Terminal Deoxynucleotidyl Transferase Nick-end Labeling Research Articles

Caspase-3-dependent apoptosis in cardiac myxoma: not associated with human papillomavirus or Epstein–Barr virus

Cardiac myxoma is the most common tumor of the heart, has a variable clinical presentation and immunohistochemical profile. Viral infections, such as herpes simplex virus, human papillomavirus (HPV), and Epstein–Barr virus (EBV), may play an important role in the causes of cardiac myxoma. This investigation will demonstrate caspase-3-dependent apoptosis in cardiac myxoma without HPV or EBV infection. This study included 15 patients with cardiac myxoma, who were treated with surgical excision of the lesion. Data were collected on detailed clinical parameters. Terminal deoxynucleotidyl transferase nick-end labeling assay, electrophoresis, and caspase-3 immunohistochemical studies were performed to characterize apoptosis. Genechip containing 39 subtypes was used to elucidate HPV; and polymerase chain reaction to detect LMP-1 gene of EBV. The patient population comprised of eight (53%) women and seven (47%) men. The mean age of patient participants was 45 years, with an age range of 30–70 years. All patient cases were sporadic myxomas rather than familial myxomas. The patient presentations included dyspnea (53%), asymptomatic (27%), stroke (7%), chest pain (7%), and fever (7%). All lesions were located in the left atrium. The individual patient cases of myxoma did not differ in location or clinical event in terms of pathological scores, such as vascular proliferation, inflammation, cellularity, hyaline, calcification, or thrombosis. Cardiac myxoma is characterized by apoptosis through caspase-dependent pathway. HPV or EBV was not detected in any of the study patient samples. In conclusion, no viral genomes of HPV or EBV were detected in these 15 patients. This study demonstrates that caspase-3-dependent apoptosis in cardiac myxoma is not dependent on concurrence of previous HPV and/or EBV infection.

Enamel matrix derivative inhibits TNF-α–induced apoptosis in osteoblastic MC3T3-E1 cells

The purpose of this study was to examine the effect of enamel matrix derivative (EMD) on TNF-alpha-induced apoptosis in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured at an initial density of 5000/cm 2 in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and allowed to adhere for 24 hours. Medium was then changed into DMEM with 0.5% FBS. After 16 hours, cells were treated with EMD (100 microg/mL) alone, tumor necrosis factor alpha (TNF-alpha) (20 ng/mL) alone, transforming growth factor beta 1 (TGF-beta1) (10 ng/mL) alone, TNF-alpha plus TGF-beta1, or TNF-alpha plus EMD. Cells cultured with DMEM and 0.5% FBS served as control. Following 24-hour incubation, apoptosis was assessed by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay, and quantified by cell death enzyme-linked immunosorbent assay (ELISA). Both TUNEL assay and cell death ELISA show that TNF-alpha induces apoptosis in MC3T3-E1 cells. TNF-alpha increases cell death by approximately 2-fold, which is attenuated by both EMD and TGF-beta1. Like TGF-beta1, EMD protects osteoblasts from inflammation-induced apoptosis.

Apoptosis in primary cardiac tumours.

Apoptosis, or programmed cell death, is now recognised as an important cellular event during both normal development and specific disease progression. Apoptosis has been suggested to play a critical role in several cardiovascular diseases, but has not yet been identified as a major influence in primary cardiac tumours. A retrospective review of the achieved material at Chang Gung Memorial Hospital revealed seven patients with cardiac myxoma and one with a tumour originating from the crista terminalis, from January 2002 to December 2002. The medical chart, surgical pathology reports and microscopic slides were available in all cases. All patients, including eight cardiac myxomas and one tumour from crista terminalis, were assessed for apoptosis by terminal deoxynucleotidyl transferase nick-end labelling assay. In this study, apoptosis is well documented in all seven myxoma and has even been reported in tumour from the crista terminalis. Interestingly, apoptosis appears related to the nature of the cell properties rather than the incidence of embolism. In conclusion, apoptosis is important in the progression of the primary cardiac tumours, but the mechanism of cardiac tumour regression still remains uncertain.

Cellular Proliferative Fraction Measured With Topoisomerase IIα Predicts Malignancy in Endocrine Pancreatic Tumors

Abstract Context.—Endocrine pancreatic tumors (EPTs) are rare lesions with varying biological behavior. Establishing malignancy is a challenge for clinicians and pathologists. Objective.—To establish the role of proliferative, apoptotic, angiogenic, and hormonal markers as predictors of malignancy in EPTs. Design.—Paraffin-embedded EPT samples were studied for prognostic markers. Patients.—Twenty-one consecutive patients with a diagnosis of EPT. Main Outcome Measures.—The proliferative fraction (topoisomerase IIα), microvascular density (CD34), vascular endothelial growth factor expression, and estrogen receptor-beta (ERβ) expression were studied by immunohistochemistry on all EPTs. Apoptosis was also assessed with terminal deoxynucleotidyl transferase nick-end labeling. Results.—We identified 13 benign and 8 malignant tumors. Topoisomerase IIα was significantly increased in malignant tumors (P = .001), while there were no differences in apoptosis, microvascular density, or vascular endothelial growth factor expression in association with malignancy. No correlation could be identified between microvascular density and vascular endothelial growth factor expression, and ERβ was not detected. A receiver operating characteristic curve for topoisomerase IIα disclosed that above a labeling index of 13, the test had 88% sensitivity and 100% specificity for predicting malignancy. Conclusion.—Cellular proliferation measured with topoisomerase IIα is a simple prognostic marker for malignancy in EPTs, unlike apoptosis, angiogenesis, or the presence of ERβ, which were not associated with malignant behavior. These findings designate a defined field for future research on endocrine pancreatic carcinogenesis and a possible target for chemotherapeutic agents.

Cellular Proliferative Fraction Measured With Topoisomerase IIα Predicts Malignancy in Endocrine Pancreatic Tumors

Abstract Context.—Endocrine pancreatic tumors (EPTs) are rare lesions with varying biological behavior. Establishing malignancy is a challenge for clinicians and pathologists. Objective.—To establish the role of proliferative, apoptotic, angiogenic, and hormonal markers as predictors of malignancy in EPTs. Design.—Paraffin-embedded EPT samples were studied for prognostic markers. Patients.—Twenty-one consecutive patients with a diagnosis of EPT. Main Outcome Measures.—The proliferative fraction (topoisomerase IIα), microvascular density (CD34), vascular endothelial growth factor expression, and estrogen receptor-beta (ERβ) expression were studied by immunohistochemistry on all EPTs. Apoptosis was also assessed with terminal deoxynucleotidyl transferase nick-end labeling. Results.—We identified 13 benign and 8 malignant tumors. Topoisomerase IIα was significantly increased in malignant tumors (P = .001), while there were no differences in apoptosis, microvascular density, or vascular endothelial growth fac...

Ibuprofen inhibition of tendon cell proliferation and upregulation of the cyclin kinase inhibitor p21 CIP1

Sports-related tendon injuries are commonly treated with nonsteroidal antiinflammatory drugs. This study was designed to determine the in vitro effect of ibuprofen on the proliferation of tendon cells intrinsic to rat Achilles tendon. Furthermore, the existence of a correlation between this effect and the expression of the cyclin kinase inhibitor p21 CIP1 and retinoblastoma (Rb) protein was also examined. Using cultured tendon cells, cell viability was evaluated by MTT assay. To determine whether apoptosis was related to the effect of ibuprofen, terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay was used. The mitotic index (MI) was calculated from the number of cells in the mitotic phase as stained and identified by propidium iodide. The mRNA expression of p21 CIP1 was determined by reverse transcription-polymerase chain reaction (RT-PCR). Protein expressions of p21 CIP1 and Rb protein were determined by Western blot analysis. A dose-dependent decrease in the cellularity of tendon cells by ibuprofen was demonstrated by MTT assay ( p<0.001). However, TUNEL assay revealed no evidence of apoptosis. Ibuprofen dose-dependently reduced the MI ( p<0.001). Upregulation of p21 CIP1 both at the levels of mRNA expression and protein was revealed from RT-PCR and Western blot analyses. The inhibition of Rb protein phosphorylation was also noted in ibuprofen-treated cells. In conclusion, ibuprofen inhibits tendon cell proliferation in a process that is probably mediated by the upregulation of p21 CIP1 and reduced phosphorylation of Rb protein.

The Effect of Ovariectomy and Estradiol on Rabbit Bladder Smooth Muscle Contraction and Morphology

The bladder can be considered a target organ for the actions of estrogen. Decreases in circulating estrogen after menopause have been associated with bladder dysfunctions, including incontinence and detrusor instability. We determined the effects of estrogen on rabbit bladder contractile function and morphology. Female New Zealand White rabbits were ovariectomized or sham operated and treated with vehicle or estradiol (1 mg/kg weekly) for 5 weeks. Serum estradiol concentration was monitored every 2 weeks. After treatment each rabbit was anesthetized, the bladder was catheterized, cystometry was performed, and the bladder was removed for contractile and morphological studies. Apoptosis in paraffin embedded rabbit bladder tissue was detected using in situ end-labeling, specifically terminal deoxynucleotidyl-transferase nick end labeling or the TUNEL assay. Ovariectomy resulted in a 50% decrease in circulating estrogen and estradiol treatment resulted in a 5-fold increase. Ovariectomy had no significant effects on bladder capacity, micturition pressure or bladder weight; whereas estradiol treatment resulted in significant increases in bladder capacity and bladder weight. Ovariectomy resulted in a decreased rate of tension generation in response to field stimulation, carbachol and KCl. Estradiol resulted in increased contractile responses to FS and carbachol, and an increased rate of tension generation for carbachol and KCl. Histologically ovariectomy resulted in significant urothelial apoptosis, which was not present in the sham operated or estradiol treated groups. Estradiol treatment resulted in the appearance of large cytoplasmic vacuoles in the urothelium and significant smooth muscle hypertrophy. These findings demonstrate that bladder function and structure can be significantly affected by modulating the circulating estrogen level. In addition, estrogen given in pharmacological doses can have a significant hypertrophic effect on bladder smooth muscle, resulting in increased contractile function.

Prevalence of necrosis in C2-ceramide-induced cytotoxicity in NB16 neuroblastoma cells.

The mechanism of cell death triggered by C2-ceramide was investigated using the NB16 neuroblastoma cell line. Treatment of NB16 cells with 20 microM C2-ceramide for 20 h resulted in approximately 75% loss of cell viability, but only 25% of cells were scored as apoptotic based on terminal deoxynucleotidyl transferase nick-end labeling. Ultrastructural analysis revealed early development of necrotic cytoplasmic vacuolization. After 20 h of treatment with C2-ceramide, the majority of cells possessed necrotic morphology with pronounced cytoplasmic vacuolization and without any nuclear changes, although a quarter of the cell population also exhibited clear perinuclear chromatin condensation characteristic of apoptosis. Flow cytometric analysis of cells labeled with both annexin V and propidium iodide showed the rapid accumulation of C2-ceramide-treated cells in the necrotic/late apoptotic fraction. In contrast, cells treated with tumor necrosis factor alpha plus cycloheximide (TNFalpha + CHX) first appeared in the early apoptotic fraction and then accumulated in the necrotic/late apoptotic fraction. Both C2-ceramide and TNFalpha + CHX increased caspase 8- and 3-like activities in cytosolic extracts; however, treatment of cells with the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected NB16 cells from TNFalpha + CHX-induced cell death but did not prevent C2-ceramide cytotoxicity. Although C2-ceramide triggered apoptosis in a fraction of the cells, cell death in the population was primarily caused by necrosis. Thus, C2-ceramide does not faithfully mimic the effects of apoptotic ligands such as TNFalpha, which are thought to be mediated by an accumulation of endogenous ceramide. The inhibition of phosphatidylcholine synthesis is a target for C2-ceramide-mediated cytotoxicity, and this work suggests that other agents that kill cells by inhibiting this pathway may also use a mixture of mechanisms, including necrosis as well as apoptosis.

Effects of hypoxia, hypoglycemia, and muscle shortening on cell death in the sheep ductus arteriosus.

After birth, constriction of the full-term ductus arteriosus produces ischemic hypoxia, caspase activation, DNA fragmentation (>70% of cell nuclei are positive by the terminal deoxynucleotidyl transferase nick-end labeling [TUNEL] technique), and permanent ductus closure. In contrast, the preterm ductus frequently fails to develop these changes. We used the TUNEL technique to examine rings of fetal ductus arteriosus (incubated for 24 h at different oxygen and glucose concentrations) to determine the roles of 1) constriction and shortening, 2) hypoxia, and 3) hypoglycemia in producing cell death. Under controlled conditions, late-gestation ductus rings had a low rate of TUNEL-positive staining (0.6 +/- 0.9%) that did not change during muscle shortening. Although hypoxia (6.9 +/- 3.5%) and hypoglycemia (2.4 +/- 1.9%) increased the incidence of TUNEL-positive staining, only the combination of hypoxia-plus-hypoglycemia increased the incidence to the range found in vivo (83 +/- 9.5%). The combination of hypoxia-plus-hypoglycemia was associated with an oligonucleosomal pattern of DNA fragmentation. Under the same experimental conditions, the preterm ductus was capable of developing a similar degree of TUNEL-positive staining as found at term. Although caspase-3 and caspase-7 were activated in rings exposed to hypoxia-plus-hypoglycemia, a nonselective caspase inhibitor, Z-VAD.FMK (which inhibited caspase-3 and caspase-7 cleavage in the rings), did not diminish the degree of TUNEL-positive staining. We hypothesize that the preterm ductus is capable of developing an extensive degree of cell death, if it can develop the same degree of hypoxia and hypoglycemia found in the full-term newborn ductus. We also hypothesize that cell death in the ductus wall may involve pathways that are not dependent on caspase-3 or -7 activation.

Induction of apoptosis of articular chondrocytes and suppression ofarticular cartilage proteoglycan synthesis by heat shock

We investigated cellular and matrix responses ofarticular cartilage to heat shock. Rat articular cartilage waspretreated at 37°C for 24 h before being exposed to 48°C for10min and subsequently incubated at 37°C for 1, 2, 4, 7, 10,and 14 days. Following heat shock, a terminal deoxynucleotidyltransferase nick end labeling assay showed that articularchondrocyte apoptosis appeared at day 1, peaked at day 7,and declined by day 14. Analysis by transmission electronmicroscopy confirmed that the chondrocytes had characteristicmorphological features of apoptosis; immunohistochemicalstaining revealed that caspase-3 activity inchondrocytes increased, 3-B-3-positive articular chondrocytesdecreased in number, and the expression of 3-B-3 nativeepitope in articular chondrocytes was reduced. Safranin-Ostaining revealed that depletion of proteoglycans in the matrixwas not found in any group. Morphological and biochemicalevidence from this study suggested that heat shock at 48°Cinduced articular chondrocyte apoptosis and suppressedproteoglycan synthesis of articular cartilage in vitro. Thisstudy thus provides evidence of the onset of osteoarthritisinduced by heat shock and a basis for choosing a temperatureat which malignant bone tumor cells can be killed with minimaldamage to articular cartilage.

Suppression of Fas-mediated signaling pathway is involved in zinc inhibition of ethanol-induced liver apoptosis.

Apoptosis is critically involved in hepatic pathogenesis induced by acute alcohol exposure. This study was undertaken to test the hypothesis that zinc interferes with an important Fas ligand-mediated pathway in the liver, leading to the inhibition of ethanol-induced apoptosis. Male 129/Sv(PC)J mice were injected subcutaneously with ZnSO4 (5 mg of Zn ion/kg) in 12-hr intervals for 24 hr before intragastric administration of ethanol (5 g/kg) in 12-hr intervals for 36 hr. Ethanol-induced apoptosis in the liver was detected by a terminal deoxynucleotidyl transferase nick-end labeling assay and was further confirmed by electron microscopy. The number of apoptotic cells in the livers pretreated with zinc was significantly decreased, being only 15% of that found in the animals treated with ethanol only. Characteristic apoptotic morphological changes observed by electron microscopy were also inhibited by zinc. Importantly, zinc inhibited ethanol-induced activation of caspase-3, the primary executioner protease responsible for alcohol-induced liver apoptosis, and caspase-8 as determined by enzymatic assay. Immunohistochemical analysis revealed that zinc inhibited ethanol-induced endogenous Fas ligand activation, which is a key component in signaling pathways leading to hepatic caspase-8 and subsequent caspase-3 activation and apoptosis. These results demonstrate that zinc is a potent inhibitor of acute ethanol-induced liver apoptosis, and this effect occurs primarily through zinc interference with Fas ligand pathway and the suppression of caspase-3.

Flow cytometric detection of spontaneous apoptosis in human breast cancer using the TUNEL-technique

Microscopic detection of structural alterations is the most reliable method to identify apoptotic cells, which however, does not allow any correlation with cell cycle phases. Discrimination of individual cells within solid human tumors undergoing apoptotic death is possible by flow cytometry where apoptotic cells appear in a hypodiploid sub G0/1-peak as a consequence of partial DNA loss. To refer induction of apoptosis to cell cycle phases we adopted the terminal deoxynucleotidyl transferase nick-end-labelling (TUNEL) technique to flow cytometry which enables the detection of cellular DNA content and DNA fragmentation by multiparametric analysis. One thousand seven hundred human breast carcinomas were screened. In 40 cases (2.3%) of 1700 carcinomas we detected a hypodiploid sub -G0/1 apoptotic peak. The spontaneous apoptotic fractions within individual tumors ranged between 1.5 and 25%. A correlation (r 2=0.78) was found between apoptotic cells in sub-G0/1-peak measured by DNA-cytometry and TUNEL positive cells measured by multiparametric cytometry, because TUNEL reaction signed also cells with strand breaks. High proliferation indices correspond well (r 2=0.807) with the increased amount of TUNEL positive cells. Multiparametric flow cytometry for the combined determination of DNA-content and DNA-fragmentation by TUNEL offers not only the advantage of a higher apoptosis sensitivity but also enables the quantification of DNA fragmentation related to any cell cycle phase.

TGF-β1 inhibits multiple caspases induced by TNF-α in murine osteoblastic MC3T3-E1 cells

Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine that induces apoptosis in a number of cell systems, including osteoblasts. Transforming growth factor β1 (TGF-β1) is an abundant growth factor that is known to stimulate bone formation. This study was designed to examine the role of TGF-β1 on TNF-α-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 20 ng/ml of TNF-α, 10 ng/ml of TGF-β1, or combination, for 6 h. TNF-α exerted a variety of effects on the apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that TNF-α upregulated the mRNA levels of caspase-1, -7, -11, -12, and FAS. Western blot analysis showed enhanced processing of caspase-1, -7, -11, and -12, with the appearance of their activated enzymes 24 h after TNF-α treatment. In addition, caspase-3-like activity was significantly activated following TNF-α treatment. Levels of cleaved poly(ADP–ribose) polymerase and FAS protein were also elevated by TNF-α. Finally, Hoechst staining, terminal deoxynucleotidyl-transferase nick-end labeling (TUNEL) assay, and oligonucleosome ELISA all indicated that TNF-α induced apoptosis. In contrast, the addition of TGF-β1 attenuated all of the aforementioned effects of TNF-α. Our results demonstrate that TGF-β1 can decrease TNF-α-induced apoptosis in murine osteoblasts at least in part by attenuating TNF-α-induced caspase gene expression.

Apoptosis in Lymph Node Granulomas from White-tailed Deer ( Odocoileus virginianus) Experimentally Infected with Mycobacterium bovis

Apoptosis is a morphologically and biochemically distinct mechanism of cell death seen in many physiological conditions as well as in various infectious diseases. To examine apoptosis in tuberculous white-tailed deer, 32 deer were each given an intra-tonsillar injection of 300 colony-forming units of Mycobacterium bovis. Medial retropharyngeal lymph nodes were collected at 15, 28, 42, 56, 89, 180, 262 and 328 days after inoculation. Microscopical sections of lymph nodes were labelled for apoptotic cells by the terminal deoxynucleotidyl transferase nick end labelling (TUNEL) method. TUNEL, and other morphological changes within developing granulomas, were analysed and quantified by computerized image analysis. TUNEL within granulomas was greatest 28 days after inoculation and had declined to negligible levels by 328 days. Granuloma enlargement was due primarily to an increase in size of the caseo–necrotic core of the granuloma and not to increased inflammatory cellular infiltrate. These findings suggested that cell death within M. bovis -induced granulomas in white-tailed deer was due mainly to mechanisms other than apoptosis.

Blockade of Platelet-Derived Growth Factor Receptor-β Pathway Induces Apoptosis of Vascular Endothelial Cells and Disrupts Glomerular Capillary Formation in Neonatal Mice

Platelet-derived growth factor (PDGF), a potent chemotactic and proliferation factor for mesenchymal-derived cells, has been demonstrated to play critical roles in kidney development. Two receptors for PDGF, PDGFR-alpha and PDGFR-beta, have been identified and we previously analyzed the effects of blockade of PDGFR-alpha signal in neonatal mice. In the current study, we examined the role of PDGFR-beta in glomerular development by blocking PDGFR-beta signal in neonatal mice by administration of antagonistic anti-PDGFR-beta monoclonal antibody. Unlike the mice injected with anti-PDGFR-alpha antibody, the mice injected daily with anti-PDGFR-beta antibody could be kept alive at least for 2 weeks after birth but showed severe disruption of the glomerular structure, whereas no apparent deformation was observed in the collecting ducts. In the disrupted glomeruli, the number of the mesangial cells was reduced markedly. Electron microscopic analysis and immunohistochemical studies with terminal deoxynucleotidyl transferase nick-end labeling staining revealed that the capillary endothelial cells of the glomeruli in the outer cortex region underwent apoptosis. However, the glomeruli located near the medulla were less affected. Because PDGFR-beta is not expressed in the endothelial cells, the effects of the blockade of PDGFR-beta might have caused glomerular endothelial cell apoptosis by inducing the loss of mesangial cells and/or pericytes.

Feline Immunodeficiency Virus Infection Is Characterized by B7+CTLA4+T Cell Apoptosis

The B7.1 and B7.2 costimulatory molecules on antigen-presenting cells provide second signals for regulating T cell immune responses via CD28 and cytotoxic T lymphocyte antigen 4 (CTLA4) on T cells. CD28 signals cell proliferation, whereas CTLA4 signals for anergy or apoptosis, terminating the immune response. Because T cell apoptosis and immunodeficiency is a characteristic of feline immunodeficiency virus (FIV)-infected cats, it is possible that negative T cell signaling via B7 and CTLA4 may be favored in these cats. Flow cytometry revealed high percentages of CD8+ and CD4+ cells expressing B7.1, B7.2, and CTLA4 in lymph nodes of FIV-positive cats and a large fraction of CTLA4+ T cells coexpressing B7.1 and B7.2. Three-color analysis with anti-B7.1, anti-B7.2, or anti-CTLA4 and TUNEL (terminal deoxynucleotidyl transferase nick-end-labeling) analysis revealed that apoptosis was a characteristic of B7.1+ B7.2+ CTLA4+ T cells. These data support the hypothesis that lymph node apoptosis and immune deterioration in FIV-infected cats results from chronic B7.1- and/or B7.2-CTLA4-mediated T-T interactions.

Delayed cytotoxic effects of endothelin-1 on neonatal rat cardiomyocytes during hypoxia: the role of endothelin-A receptor

Endogenous endothelin-1 (ET-1) has been shown to play an important role in various cardiac ischemic conditions. However, the pathophysiological mechanism by which ET-1 is involved in these conditions is incompletely understood. Therefore, we investigated the role of ET-1, and ETA receptor antagonism during simulated hypoxia in cardiac cells. Neonatal rat cardiomyocytes were treated with ET-1 before exposure to a 120 min period of hypoxia. Measurements of lactate dehydrogenase (LDH) release were performed after incubation period of 24 h. Cardiomyocyte damage was also evaluated by using morphological technique: staining cells with α-sarcomeric actin, and nuclei by in situ terminal deoxynucleotidyl transferase nick-end (TUNEL) labeling. Ultrastructural changes were evaluated using electron microscopy. The role of ETA antagonism was evaluated by pretreatment with BQ-123. Intracellular Ca2+ changes were assessed by indo-1 fluorescence microscopy. Hypoxia increased LDH release by 4 folds. Exposure to ET-1 (0.1 μM) before hypoxia resulted in a 6-fold increase in LDH release (p<0.05 vs hypoxia alone). Pretreatment with BQ-123 before hypoxia significantly attenuated LDH release. Morphological assays revealed a severe cytotoxic effect induced by ET-1. Pretreatment with BQ-123 (5 μM) prevented the cytotoxic effects. The results were confirmed by electron microscopy. Exposure of myocytes to ET-1 (10–100 nM) caused an increase in the concentration of intracellular free Ca2+ in a concentration-dependent manner. The effect was completely inhibited by pretreatment with the ETA antagonist BQ-123 at a concentration of 5 μM. ET-1 augments cytotoxic damage in cardiac cells 24 h after exposure to simulated hypoxia. The effect is mediated through activation of the ETA receptor.

A study on apoptosis induced by As₂O₃ for small cell lung cancer

To study apoptosis of small cell lung cancer (SCLC) cell induced by As₂O₃ and enhancement effect of interferon (IFN) on As₂O₃. DNA gel electrophoresis, terminal deoxynucleotidyl transferase nick end labeling (TUNEL) and immunocytochemistry were used to detect the apoptosis induced by As₂O₃ with different doses and time and the expression of Bcl-2 or PCNA. Within certain extent of concentration, As₂O₃ could induce apoptosis of SCLC cells. And apoptotic cells increased and expression of Bcl 2 and PCNA decreased with time prolonged or dose increased. The apoptosis inducing effect of As₂O₃ combined with IFN was much higher than that of As₂O₃ alone. As₂O₃ may induce apoptosis of SCLC cells and the efficiency of apoptosis is related to dose and time. IFNα-2b might enhance the effect of apoptosis on SCLC induced by As₂O₃.

Apoptosis in cerebrum of macular mutant mouse.

Neuronal cell death in the brain of macular mutant mouse, a model of copper metabolism abnormality, has features of both apoptosis and necrosis. Apoptotic cells were morphologically identified by the terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) method and electron microscopy. Numerous TUNEL-positive cells were identified in the cerebral cortex, hippocampus and thalamus of the hemizygotes after postnatal day 11. Ultramicroscopic studies confirmed that a number of cells had apoptotic features characterized by condensation and segregation of the nuclei. Furthermore, genomic DNA gel electrophoresis revealed a laddering pattern in the hemizygous brain. Starvation, which produced a low body weight in normal mice similar to that seen in the hemizygotes, did not result in an increase of TUNEL-positive cells. We also found that there was no increase of apoptotic cells in the brains of heterozygotes and copper-supplemented hemizygotes. Immunocytochemical analysis revealed that the distribution of copper/zinc superoxide dismutase-containing cells differed from that of TUNEL-positive cells. These findings suggest that copper deficiency is a key factor triggering apoptosis in the brain of macular mutant mouse through a mechanism different from suppression of antioxidant action of the dismutase. The improved survival period of the copper-supplemented hemizygotes may be attributed, in part, to inhibition of excessive neuronal apoptosis identified in the late stage of the disease.

Role of fas ligand in uveal melanoma-induced liver damage.

Uveal melanoma, the most common adult intraocular malignancy, metastasizes preferentially to the liver. Areas of cell death surrounding uveal melanoma metastases were observed in the livers of mice. We hypothesized that uveal melanoma cells might express Fas ligand (FasL), facilitating FasL-mediated apoptosis of Fas-expressing hepatocytes. To determine whether Fas ligand (FasL)-expressing human uveal melanoma cells induce apoptosis of human hepatocytes in vitro and in vivo. Human uveal melanoma cell lines were assayed for FasL expression by flow cytometry and immunohistology. A human hepatocyte cell line was assayed for Fas expression by flow cytometry. Apoptosis of hepatocytes was detected by annexin V staining in vitro, and terminal deoxynucleotidyl transferase nick end labeling (TUNEL) in vivo. Human uveal melanoma cell lines expressed FasL, as determined by flow cytometry and immunohistology. Human hepatocytes were Fas-positive by flow cytometry. In vitro, annexin V staining revealed that human uveal melanoma cells induced apoptosis of human hepatocytes. TUNEL staining of liver metastases revealed apoptosis of murine hepatocytes in contact with metastatic human uveal melanoma cells. FasL-induced apoptosis of hepatocytes in contact with FasL-positive human uveal melanoma cells may contribute to hepatic failure during metastatic disease.