Transferase biotin-dUTP Nick End Labeling Research Articles

Survivin expression in canine epidermis and in canine and human cutaneous squamous cell carcinomas

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is ubiquitously expressed during tissue development, undetectable in most normal tissues, but re-expressed in most cancers, including skin malignancies. Expression of survivin was evaluated retrospectively in 19 canine cutaneous squamous cell carcinomas (SCCs; one in situ; 16 well differentiated; one invasive, one lymph node metastasis) and 19 well differentiated SCCs from human beings. Seven specimens of normal canine skin were included. Immunohistochemical expression of full-length survivin was determined using a commercially available antibody. In addition, apoptotic rate [Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labelling index (TUNEL) index] and mitotic index (MI), counting mitoses in 10 high power fields (HPF), were determined. Scattered survivin positive nuclei were identified in the epidermal basal cell layer of normal canine skin. Nuclear survivin expression was identified in 18 of 19 human and in all canine SCCs, mainly along the base of the tumour cell population. Cytoplasmic survivin expression was rarely observed in human SCCs and in 84.2% of canine SCCs. The TUNEL index ranged from 0.1 to 2.6 in human beings and from 7.5 to 69.4 in dogs, while MIs ranged from 0 to 4 in human beings and dogs. No correlation was found between survivin expression and apoptotic or mitotic rates. Canine and human tumours showed similar nuclear survivin expression, indicating similar functions of the molecule. We demonstrated survivin expression in normal adult canine epidermis. Increased nuclear survivin expression in pre-neoplastic and neoplastic lesions demonstrates a possible association of survivin with development of SCCs in human beings and dogs.

Antiapoptotic properties of recombinant human erythropoietin protects against tubular cyclosporine toxicity

During the early post transplant period, the tubular epithelium is the main target of injuries including ischemia reperfusion and toxicity effects from calcineurin inhibitors. Taking into account the tissue protective effects of erythropoietin mediated through its antiapoptotic properties, we tested whether administration of recombinant human erythropoietin protects against acute cyclosporine nephrotoxicity. Four groups of five rats were intraperitoneally treated over 28 days with 100 UI/Kg/48 h Epoetin β (15 mg/kg/day CsA diluted in olive oil, 100 UI/Kg/48 h Epoetin β + 15 mg/kg/day CsA, or olive oil. Histological changes due to tubular necrosis were evaluated with Masson’Trichrome staining. Apoptotic nuclei in kidneys were detected using the Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) method. Phospho-Akt, Akt, cleaved caspase 3 and non cleaved caspase 3 expression were evaluated using immunblotting. We demonstrate that recombinant human erythropoietin (epoetin β) improves renal function and protects against acute tubular injury. Our data suggest that this nephroprotective effect is mediated by Akt activation and inhibition of tubular apoptosis. Indeed, western blotting analysis of caspase 3 cleavage and Akt phosphorylation demonstrates that rhEPO activate Akt signaling and inhibits caspase 3 cleavage induced by CsA. TUNEL staining confirms that rhEPO inhibits CsA-induced tubular apoptosis. In conclusion, we describe here a new potential target of recombinant human erythropoietin and our results provide an interesting framework for further nephroprotective therapies based on recombinant human erythropoietin.

A correlation between growth rate, apoptosis, and tumor necrosis factor‐α in umbilical cord blood cells infected with two strains of Mycobacterium tuberculosis

Using umbilical cord blood (UCB) cells, it was demonstrated that three virulent isolates, two highly transmissible clinical isolates and the virulent laboratory strain, demonstrated rapid growth in the UCB cells, which was significantly faster than the growth rate observed for a unique isolate. There was also a significant increase in the amount of tumor necrosis factor (TNF)-a elicited from the UCB cells after infection with the unique isolate compared to the hypervirulent isolates. This study investigated whether neutralization or addition of TNF-a within this system would alter growth rates and apoptosis. Ten UCB samples were obtained for these experiments and adherent cells were isolated. Two clinical isolates, one virulent and one unique, were used. Colony-forming units were assessed at 3 hours postinfection (Day 0) and on Day 7 to generate growth ratios. TNF-a antibody or exogenous TNF-a was added after the 3-hour incubation period. Viability of the UCB cells was assessed. Apoptosis was measured using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining. There was no significant difference in the growth ratio for the virulent strain regardless of the presence of TNF-a antibody. There was a significant increase for the nonvirulent strain after the addition of antibody. There was an increase in viability for the UCB cells in the presence of antibody, suggesting a decrease in TNF-a–dependent apoptosis. Addition of exogenous TNF-a to the UCB cells after infection with the virulent strain decreased the growth ratio with a significant increase in apoptosis. The TNF-a response of the UCB cells is related to the infecting strain and the intracellular growth of the strain of Mycobacterium tuberculosis is not directly controlled by the level of TNF-a. This cytokine is at the start of a powerful cascade of transcription factors with numerous pleiotropic effects; consequently abrogating/enhancing a single direct outcome was difficult, with only slight alterations in growth.

Dexrazoxane-afforded protection against chronic anthracycline cardiotoxicity in vivo: effective rescue of cardiomyocytes from apoptotic cell death

Background:Dexrazoxane (DEX, ICRF-187) is the only clinically approved cardioprotectant against anthracycline cardiotoxicity. It has been traditionally postulated to undergo hydrolysis to iron-chelating agent ADR-925 and to prevent anthracycline-induced oxidative stress, progressive cardiomyocyte degeneration and subsequent non-programmed cell death. However, the additional capability of DEX to protect cardiomyocytes from apoptosis has remained unsubstantiated under clinically relevant in vivo conditions.Methods:Chronic anthracycline cardiotoxicity was induced in rabbits by repeated daunorubicin (DAU) administrations (3 mg kg−1 weekly for 10 weeks). Cardiomyocyte apoptosis was evaluated using TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling) assay and activities of caspases 3/7, 8, 9 and 12. Lipoperoxidation was assayed using HPLC determination of myocardial malondialdehyde and 4-hydroxynonenal immunodetection.Results:Dexrazoxane (60 mg kg−1) co-treatment was capable of overcoming DAU-induced mortality, left ventricular dysfunction, profound structural damage of the myocardium and release of cardiac troponin T and I to circulation. Moreover, for the first time, it has been shown that DEX affords significant and nearly complete cardioprotection against anthracycline-induced apoptosis in vivo and effectively suppresses the complex apoptotic signalling triggered by DAU. In individual animals, the severity of apoptotic parameters significantly correlated with cardiac function. However, this effective cardioprotection occurred without a significant decrease in anthracycline-induced lipoperoxidation.Conclusion:This study identifies inhibition of apoptosis as an important target for effective cardioprotection against chronic anthracycline cardiotoxicity and suggests that lipoperoxidation-independent mechanisms are involved in the cardioprotective action of DEX.

Hepatocyte growth factor suppresses tumor cell apoptosis in nasopharyngeal carcinoma by upregulating Bcl-2 protein expression

Hepatocyte growth factor (HGF) is a multifunctional cytokine, but cell apoptosis related to HGF in nasopharyngeal carcinoma (NPC) and the potential mechanisms involved have not yet been identified. In this study, we aimed at determining whether HGF is a potent inhibitor of cell apoptosis in NPC, and tried to find out which antiapoptotic or proapoptotic protein is involved in this process. A hundred and forty-seven cases of NPC and CNE-1, CNE-2 NPC cell line were collected. Expression of HGF, Bcl-2 and Bax in tumor tissues was investigated by immunohistochemical staining, and the apoptotic index was evaluated using the Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) method in all of the NPC cases. NPC cells were treated with HGF (25 ng/ml), followed by an assay for cell viability and apoptosis, as well as by an expression analysis of Bcl-2 and Bax using immunostaining and Western blot. The presence of Epstein-Barr virus (EBV) was also detected in NPC cells using polymerase chain reaction (PCR) for Bam HI W fragment. In tumor cells, expression of HGF was strong in 51% (75/147) of the NPC cases. In stromal cells, expression of HGF was also strong in 78.9% (116/147) of the cases. Strong expression of HGF in tumor cells and stromal cells was significantly associated with decreased apoptotic index, advanced clinical stage, lymph node metastasis and high-expression of Bcl-2. In vitro, exogenous HGF was found to promote cell growth, to suppress cell apoptosis and to upregulate the expression of Bcl-2 in NPC cells without EBV infection. HGF is a potent inhibitor of cell apoptosis in NPC by upregulating Bcl-2 through both autocrine and paracrine EBV-independent pathways. HGF might be a potential marker for the prognosis of NPC.

Expression of the macrophage antigen CD163 in rectal cancer cells is associated with early local recurrence and reduced survival time

Expression of the macrophage antigen CD163 in breast cancer cells is recently shown to be related to early distant recurrence and shortened survival. In this study, 163 patients with rectal cancer, included in the Swedish rectal cancer trial and followed up for a median of 71 months, were examined for the expression of CD163 in the primary tumors. The cancer cells expressed CD163 in the primary tumors in 23% (n = 32) of the patients. In pretreatment biopsies from 101 patients, 10 had CD163-positive cancers and these patients had earlier local recurrence (p < 0.044) and reduced survival time (p < 0.045) compared with those with CD163-negative tumors. When studying surgical specimens from 61 patients randomized to preoperative irradiation (5 x 5 Gy delivered in 1 week), it was found that 31% were CD163 positive whereas the corresponding figure was only 17% for 78 patients who were nonirradiated (p < 0.044), which tentatively may be consistent with X-rays inducing fusion. In CD163-positive tumors there was a reduced apoptotic activity as measured with the Termina deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) technique (p = 0.018). There tended also to be an increased proliferation activity measured as an expression of Ki-67 non significant (NS). It is concluded that primary rectal cancers may express CD-163, and this phenotypic macrophage trait is related to early local recurrence, shorter survival time and reduced apoptosis. Furthermore, the expression of CD163 is more common after irradiation.

Statins inhibit the growth of variant human embryonic stem cells and cancer cells in vitro but not normal human embryonic stem cells

Statins inhibit proliferation of various human cancer cell lines in vitro. As human embryonic stem cells (hESCs) possess neoplastic-like properties we have evaluated the role of various statins on karyotypically normal hESCs (HES3 and BG01), abnormal hESCs (BG01V) and breast adenocarcinoma cells (MCF-7) to evaluate whether the mode of action of the statins was via a stemness pathway. All cell lines were treated with simvastatin, pravastatin, lovastatin and mevastatin (1 micromol x L(-1) to 20 micromol x L(-1)) up to 7 days and their effects on cell proliferation, cell cycle, apoptosis and pluripotency studied. All four statins did not inhibit HES3 and BG01 proliferation, but BG01V and MCF-7 were inhibited by simvastatin, lovastatin and mevastatin. These inhibitory effects were reversed by the endogenous isoprenoids, farnesylpyrophosphate and geranylgeranylpyrophosphate. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and cell cycle assay confirmed apoptosis in BG01V and MCF-7. Stem cell surface markers [stage-specific embryonic antigen-4, tumour rejection antigen-1-81, octamer-4 (OCT-4)] were expressed in HES3 and BG01, but not in BG01V cells, even after prolonged treatment with simvastatin. In BG01V and MCF-7, the pro-apoptotic Bcl-2-associated X protein genes were up-regulated, while the antiapoptotic BCL2 and SURVIVIN genes were down-regulated. Expression of the stemness-related genes namely, the growth differentiation factor-3, NANOG and OCT-4 was decreased in BG01V compared with BG01 and HES3. Normal hESCs were resistant to prolonged exposure to statins over a range of doses, compared with BG01V and MCF-7, probably because of genetic and behavioural differences. The statins not only have anti-cancer properties but can suppress abnormal hESCs thus promoting growth of normal hESCs in vitro.

Effect of TRA-8 Anti-Death Receptor 5 Antibody in Combination With Chemotherapy in an Ex Vivo Human Ovarian Cancer Model

To investigate the cytotoxicity of TRA-8, an antibody that specifically binds death receptor 5, alone and in combination with chemotherapy, using an ex vivo human ovarian cancer model. Twenty-six ovarian cancer specimens were obtained during ovarian cancer debulking, and tumor slices were prepared with the Krumdieck tissue slicer. The tumor slices were exposed to varying concentrations of TRA-8, carboplatin/paclitaxel, or the combination of TRA-8 and chemotherapy. Using nonlinear modeling, dose-response curves and IC50 values were generated for specimens treated with TRA-8. The additive and synergistic cytotoxic effects of chemotherapy combination with TRA-8 were evaluated in specimens. In addition to adenosine triphosphate viability assays, the treated and untreated slices were assessed by immunohistochemistry to confirm apoptosis induction. Specimens from 13 patients yielded TRA-8-induced IC50 values. Of these specimens, 15% were found to be sensitive to TRA-8-induced cytotoxicity at IC50 doses less than 500 ng/mL. Specimens from 13 patients underwent combination treatment with TRA-8 and carboplatin/paclitaxel. Of these specimens, 77% exhibited additive cytotoxicity in comparison with those treated with either agent alone, whereas 15% exhibited synergistic cytotoxicity. Immunohistochemical analysis of terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling and cleaved caspase 3 staining demonstrated a dose-dependent increase in apoptosis with the combination treatment. This study demonstrates the efficacy of the death receptor monoclonal antibody TRA-8 in combination with conventional chemotherapy in an ex vivo human ovarian cancer model. This model can be used to assess cytotoxicity of novel agents in combination with chemotherapy in ovarian cancer.

Inhibition of cell death results in hyperganglionosis: implications for enteric nervous system development

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC) that delaminate from the neural tube and undergo extensive migration and proliferation in order to colonize the entire length of the gut and differentiate into many millions of neurons and glial cells. Although apoptotic programmed cell death is an essential physiological process during development of the majority of the vertebrate nervous system, apoptosis within early ENS development has not been comprehensively investigated. The aim of this study was to determine the presence and extent of apoptosis within the vagal NCC population that gives rise to most of the ENS in the chick embryo. We demonstrated that apoptotic cells, as shown by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and active caspase-3 immunoreactivity, are present within an electroporated green fluorescent protein (GFP) and human natural killer-1 (HNK-1) immunopositive NCC population migrating from the vagal region of the neural tube to the developing foregut. Inhibition of caspase activity in vagal NCC, by electroporation with a dominant-negative form of caspase-9, increased the number of vagal NCC available for ENS formation, as shown by 3-dimensional reconstruction of serial GFP or HNK-1 labelled sections, and resulted in hyperganglionosis within the proximal foregut, as shown by NADPH-diaphorase whole gut staining. These findings suggest that apoptotic cell death may be a normal process within the precursor pool of pre-enteric NCC that migrates to the gut, and as such it may play a role in the control of ENS formation.

Oncolytic Reovirus Effectively Targets Breast Cancer Stem Cells

Recent evidence suggests that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are resistant to current anticancer therapies. Hence, novel cancer therapies will need to be tested for both tumor regression and CSC targeting. Herein we show that oncolytic reovirus that induces regression of human breast cancer primary tumor samples xenografted in immunocompromised mice also effectively targets and kills CSCs in these tumors. CSCs were identified based on CD24(-)CD44(+) cell surface expression and overexpression of aldehyde dehydrogenase. Upon reovirus treatment, the CSC population was reduced at the same rate as non-CSCs within the tumor. Immunofluorescence of breast tumor tissue samples from the reovirus- and mock-treated mice confirmed that both CSCs and non-CSCs were infectible by reovirus, and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay showed that both populations died by apoptosis. Ras, which has been shown to mediate reovirus oncolysis, was found to be present at similar levels in all cell types, and this is consistent with their comparable sensitivity to reovirus. These experiments indicate that oncolytic reovirus has the potential to induce tumor regression in breast cancer patients. More important, the CSC population was equally reduced and was as susceptible to reovirus treatment as the non-CSC population.

Puerarin protects against ischemic brain injury in a rat model of transient focal ischemia

Objectives: This study examines the efficacy of puerarin, a drug used in traditional Chinese medicine, in attenuating ischemic brain injury after cerebral ischemia and reperfusion, and explores possible mechanisms underlying neuroprotective effects.Methods: The animal model of ischemia/reperfusion injury was induced by middle cerebral artery occlusion for 2 hours followed by up to 72 hour reperfusion. The rats were randomly assigned into four groups (n=6/group): puerarin at 100, 200 and 400 mg/kg or saline, administered intraperitoneally. Neurological outcome and infarct volume by 2% triphenyl tetrazolium chloride staining were determined 72 hours after reperfusion. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining was used to detect the cell damage of brains (n=5/group). Erythropoietin activation was detected by enzyme-linked immunosorbent assay (n=5/group).Results: Compared with the vehicle saline group, puerarin decreased infarction volume at doses of 200 mg/kg (p=0.045) and 400 mg/kg (p=0.0002), but not at 100 mg/kg (p=0.387). Functional neurological outcome was improved with puerarin at 400 mg/kg (p=0.015), but not at 100 mg/kg (p=0.68) or 200 mg/kg (p=0.056). Puerarin significantly decreased the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining cells compared with the vehicle group 4, 24 and 72 hours after reperfusion. The erythropoietin activity was higher in puerarin treated group compared with the vehicle group.Discussion: Puerarin has neuroprotection effects in rats at doses of 200 and 400 mg/kg, administered intraperitoneally after transient middle cerebral artery occlusion which may be partly due to activation of erythropoietin activity.

Attenuation of ischemia-induced rat brain injury by 2-(-2-benzofuranyl)-2-imidazoline, a high selectivity ligand for imidazoline I2 receptors

Objective: The aim of this study was to determine whether 2-(2-benzofuranyl)-2-imidazoline, an imidazoline I2 receptor ligand, could protect against cell death from brain injury and improve the functional outcome after focal cerebral ischemia in rats.Methods: Transient focal ischemia was induced by suture occlusion of the middle cerebral artery. Rats were intraperitoneally treated with a vehicle, 2-(2-benzofuranyl)-2-imidazoline or idazoxan immediately after focal ischemia. Infarct volume was assessed by 2,3,5-triphenyltrazolium chloride staining and neurobehavioral deficits were monitored. The volume of cell death in the penumbra after ischemia was determined by immunostaining using anti-cleaved caspase-3 antibody and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL).Results: Both 2-(2-benzofuranyl)-2-imidazoline and idazoxan significantly improved the neurological score compared with the vehicle at 24 hours after focal ischemia. Treatment with 2-(2-benzofuranyl)-2-imidazoline or idazoxan also significantly reduced infarct volume and the number of both caspase-3- and TUNEL-positive cells in the penumbra compared with vehicle-treated rats (p<0.01 and p<0.05, respectively).Conclusion: The results suggest the neuroprotective role of 2-(2-benzofuranyl)-2-imidazoline and idazoxan in focal cerebral ischemia, and may therefore represent useful targets for developing new treatments for stroke.

Enhanced cytotoxicity of TATp-bearing paclitaxel-loaded micelles in vitro and in vivo

Cell-penetrating peptide (TATp) was attached to the distal tips of polyethyleneglycol (PEG) moieties of polyethyleneglycol–phosphatidylethanolamine (PEG–PE) micelles loaded with paclitaxel (PCT). The TATp-modified micelles demonstrated an increased interaction with cancer cells compared to non-modified micelles resulting in a significant increase of the in vitro cytotoxicity to different cancer cells. TATp-modified PCT-loaded micelles were administered intratumorally in mice and the induction of apoptosis in tumor cells was studied after 48 h with the Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay using free PCT and TATp-free PCT-loaded PEG–PE micelles as controls. A significant apoptotic cell death was observed in tumors treated with PCT-loaded micelles modified with TATp, while the treatment with free PCT or with non-modified PCT-loaded micelles resulted in much smaller number of TUNEL-positive cells within tumors.

Mice that overexpress human heat shock protein 27 have increased renal injury following ischemia reperfusion

We previously showed that activation of the A1 adenosine receptor protected the kidney against ischemia-reperfusion injury by induction and phosphorylation of heat shock protein 27 (HSP27). Here, we used mice that overexpress human HSP27 (huHSP27) to determine if kidneys from these mice were protected against injury. Proximal tubule cells cultured from the transgenic mice had increased resistance to peroxide-induced necrosis compared to cells from wild-type mice. However, after renal ischemic injury, HSP27 transgenic mice had decreased renal function compared to wild-type mice, along with increased renal expression of mRNAs of pro-inflammatory cytokines (TNF-alpha, ICAM-1, MCP-1) and increased plasma and kidney keratinocyte-derived cytokine. Following ischemic injury, neutrophils infiltrated the kidneys earlier in the transgenic mice. Flow cytometric analysis of lymphocyte subsets showed that those isolated from the kidneys of transgenic mice had increased CD3(+), CD4(+), CD8(+), and NK1.1(+) cells 3 h after injury. When splenocytes or NK1.1(+) cells were isolated from transgenic mice and adoptively transferred into wild-type mice there was increased renal injury. Further, depletion of lymphocytes by splenectomy or neutralization of NK1.1(+) cells resulted in improved renal function in the transgenic mice following reperfusion. Our study shows that induction of HSP27 in renal tubular cells protects against necrosis in vitro, but its systemic increase counteracts this protection by exacerbating renal and systemic inflammation in vivo.

The 27-kDa Heat Shock Protein Confers Cytoprotective Effects through a β2-Adrenergic Receptor Agonist-Initiated Complex with β-Arrestin

Heat shock proteins represent an emerging model for the coordinated, multistep regulation of apoptotic signaling events. Although certain aspects of the biochemistry associated with heat shock protein cytoprotective effects are known, little information is found describing the regulation of heat shock protein responses to harmful stimuli. During screening for noncanonical beta adrenergic receptor signaling pathways in human urothelial cells, using mass spectroscopy techniques, an agonist-dependent interaction with beta-arrestin and the 27-kDa heat shock protein was observed in vitro. Formation of this beta-arrestin/Hsp27 complex in response to the selective beta adrenergic receptor agonist isoproterenol, was subsequently confirmed in situ by immunofluorescent colocalization studies. Radioligand binding techniques characterized a homogeneous population of the beta2 adrenergic receptor subtype expressed on these cells. Using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, immunoblot analysis and quantitation of caspase-3 activity to detect apoptosis, preincubation of these cells with isoproterenol was found to be sufficient for protection against programmed cell death initiated by staurosporine. RNA interference strategies confirmed the necessity for Hsp27 as well as both beta-arrestin isoforms to confer this cytoprotective consequence of beta adrenergic receptor activation in this cell model. As a result, these studies represent the first description of an agonist-dependent relationship between a small heat shock protein and beta-arrestin to form a previously unknown antiapoptotic "signalosome."

Elevated expression of caspase-3 inhibitors, survivin and xIAP correlates with low levels of apoptosis in active rheumatoid synovium.

IntroductionTumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a tumour necrosis factor (TNF) family member capable of inducing apoptosis in many cell types.MethodsUsing immunohistochemistry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and real-time PCR we investigated the expression of TRAIL, TRAIL receptors and several key molecules of the intracellular apoptotic pathway in human synovial tissues from various types of arthritis and normal controls. Synovial tissues from patients with active rheumatoid arthritis (RA), inactive RA, osteoarthritis (OA) or spondyloarthritis (SpA) and normal individuals were studied.ResultsSignificantly higher levels of TRAIL, TRAIL R1, TRAIL R2 and TRAIL R4 were observed in synovial tissues from patients with active RA compared with normal controls (p < 0.05). TRAIL, TRAIL R1 and TRAIL R4 were expressed by many of the cells expressing CD68 (macrophages). Lower levels of TUNEL but higher levels of cleaved caspase-3 staining were detected in tissue from active RA compared with inactive RA patients (p < 0.05). Higher levels of survivin and x-linked inhibitor of apoptosis protein (xIAP) were expressed in active RA synovial tissues compared with inactive RA observed at both the protein and mRNA levels.ConclusionsThis study indicates that the induction of apoptosis in active RA synovial tissues is inhibited despite stimulation of the intracellular pathway(s) that lead to apoptosis. This inhibition of apoptosis was observed downstream of caspase-3 and may involve the caspase-3 inhibitors, survivin and xIAP.

Aminoguanidine impedes human pancreatic tumor growth and metastasis development in nude mice

To study the action of aminoguanidine on pancreatic cancer xenografts in relation to cell proliferation, apoptosis, redox status and vascularization. Xenografts of PANC-1 cells were developed in nude mice. The animals were separated into two groups: control and aminoguanidine treated. Tumor growth, survival and appearance of metastases were determined in vivo in both groups. Tumors were excised and ex vivo histochemical studies were performed. Cell growth was assessed by Ki-67 expression. Apoptosis was studied by intratumoral expression of B cell lymphoma-2 protein (Bcl-2) family proteins and Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (Tunel). Redox status was evaluated by the expression of endothelial nitric oxide synthase (eNOS), catalase, copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and glutathione peroxidase (GPx). Finally, vascularization was determined by Massons trichromic staining, and by VEGF and CD34 expression. Tumor volumes after 32 d of treatment by aminoguanidine (AG) were significantly lower than in control mice (P < 0.01). Median survival of AG mice was significantly greater than control animals (P < 0.01). The appearance of both homolateral and contralateral palpable metastases was significantly delayed in AG group. Apoptotic cells, intratumoral vascularization (trichromic stain) and the expression of Ki-67, Bax, eNOS, CD34, VEGF, catalase, CuZnSOD and MnSOD were diminished in AG treated mice (P < 0.01), while the expression of Bcl-2 and GPx did not change. The antitumoral action of aminoguanidine is associated with decreased cell proliferation, reduced angiogenesis, and reduced expression of antioxidant enzymes.

Apoptosis and Expression of AQP5 and TGF-β in the Irradiated Rat Submandibular Gland

To evaluate the effect of X-ray irradiation on apoptosis and change of expression of aquaporin 5 (AQP5) and transforming growth factor-β(TGF-β) in the rat submandibular gland (SMG). SMGs of 120 male Sprague-Dawley rats were irradiated with a single X-ray dose (3, 10, 20, or 30 Gy). At the early and late post-irradiation phase, apoptosis was measured by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) method, and expression of AQP5 and TGF-β was determined by immunohistochemical staining. At the late post-irradiation phase, increased apoptosis was evident and marked decreases of expression of AQP5 expression by acinar cells and TGF-β expression by ductal cells were evident. Apoptosis after X-ray irradiation develops relatively late in rat SMG. Irradiation reduces AQP5 and TGF-β expression in different SMG cell types.

Extracellular HSP70 blocks CD40L‐induced apoptosis and tubular formation in endothelial cells

Recent studies have shown that CD40, a key player in angiogenesis and tubular formation, is an extracellular receptor of the heat shock protein 70 (HSP70)-peptide complex in endothelial cells. The aim of the present study was to determine the effect of extracellular HSP70 treatment on CD40L-suppressed apoptosis and CD40L-induced tubular formation in human umbilical vein endothelial cells (HUVEC). The apoptotic index of CD40L-stimulated HUVEC with or without recombinant human HSP70 was evaluated using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay analysis. Binding of HSP70-peptide complex to CD40 on HUVEC was determined by double-labeling immunofluorescence methods. To evaluate the biological activity of CD40 engagement pretreated with rhHSP70 (0.5, 1 and 3 ng/mL), the extent of new capillary-like networking structure (tubular formation) formation in HUVEC was counted using an Olympus digital camera. Vascular invasion into MNK-28 cell clusters was assessed by counting the number of tubular structures extending from the HUVEC into growth factor-depleted Matrigel. Scores for CD34, HSP70 and CD40L expression levels in gastric cancer tissues were determined by immunostaining. CD40L stimulation inhibited vincristine-induced apoptosis of HUVEC in a dose-dependent manner. Extracellular HSP70 treatment significantly blocked the inhibition of apoptosis by CD40L in HUVEC exposed to vincristine. HSP70-peptide complex bound to CD40 on HUVEC. Extracellular HSP70 treatment also significantly reduced CD40L-induced tubular formation in a dose-dependent manner. HSP70 treatment also suppressed invasive tubular formation into MKN-28 cells clusters by CD40L-activated HUVEC. There was a significant relationship between CD40L expression levels and microvessel density; however, the relationship between HSP70 expression level and microvessel density in gastric cancer tissues was not significant. Extracellular HSP70 treatment blocks CD40L inhibition of apoptosis and CD40L induction of tubular formation in HUVEC.

Excitatory tonus is required for the survival of granule cell precursors during postnatal development within the cerebellum

In addition to protective effects within the adult central nervous system (CNS), in vivo application of N-methyl-d-aspartate inhibitors such as (+) MK-801 have been shown to induce neurodegeneration in neonatal rats over a specific developmental period. We have systematically mapped the nature and extent of MK-801-induced neurodegeneration throughout the neonatal murine brain in order to genetically dissect the mechanism of these effects. Highest levels of MK-801-induced neurodegeneration are seen in the cerebellar external germinal layer; while mature neurons of the internal granule layer are unaffected by MK-801 treatment. Examination of external germinal layer neurons by electron microscopy, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and bromodeoxyuridine (BrdU) labeling, and caspase-3 activation demonstrate that these neurons die through the process of programmed cell death soon after they exit from the cell cycle. Significantly, ablation of caspase-3 activity completely inhibited the MK-801-induced (and developmental) programmed cell death of external germinal layer neurons. Similar to caspase-3, inactivation of muscarinic acetylcholine receptors in vivo using scopolamine inhibited MK-801-induced programmed cell death. By contrast, the GABAergic agonist diazepam, either alone or in combination with MK-801, enhanced programmed cell death within external germinal layer neurons. These data demonstrate that, in vivo, cerebellar granule neurons undergo a dramatic change in intracellular signaling in response to molecules present in the local cellular milieu during their first 24 h following exit from the cell cycle.