant for ARE-like-2 sequence of osteocalcin promoter. These data suggest that Nrf2 may negatively regulate cellular differentiation through inhibition of the Runx2-dependent transcriptional activity in osteoblasts.

Effects of Chymase on the Macular Region in Monkeys and Porcine Müller Cells: Probable Involvement of Chymase in the Onset of Idiopathic Macular Holes

Objectives: To investigate chymase involvement in idiopathic macular hole onset, the effects of chymase on monkey eyes and cultured Müller cells were investigated. Methods: Immunohistochemistry using antinestin and antiglial fibrillary acidic protein antibodies was performed in a normal monkey eye. After chymase was injected into the monkey vitreous, histological changes in the retina were evaluated using the TdT-mediated dUTP nick-end labeling (TUNEL) assay. Expression of c-kit, a stem cell factor receptor, and nestin was examined in porcine Müller cells cultured with basic fibroblast growth factor. The effects of chymase on proliferation and TUNEL staining in Müller cells were also examined. Results: The number of nestin and glial fibrillary acidic protein-positive cells was higher in the macula than in other regions. Thickening of the posterior hyaloid membrane and some apoptotic cells were found in the macula of chymase-treated eyes. The expression of c-kit and nestin in Müller cells was shown and enhanced when cultured with basic fibroblast growth factor. Exposure to chymase inhibited Müller cell proliferation and produced TUNEL-positive cells. Conclusions: There might be Müller cells possessing atypical properties near the macular region and chymase might cause fibrosis and apoptosis through these cells. These findings suggest that increased chymase activity may result in idiopathic macular hole onset.

A Novel Platinum Compound Inhibits Constitutive Stat3 Signaling and Induces Cell Cycle Arrest and Apoptosis of Malignant Cells

Previous studies have established constitutive activation of Stat3 protein as one of the molecular changes required for tumorigenesis. To develop novel therapeutics for tumors harboring constitutively active Stat3, compounds from the NCI 2000 diversity set were evaluated for inhibition of Stat3 DNA-binding activity in vitro. Of these, a novel platinum (IV) compound, IS3 295, interacted with Stat3 and inhibited its binding to specific DNA-response elements. Further analysis suggested noncompetitive-type kinetics for the inhibition of Stat3 binding to DNA. In human and mouse tumor cell lines with constitutively active Stat3, IS3 295 selectively attenuated Stat3 signaling, thereby inducing cell growth arrest at G0/G1 phase and apoptosis. Moreover, in transformed cells, IS3 295 repressed expression of cyclin D1 and bcl-xL, two of the known Stat3-regulated genes that are overexpressed in malignant cells, suggesting that IS3 295 mediates anti-tumor cell activity in part by blocking Stat3-mediated sub-version of cell growth and apoptotic signals. Together, our findings provide evidence for the inhibition of Stat3 activity and biological functions by IS3 295 through interaction with Stat3 protein. This study represents a significant advance in small molecule-based approaches to target Stat3 and suggests potential new applications for platinum (IV) complexes as modulators of the Stat3 pathway for cancer therapy.

Protection by Food‐derived Antioxidants from UV‐A1‐Induced Photodamage, Measured Using Living Skin Equivalents¶

ABSTRACTIn a study of biomarkers of ultraviolet‐A1 radiation (UV‐A1)‐induced skin damage, living skin equivalent cultures (LSE) were treated with the antioxidants hesperetin and quercetin‐3‐glucoside and irradiated with 25 or 50 J/cm2 UV‐A1. Changes in the following biomarkers were measured; Interleukin 1‐alpha (IL‐1α), Heme Oxygenase‐1 (HO‐1), TdT‐mediated dUTP nick end labeling (TUNEL) and 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG). IL‐1α and HO‐1 were analyzed by realtime PCR, Western blot, enzyme‐linked immunosorbent assay (ELISA) and immunohistochemistry. TUNEL and 8‐OHdG were determined by (immuno)histochemical techniques. Sections were stained with hematoxylin and eosin (H&amp;E). UV‐A1 induced keratinocyte and fibroblast vacuolation and nuclear pyknosis, intense TUNEL staining of fibroblasts and increased staining of cells and nuclei for 8‐OHdG. Lesser or marginal increases in intensity followed staining for HO‐1 and IL‐1α. The IL‐1α increase was confirmed by ELISA assays of the medium supernatants. Hesperetin and quercetin‐3‐glucoside reduced changes in H&amp;E, 8‐OHdG, TUNEL and IL‐1α. Quercetin‐3‐glucoside reduced the amount of IL‐1α in LSE media. These observations support the use of the selected biomarkers to monitor UV‐A1 damage and provide evidence that dietary ingredients could reduce ultraviolet‐A radiation‐induced damage.

Comparison of Staining Characteristics of Apoptotic Markers

The tdt-mediated dUTP nick end labeling (TUNEL) assay was used as a standard to compare three antibodies, DNA fragmentation factor, caspase, and Fas, as potential makers in the apoptotic cell pathway. Paraffin sections from normal human, simian, rat and mouse tissues were used consisting of colon, pancreas, and lung for each species. DNA fragmentation factor proved to be a comparable marker to the TUNEL assay but lacked specificity, resulting in the labeling of nonapoptotic cells. Full-length caspase-3 and Fas antibodies did label some apoptotic cells, although neither showed any correlation with TUNEL staining in any tissue types. The ApoTAg TUNEL kit displayed the most consistent and specific apoptotic cell staining with the least amount of background. (The J Histotechnol 28:29, 2005)Submitted: August 12, 2004; Accepted with revisions: December 9, 2004

Effects of postnatal nicotine exposure on apoptotic markers in the developing piglet brain

Exposure to cigarette smoke is a risk factor for the sudden infant death syndrome (SIDS), but the ability to distinguish between the neuropathological effects of pre- versus postnatal exposure is limited in the clinical setting. To test whether postnatal nicotine exposure could contribute to the increased neuronal expression of apoptotic markers that we have previously observed in SIDS infants, as well as including study of gender influences, we developed a piglet model to mimic passive smoking in the early postnatal period. Piglets were exposed to nicotine (2 mg/kg/day infused via an implanted osmotic minipump) within 48 h of birth until the age of 13–14 days, when the brain was collected for study. Four piglet groups included: control females ( n=7), control males ( n=7), nicotine females ( n=7), and nicotine males ( n=7). Apoptotic markers included immunohistochemistry for activated caspase-3, and for DNA fragmentation or terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) in seven nuclei of the brainstem caudal medulla and two subregions of the hippocampus (CA4 and dentate gyrus). Among control females compared with males, there was less active caspase-3 and less TUNEL in the dorsal motor nucleus of vagus (DMNV), and there was less TUNEL in the nucleus of the spinal trigeminal tract (NSTT). Compared with controls, nicotine-exposed male piglets had increased TUNEL staining in the cuneate nucleus ( P=0.05), and increased active caspase-3 in the hypoglossal, gracile and dentate gyrus ( P<0.05 for each). Nicotine-exposed females showed no change in TUNEL staining in any of the nuclei studied, but increased active caspase-3 in the hypoglossal, DMNV and NSTT ( P<0.05 for each). These results show for the first time that postnatal nicotine exposure can lead to an increase in apoptotic markers in the brain. In piglets, these effects showed regional and gender-specific differences, suggesting that passive, postnatal nicotine exposure may be responsible for some neuropathological changes observed in infants dying from SIDS.

Protection by Food-derived Antioxidants from UV-A1–Induced Photodamage, Measured Using Living Skin Equivalents¶

Abstract In a study of biomarkers of ultraviolet-A1 radiation (UV-A1)-induced skin damage, living skin equivalent cultures (LSE) were treated with the antioxidants hesperetin and quercetin-3-glucoside and irradiated with 25 or 50 J/cm2 UV-A1. Changes in the following biomarkers were measured; Interleukin 1-alpha (IL-1α), Heme Oxygenase-1 (HO-1), TdT-mediated dUTP nick end labeling (TUNEL) and 8-hydroxy-2′-deoxyguanosine (8-OHdG). IL-1α and HO-1 were analyzed by real-time PCR, Western blot, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. TUNEL and 8-OHdG were determined by (immuno)histochemical techniques. Sections were stained with hematoxylin and eosin (H&E). UV-A1 induced keratinocyte and fibroblast vacuolation and nuclear pyknosis, intense TUNEL staining of fibroblasts and increased staining of cells and nuclei for 8-OHdG. Lesser or marginal increases in intensity followed staining for HO-1 and IL-1α. The IL-1α increase was confirmed by ELISA assays of the medium supernatants. Hespe...

Protection by Food-derived Antioxidants from UVA1-induced Photodamage, Measured Using Living Skin Equivalents

In a study of biomarkers of ultraviolet-A1 radiation (UV-A1)-induced skin damage, living skin equivalent cultures (LSE) were treated with the antioxidants hesperetin and quercetin-3-glucoside and irradiated with 25 or 50 J/cm2 UV-A1. Changes in the following biomarkers were measured; Interleukin 1-alpha (IL-1alpha), Heme Oxygenase-1 (HO-1), TdT-mediated dUTP nick end labeling (TUNEL) and 8-hydroxy-2'-deoxyguanosine (8-OHdG). IL-1alpha and HO-1 were analyzed by real-time PCR, Western blot, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. TUNEL and 8-OHdG were determined by (immuno)histochemical techniques. Sections were stained with hematoxylin and eosin (H&E). UV-A1 induced keratinocyte and fibroblast vacuolation and nuclear pyknosis, intense TUNEL staining of fibroblasts and increased staining of cells and nuclei for 8-OHdG. Lesser or marginal increases in intensity followed staining for HO-1 and IL-1alpha. The IL-1alpha increase was confirmed by ELISA assays of the medium supernatants. Hesperetin and quercetin-3-glucoside reduced changes in H&E, 8-OHdG, TUNEL and IL-1alpha. Quercetin-3-glucoside reduced the amount of IL-1alpha in LSE media. These observations support the use of the selected biomarkers to monitor UV-A1 damage and provide evidence that dietary ingredients could reduce ultraviolet-A radiation-induced damage.

Small interfering RNA targeting Fas protects mice against renal ischemia-reperfusion injury

Fas-mediated apoptosis has been suggested to contribute to tubular cell death after renal ischemia-reperfusion injury. Here we investigate whether small interfering RNA (siRNA) duplexes targeting Fas protect mice from acute renal failure after clamping of the renal artery. Renal ischemia-reperfusion injury was induced by clamping the renal vein and artery for 15 or 35 min. Mice were treated before or after ischemia with siRNA targeting Fas or a control gene, administered by hydrodynamic injection, low-volume renal vein injection, or both. Treated mice were evaluated for renal Fas protein and mRNA expression, tissue histopathology, and apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining. Blood urea nitrogen and survival were monitored in mice in which the contralateral kidney had been removed. A single hydrodynamic injection of Fas siRNA reduced Fas mRNA and protein expression in the kidney 4-fold. Kidneys from mice that received Fas siRNA two days earlier had substantially less renal tubular apoptosis by TUNEL staining and less tubular atrophy and hyaline damage. Whereas 90% of mice pretreated with saline or GFP siRNA died, only 20% of Fas-siRNA-pretreated animals died. The same survival advantage was provided by a single low-volume Fas siRNA injection into the renal vein. Moreover, postischemic injection through the renal vein protected 38% of mice from death. This study confirms the importance of Fas-mediated apoptosis in renal ischemia-reperfusion injury. Silencing Fas by systemic or local catheterization holds therapeutic promise to limit ischemia-reperfusion injury.

RyR2 and Calpain-10 Delineate a Novel Apoptosis Pathway in Pancreatic Islets

Cells are programmed to die when critical signaling and metabolic pathways are disrupted. Inhibiting the type 2 ryanodine receptor (RyR2) in human and mouse pancreatic beta-cells markedly increased apoptosis. This mode of programmed cell death was not associated with robust caspase-3 activation prompting a search for an alternative mechanism. Increased calpain activity and calpain gene expression suggested a role for a calpain-dependent death pathway. Using a combination of pharmacological and genetic approaches, we demonstrated that the calpain-10 isoform mediated ryanodine-induced apoptosis. Apoptosis induced by the fatty acid palmitate and by low glucose also required calpain-10. Ryanodine-induced calpain activation and apoptosis were reversed by glucagon-like peptide or short-term exposure to high glucose. Thus RyR2 activity seems to play an essential role in beta-cell survival in vitro by suppressing a death pathway mediated by calpain-10, a type 2 diabetes susceptibility gene with previously unknown function.

Transient Cerebral Ischemia Induces Aberrant Neuronal Cell Cycle Re-entry and Alzheimer's Disease-like Tauopathy in Female Rats

Aberrant mitosis occurs in many tauopathy-related neurodegenerative diseases and is believed to precede the formation of neurofibrillary tangles. In this study, we report for the first time that transient cerebral ischemia induces aberrant mitotic proteins and hyperphosphorylation of tau protein with neurofibrillary tangle-like conformational epitopes in adult female rat cortex. Following transient cerebral ischemia in rats, initiation of apoptosis precedes and is potentially integrated with subsequent aberrant mitosis and tau hyperphosphorylation. Furthermore, inhibition of mitosis-related cyclin-dependent kinases (Cdks) by roscovitine significantly reduced the hyperphosphorylation of tau. Administration of the female sex steroid and potent neuroprotective agent, 17beta-estradiol, reduced ischemia-reperfusion-induced cerebral damage and the subsequent aberrant mitosis and tauopathies. These results provide a neuropathological basis for the higher prevalence of dementia in stroke patients and support the hypothesis that apoptosis and aberrant mitosis are integrated pathological events in neurons that may play a critical role in the development of Alzheimer's disease and other tauopathy-related neuropathology.

Epigallocatechin-3-gallate Inhibits Epidermal Growth Factor Receptor Signaling Pathway: EVIDENCE FOR DIRECT INHIBITION OF ERK1/2 AND AKT KINASES

Epidermal growth factor receptor (EGFR) activation is absolutely required for cervical cell proliferation. This suggests that EGFR-inhibitory agents may be of therapeutic value. In the present study, we investigated the effects of epigallocatechin-3-gallate (EGCG), a bioactive green tea polyphenol, on EGFR signaling in cervical cells. EGCG inhibits epidermal growth factor-dependent activation of EGFR, and EGFR-dependent activation of the mitogen-activated protein kinases ERK1/2. EGCG also inhibits EGFR-dependent AKT activity. The EGCG-dependent reduction in ERK and AKT activity is associated with reduced phosphorylation of downstream substrates, including p90RSK, FKHR, and BAD. These changes are associated with increased p53, p21(WAF-1), and p27(KIP-1) levels, reduced cyclin E level, and reduced CDK2 kinase activity. Consistent with these findings, flow cytometry and TUNEL (terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling) staining revealed EGCG-dependent G(1) arrest. Moreover, sustained EGCG treatment caused apoptotic cell death. In addition to inhibiting EGFR, cell-free studies demonstrated that EGCG directly inhibits ERK1/2 and AKT, suggesting that EGCG acts simultaneously at multiple levels to inhibit EGF-dependent signaling. Importantly, the EGCG inhibition is selective, as EGCG does not effect the EGFR-dependent activation of JNK. These results suggest that EGCG acts to selectively inhibit multiple EGF-dependent kinases to inhibit cell proliferation.

Relationship between tumour endothelial cell apoptosis and tumour blood flow shutdown following treatment with the antivascular agent DMXAA in mice.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is currently undergoing clinical evaluation as an antivascular agent for the treatment of cancer. We have previously demonstrated that DMXAA induces apoptosis of vascular endothelial cells in murine tumour sections and in a breast carcinoma biopsy from one patient in a Phase I trial. We wished to determine the tissue selectivity of this effect and its relationship to induced blood flow changes. Mice with Colon 38 tumours were treated with DMXAA and tissues were examined for apoptosis by TdT-mediated dUTP nick-end labelling (TUNEL). Hoechst 33342 was used to stain functional vessels, with the loss of stained vessels used as a measure of tumour vascular collapse. Treatment with DMXAA at 25 mg kg−1, its maximum tolerated dose (MTD), showed, after 3 h, a 12-fold increase in TUNEL staining of tumour vascular endothelial cells. In contrast, tissue from the heart, brain, liver and spleen showed no increase. Induction of apoptosis in tumour tissue was both dose-dependent, observable at doses as low as 5 mg kg−1, and time-dependent. Apoptosis was significantly lower in Colon 38 tumours of mice, with a targeted disruption in the TNF gene (TNF−/−), or in the TNF receptor 1 gene (TNFR−/−), as compared with that in wild-type mice. Increasing the DMXAA dose to 50 mg kg−1 in these knockout mice raised tumour apoptosis to a level comparable to that induced in wild-type mice given DMXAA at the MTD. For all the data, a significant correlation (r=0.94; P<0.001) was found between logarithmic percentage apoptosis induction and the logarithmic density of Hoechst-stained vessels. These results suggest that blood flow inhibition caused by DMXAA is tumour tissue-specific and is a consequence of induction of apoptosis in tumour vascular endothelial cells.

Modes of cell death in rat liver after monocrotaline exposure.

Monocrotaline (MCT) is a pyrrolizidine alkaloid (PA) plant toxin that produces sinusoidal endothelial cell (SEC) injury, hemorrhage, fibrin deposition, and coagulative hepatic parenchymal cell (HPC) oncosis in centrilobular regions of rat livers. Cells with apoptotic morphology have been observed in the livers of animals exposed to other PAs. Whether apoptosis occurs in the livers of MCT-treated animals and whether it is required for full manifestation of pathological changes is not known. To determine this, rats were treated with 300 mg MCT/kg, and apoptosis was detected by transmission electron microscopy and the TUNEL (TdT-mediated dUTP nick end labeling) assay. MCT produced significant apoptosis in the liver by 4 h after treatment. To determine if MCT kills cultured HPCs by apoptosis, HPCs were isolated from the livers of rats and exposed to MCT. MCT caused a concentration-dependent release of alanine aminotransferase (ALT), a marker of HPC injury. Furthermore, caspase 3 was activated and TUNEL staining increased in MCT-treated HPCs. MCT-induced TUNEL staining and release of ALT into the medium were completely prevented by the pancaspase inhibitors z-VAD.fmk and IDN-7314, suggesting that MCT kills cultured HPCs by apoptosis. To determine if caspase inhibition prevents MCT-induced apoptosis in the liver, rats were cotreated with MCT and IDN-7314. IDN-7314 reduced MCT-induced TUNEL staining in the liver and release of ALT into the plasma. Morphometric analysis confirmed that IDN-7314 reduced HPC oncosis in the liver by approximately 50%. Inasmuch as HPC hypoxia occurred in the livers of MCT-treated animals, upregulation of the hypoxia-regulated cell-death factor, BNIP3 (Bcl2/adenovirus EIB 19kD-interacting protein 3), was examined. BNIP3 was increased in the livers of mice treated 24 h earlier with MCT. Results from these studies show that MCT kills cultured HPCs by apoptosis but causes both oncosis and apoptosis in the liver in vivo. Furthermore, caspase inhibition reduces both apoptosis and HPC oncosis in the liver after MCT exposure.