Terminal-deoxynucleotidyl Nick End Labeling Research Articles

Ruxolitinib altered IFN-β induced necroptosis of human dental pulp stem cells during osteoblast differentiation

ObjectiveThis study aimed to evaluate the role of ruxolitinib in the interferon beta (IFN-β) mediated osteoblast differentiation using human dental pulp stem cells (hDPSCs). DesignhDPSCs from five deciduous teeth of healthy patients were stimulated by adding human recombinant IFN-β protein (1 or 2 ng/ml) to the osteogenic differentiation induction medium. Substrate formation was determined using Alizarin Red staining, calcium concentration, and osteoblast marker expression levels. Ruxolitinib was used to inhibit the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway. Apoptosis was detected using terminal deoxynucleotidyl nick-end labeling (TUNEL) staining, and necroptosis was detected using propidium iodide staining and phosphorylated mixed lineage kinase domain-like protein (pMLKL) expression. ResultsIn the IFN-β-treated group, substrate formation was inhibited by a reduction in alkaline phosphatase (ALP) expression in a concentration-dependent manner. Although the proliferation potency was unchanged between the IFN-β-treated and control groups, the cell number was significantly reduced in the experimental group. TUNEL-positive cell number was not significantly different; however, the protein level of necroptosis markers, interleukin-6 (IL-6) and pMLKL were significantly increased in the substrate formation. Cell number and ALP expression level were improved in the group administered ruxolitinib, a JAK-STAT inhibitor. Additionally, ruxolitinib significantly suppressed IL-6 and pMLKL levels. ConclusionRuxolitinib interfered with the IFN-β-mediated necroptosis and osteogenic differentiation via the JAK-STAT pathway.

Astragaloside IV Inhibits Mitochondrial-Dependent Apoptosis of the Dorsal Root Ganglion in Diabetic Peripheral Neuropathy Rats Through Modulation of the SIRT1/p53 Signaling Pathway.

PurposeTo investigate the effect of astragaloside IV (AS-IV) on mitochondrial-dependent apoptosis in the dorsal root ganglion of diabetic peripheral neuropathy (DPN) rats through the SIRT1/p53 pathway.MethodsDiabetic rat model was induced by high-carbohydrate/high-fat diet and intraperitoneal injection of STZ. Diabetic rats were divided into three groups (n =16 per group): DPN group, AS-IV group (60mg/kg/d) and α-lipoic acid (ALA) group (60mg/kg/d). Weight and blood glucose levels were monitored every 4 weeks for 12 weeks. DPN was evaluated using the Von Frey Filaments Test and nerve conduction velocity. The dorsal root ganglia of rats were isolated and the pathological changes of mitochondria were observed by electron microscopy. The activity of mitochondrial electron transport chain complex, mitochondrial membrane potential, malonaldehyde (MDA) and glutathione (GSH) levels were measured. Neural apoptosis was detected using the Terminal Deoxynucleotidyl Nick-End Labeling (TUNEL) assay kit. The cleaved caspase-3, major proteins in the SIRT1/p53 pathway, including SIRT1, acetyl p53, Drp1, BAX, and BCL-2, were detected using immunohistochemistry and Western blot. Gene expression of major proteins in the SIRT1/p53 pathway was also detected.ResultsAfter 12 weeks of treatment, AS-IV and ALA did not significantly affect body weight or fasting glucose levels, but reduced mechanical abnormal pain in DPN and improved nerve conduction velocity. AS-IV and ALA increased the level of GSH and decreased the level of MDA. Both AS-IV and ALA can reduce mitochondrial damage, improve mitochondrial electron transport chain complex activity and mitochondrial membrane potential, and reduce the percentages of positive cells with DNA fragmentation and the expression of cleaved caspase-3 protein. AS-IV and ALA up-regulated the expression of SIRT1 and down-regulated the expression of acetyl-p53, Drp1 and the ratio of BAX to BCL-2. Changes in gene expression were similar.ConclusionAS-IV can reduce the occurrence of mitochondrial-dependent apoptosis by regulating the SIRT1/p53 pathway. It has a similar therapeutic effect as ALA and is therefore a promising drug for the potential treatment of DPN.

Some ‘white’ oocytes undergo atresia and fail to mature during the reproductive cycle in female genetically improved farmed tilapia (Oreochromis niloticus)

Low fecundity and unsynchronized spawning cycles in tilapia contribute to low broodstock productivity, a major factor limiting tilapia culture production and expansion. Previous studies have reported that ‘white’ oocytes in the ovarian tissue of tilapia continue to develop to maturity during the next spawning period. To test this hypothesis we use hematoxylin-eosin staining, terminal deoxynucleotidyl nick-end labeling, and electron microscopy to analyze development of ‘white’ oocytes in genetically improved farmed tilapia (GIFT: Oreochromis niloticus) females over time (1, 7, 13, 19, and 25 days) during one reproductive cycle, and real-time PCR to identify expression of autophagy- and apoptosis-related genes in gonad tissue. Only some of these ‘white’ oocytes developed to maturity and discharge, while many underwent atresia during development. Atretic follicles were detected at various stages of oocyte development, and the number of ‘white’ oocytes continued to increase during the reproductive cycle. On days 19 and 25 during one reproductive cycle, many apoptotic granulosa cells were distributed around stage IV and V oocytes; the integrity of granulosa cells was poor, organelles were deformed and ruptured, and the number of autophagic lysosomes was significantly increased. We detect increased transcript levels of Caspase-3 and Becline-1 and a reduced Bcl-2/Bax ratio in ovarian tissues on day 25 of culture. Our results show that autophagy and apoptosis of granulosa cells participate in the atresia of GIFT follicles. These findings provide a theoretical basis for further improving the reproductive efficiency of tilapia and for reducing follicular atresia.

The role of glycogen synthase kinase-3β phosphorylation in the protective effects of preconditioning with endoplasmic reticulum stress induced by low dose tunicamycin in myocardial ischemia-reperfusion injury

Objective To evaluate the role of glycogen synthase kinase-3beta phosphorylation (p-GKS-3β) in the protective effects of preconditioning with endoplasmic reticulum stress (ERS) induced by tunicamycin (TM) against myocardial ischemia reperfusion (I/R) injury. Methods Forty Sprague-Dawley rats were randomly divided into four groups: Sham, TM [Rats were treated by TM (0.6 mg/kg) at 30 min before model established], I/R, and TM+ LY [Rats were treated by TM (0.6 mg/kg) and protein kinase B (Akt) inhibitor LY294002 (10 mg/kg) before model established]. The models of I/R were established by occlusion of the left anterior descending coronary artery for 0.5 h followed by reperfusion for 2 h. Blood samples were withdrawn from carotid artery after thoracotomy and 2 h after reperfusion. The levels of creatine kinase isozyme (CK-MB) and lactic dehydrogenase (LDH) were detected. The expression of phosphorylated Akt (p-Akt) and p-GSK-3β in the heart at the end of the I/R was examined by Western blotting in all groups. The myocardial apoptosis index (AI) was measured using terminal deoxynucleotidyl nick-end labeling in all groups. Results The levels of CK-MB, LDH and AI in TM+ LY group were higher than TM group[(987±103) U/L vs. (746±99) U/L, t=2.945, P=0.006; (4 761±636) U/L vs. (3 681±518) U/L, t=2.789, P=0.008; (18.7±2.9)% vs. (14.4±2.8)%, t=2.864, P=0.007; respectively] at 2 h after reperfusion. The expressions of p-Akt, p-GSK-3β protein in TM group (0.613±0.068, 0.717±0.086 respectively) and TM+ I/R group (0.364±0.046, 0.452±0.053) were higher than I/R group (0.251±0.032, 0.232±0.031), but expressions in TM group were higher than TM+ LY group at 2 h after reperfusion (t=2.925, P=0.006; t=2.858, P=0.007; respectively). Conclusion Preconditioning with ERS induced by low dose TM attenuates I/R injury, which may be associated with GSK-3β phosphorylation level by Akt-GSK-3β pathway in rats. Key words: Myocardial ischemia reperfusion injury; Endoplasmic reticulum stress; Preconditioning; Glycogen synthase kinase-3β

Preconditioning with endoplasmic reticulum stress induced by low dose tunicamycin attenuates myocardial inflammation in myocardial ischemia-reperfusion injury

Objective To evaluate the protective effects of preconditioning with endoplasmic reticulum stress (ERS) induced by tunicamycin (TM) against myocardial inflammation in myocardial ischemia reperfusion (I/R) injury. Methods Forty Sprague-Dawley rats were randomly divided into four groups: Sham, TM, I/R, and TM+ I/R. The model of I/R were established by occlusion of the left anterior descending coronary artery for 0.5 h followed by reperfusion for 2 h in I/R group and TM+ I/R group. TM group and TM+ I/R group were treatmented by TM 0.6 mg/kg at 30 min before model established. Blood samples were withdrawn from carotid artery after thoracotomy and 2 h after reperfusion, to detect the levels of creatine kinase isozyme (CK-MB) and lactic dehydrogenase (LDH). The protein expression of cysteinyl aspartate-specific protease (Caspase)-12 and nuclear factor (NF)-κB p65 in heart at the end of the I/R were examined by Western blotting in all groups. The myocardial apoptosis index (AI) using terminal deoxynucleotidyl nick-end labeling were observed in all groups. Results The levels of CK-MB and LDH, AI, and expressions of Caspase-12 and NF-κBp65 protein in I/R group [(546±69) U/L, (2 094±328) U/L, (29.4±3.1)%, 0.461±0.046, and 0.287±0.032 respectively] and TM+ I/R group [(977±103) U/L, (3 198±417) U/L, (12.8±1.9)%, 0.269±0.041, and 0.153±0.031 respectively] were higher than Sham group [(559±81) U/L, (2 459±231) U/L, (2.4±0.3)%, 0.067±0.009, and 0.049±0.006 respectively] and TM group [(572±81) U/L, (2 335±241) U/L, (7.7±0.6)%, 0.149±0.031, and 0.082±0.010 respectively] at 2 h after reperfusion (P<0.05). All observed indexes in I/R group were higher than TM+ I/R group at 2 h after reperfusion(P<0.05). Conclusion Preconditioning with ERS induced by low dose TM attenuates myocardial inflammatory, which may be associated with the reduced I/R injury in rats. Key words: Myocardial ischemia reperfusion injury; Endoplasmic reticulum stress; Preconditioning; Inflammation

Protective Effects of Kaempferol against Myocardial Ischemia/Reperfusion Injury in Isolated Rat Heart via Antioxidant Activity and Inhibition of Glycogen Synthase Kinase-3β.

Objective. This study aimed to evaluate the protective effect of kaempferol against myocardial ischemia/reperfusion (I/R) injury in rats. Method. Left ventricular developed pressure (LVDP) and its maximum up/down rate (±dp/dt max) were recorded as myocardial function. Infarct size was detected with 2,3,5-triphenyltetrazolium chloride staining. Cardiomyocyte apoptosis was determined using terminal deoxynucleotidyl nick-end labeling (TUNEL). The levels of creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione/glutathione disulfide (GSH/GSSG) ratio, and tumor necrosis factor-alpha (TNF-α) were determined using enzyme linked immunosorbent assay (ELISA). Moreover, total glycogen synthase kinase-3β (GSK-3β), phospho-GSK-3β (P-GSK-3β), precaspase-3, cleaved caspase-3, and cytoplasm cytochrome C were assayed using Western blot analysis. Results. Pretreatment with kaempferol significantly improved the recovery of LVDP and ±dp/dt max, as well as increased the levels of SOD and P-GSK-3β and GSH/GSSG ratio. However, the pretreatment reduced myocardial infarct size and TUNEL-positive cell rate, as well as decreased the levels of cleaved caspase-3, cytoplasm cytochrome C, CK, LDH, MDA, and TNF-α. Conclusion. These results suggested that kaempferol provides cardioprotection via antioxidant activity and inhibition of GSK-3β activity in rats with I/R.

Transplantation of iPSc Restores Cardiac Function by Promoting Angiogenesis and Ameliorating Cardiac Remodeling in a Post-infarcted Swine Model.

Induced pluripotent stem cells (iPSc) hold significant promise for the development of cardiac regenerative therapy for myocardial infarction (MI). However, preclinical optimization and validation of large-animal models will be required before iPSc used clinically. Therefore, we aim to investigate the therapeutic potential of iPSc transplantation for MI and relative mechanisms in a post-infarcted swine model. Left anterior descending coronary artery was balloon-occluded after percutaneous transluminal angiography to generate MI (60-min no-flow ischemia). Animals were then divided into Sham, PBS control, and iPS experimental groups. The cardiac function and LV structural were assessed by dual-source computed tomography. Terminal deoxynucleotidyl nick end labeling, histology, and immunofluorescence were used to examine the effect of transplanted iPS cells on apoptosis, fibrosis, and hypertrophy. At 6 weeks, LV structural abnormality and cardiac dysfunction were less pronounced in iPSc group than in PBS group, and these improvements were accompanied by reduction of scar size. iPSc transplantation was associated with significant increase of vascular density and reduced myocardial apoptosis in the border zone of infarction, which was accompanied by the reduction in fibrosis degree. Moreover, proangiogenic and antiapoptotic factors were increased significantly in iPS group compared with PBS group. Cardiomyocyte hypertrophy was significantly attenuated by iPSc transplantation. In conclusion, these results suggested that transplantation of iPSc may result in functional recovery by promoting angiogenesis, inhibiting apoptosis, and ameliorating cardiac remodeling. This proof of concept study may provide a basis for an autologous iPSc-based therapy of MI.

Increase in phosphorylation of Akt and its downstream signaling targets and suppression of apoptosis by simvastatin after traumatic brain injury

In their previous studies, the authors found that simvastatin treatment of traumatic brain injury (TBI) in rats had beneficial effects on spatial learning functions. In the current study they wanted to determine whether simvastatin suppressed neuronal cell apoptosis after TBI, and if so, they wanted to examine the underlying mechanisms of this process. Saline or simvastatin (1 mg/kg) was administered orally to rats starting on Day 1 after TBI and then daily for 14 days. Modified Neurological Severity Scores were used to evaluate the sensory motor functional recovery. Rats were killed at 1, 3, 7, 14, and 35 days after treatment, and brain tissue was harvested for terminal deoxynucleotidyl nick-end labeling (TUNEL) staining, caspase-3 activity assay, and Western blot analysis. Simvastatin significantly decreased the modified Neurological Severity Scores from Days 7 to 35 after TBI, significantly reduced the number of TUNEL-positive cells at Day 3, suppressed the caspase-3 activity at Days 1 and 3 after TBI, and increased phosphorylation of Akt as well as Forkhead transcription factor 1, inhibitory-kappaB, and endothelial nitric oxide synthase, which are the downstream targets of the prosurvival Akt signaling protein. These data suggested that simvastatin reduces the apoptosis in neuronal cells and improves the sensory motor function recovery after TBI. These beneficial effects of simvastatin may be mediated through activation of Akt, Forkhead transcription factor 1 and nuclear factor-kappaB signaling pathways, which suppress the activation of caspase-3 and apoptotic cell death, and thereby, lead to neuronal function recovery after TBI.

Transplanted embryonic stem cells following mouse myocardial infarction inhibit apoptosis and cardiac remodeling

We have previously shown that mouse embryonic stem (ES) cells transplanted following myocardial infarction (MI) differentiate into the major cell types in the heart and improve cardiac function. However, the extent of regeneration was relatively meager compared with the observed functional improvement. Therefore, we hypothesize that mechanisms in addition to regeneration contribute to the functional improvement from ES cell therapy. In this study, we examined the effect of mouse ES cells transplanted post-MI on cardiac apoptosis, fibrosis, and hypertrophy. MI was produced by left coronary artery ligation in C57BL/6 mice. Two different mouse ES cell lines, expressing enhanced green fluorescent protein and beta-galactosidase, respectively, were tested. Post-MI intramyocardial injection of 3 x 10(4) ES cells was compared with injection of medium alone. Terminal deoxynucleotidyl nick end labeling (TUNEL), immunofluorescence, and histology were used to examine the effect of transplanted ES cells on apoptosis, fibrosis, and hypertrophy. Two weeks post-MI, ES cell-transplanted hearts exhibited a significant decrease in TUNEL-stained nuclei (mean +/- SE; MI+medium = 12 +/- 1.5%; MI+ES cells = 6.6 +/- 1%, P < 0.05). TUNEL-positive nuclei were confirmed to be apoptotic by colabeling with a caspase-3 antibody. Cardiac fibrosis was 57% less in the MI+ES cell group compared with the MI + medium group (P < 0.05) as shown with Masson's trichrome staining. Picrosirius red staining confirmed a decreased amount of collagen present in the MI+ES cell group. Cardiomyocyte hypertrophy was significantly decreased following ES cell transplantation compared with medium control animals. In conclusion, transplanted mouse ES cells in the infarcted heart inhibit apoptosis, fibrosis, and hypertrophy, thereby reducing adverse remodeling.

Oligodendrocyte Progenitor Enrichment in the Connexin32 Null-Mutant Mouse

Before the establishment of chemical synapses, neural progenitors are often coupled by connexin-mediated gap junctions providing a robust mechanism for cell-cell communication in developing brain. The present study was undertaken to determine whether alterations in junctional coupling also affect neural progenitor proliferation, survival, and differentiation in adult brain. We localized the connexin32 gap junction protein to a subset of NG2+ and platelet-derived growth factor alpha receptor+ early oligodendrocyte progenitors in the dentate gyrus of adult mice. In connexin32-deficient mice, we found an increase in the total number of proliferating nestin+ and NG2+ progenitors in the subgranular zone, hilus, and polymorphonuclear layer of the dentate gyrus in vivo and in the total number of nestin+ progenitors capable of clonogenic expansion in vitro. By bromodeoxyuridine labeling, lineage analysis, and terminal deoxynucleotidyl nick end labeling, we demonstrate that turnover of these cells is constitutively enhanced in the connexin32 knock-out dentate gyrus reflecting a dynamic defect in oligodendrogenesis in this population. Analyses of surviving bromodeoxyuridine-labeled cells at 1, 3, 7, and 28 d after injection demonstrate that this transient amplifying population fails to terminally differentiate and is deleted by an apoptotic-like mechanism within 3 d of labeling. These data provide empirical evidence to support the hypothesis that connexin expression influences adult progenitor number and specifically implicate connexin32-mediated signaling in the activation, survival, and differentiation of a subset of early oligodendrocyte progenitors in postnatal brain.

Homozygous C1qa Deficiency Alters Pulmonary Clearance of Apoptotic Cells

Clearance of dying (apoptotic) cells from inflamed tissues is an integral step in the control of inflammation, and defective processing of autoantigens present in apoptotic cells may contribute to the initiation of autoimmune syndromes. The complement system is one mechanism that has been implicated in apoptotic cell removal. C1q, the first component of the classical complement cascade, binds to apoptotic cells, potentially facilitating their removal by phagocytes. In support of this hypothesis, mice homozygous for C1qa deficiency (C1qa−/−) spontaneously accumulate apoptotic cells in the kidney, and do not effectively clear apoptotic cells during thioglycolate-induced peritonitis. Interestingly, humans homozygous for C1qa−/− develop an early and severe form of systemic lupus erythematosus. We sought to determine whether C1qa−/− delayed resolution of pulmonary inflammation through defective removal of apoptotic cells. Baseline differences between C1qa−/− mice and control mice were assessed by CBCs, BAL WBC subsets, and apoptotic cells. Inflammation in C1qa−/− mice was evaluated in BAL by measurement of WBC subsets at 24, 48, and 72 h posttreatment with 20 μg of intratracheal endotoxin lipopolysaccharide [LPS]. Spontaneous clearance of apoptotic cells in BAL was assessed at these same time points by nuclear morphology, terminal deoxynucleotidyl nick-end labeling, and propidium iodide/annexin V staining. LPS-treated, C1qa−/− mice were also challenged intratracheally with apoptotic human polymorphonuclear leukocytes (PMNs) at 72 h, and clearance was ascertained. No differences were found in baseline blood cell counts, BAL WBC subsets, or BAL apoptotic cells in C1qa−/− mice vs control mice. LPS increased pulmonary PMN, but not macrophage, influx at 48 h and 72 h in C1qa−/− mice compared to control mice. BAL from LPS-challenged C1qa−/− mice and control mice contained only small percentages of spontaneous apoptotic cells (1 to 3%), and no differences were observed between groups. However, when C1qa−/− mice were challenged intratracheally with apoptotic human PMNs, clearance was decreased. These results demonstrate that C1qa−/− impairs clearance of apoptotic cells and enhances pulmonary inflammation. The impact of dysregulated apoptotic cell removal on interstitial lung disease is as yet unknown, but may provide a basis for ongoing inflammation.EST

Stress-induced apoptosis by heat shock, UV and gamma-ray irradiation in zebrafish embryos detected by increased caspase activity and whole-mount TUNEL staining

SUMMARY: Environmental stress-induced apoptosis in zebrafish Danio rerio embryos was characterized by assaying caspase-3-like activity and whole-mount terminal deoxynucleotidyl nick-end labeling (TUNEL) staining. Severe stress conditions, such as heat shock at 39°C for 1 h, ultraviolet light at 10–100 mJ/cm2 and γ-ray irradiation at 5–20 Gy induced extensive apoptosis in embryos. Apoptotic cells were observed after the bud and 1-somite stages in normal embryos by TUNEL staining, and after stress treatment many TUNEL-positive cells were found in the enveloping and deep cell layers and the larval fin. The caspase-3-like activity increased severalfold during stress-induced apoptosis in a dose-dependent manner. These findings indicate that apoptotic pathways, mediated by caspase-3-like activity, play a major role in zebrafish embryogenesis under stress conditions.

Detection of apoptosis in kidney biopsies of patients with D+ hemolytic uremic syndrome.

In this study we have investigated the presence of apoptotic cells in renal biopsy material of seven patients with hemolytic uremic syndrome (HUS) by using an improved and stringent terminal deoxynucleotidyl nick-end labeling (TUNEL) technique. Renal biopsy material was taken in the second or third week after onset of the disease. Renal biopsy material of patients with minimal lesions nephrotic syndrome or thin basement syndrome were used as control. It has been reported that nonapoptotic cells can be labeled nonspecifically due to proteinase K pretreatment or a delay in fixation when only TUNEL technique is used. In post mortem material this delay in fixation is seen. Moreover, it has been described that mainly nonapoptotic cells that shows signs of active gene transcription can be labeled in this nonspecific way. For this reason we used the TUNEL technique in combination with a label for RNA synthesis and splicing factor (SC-35). Indeed, we found nonspecific labeling of nonapoptotic nuclei in biopsy material of HUS patients, but not in control biopsy material. By using co-labeling with RNA synthesis factor SC-35, we were able to identify true apoptotic cells. There was a significant increase (p < 0.05) in the presence of apoptotic cells in biopsy material of HUS patients compared with material of controls. About 80 % of apoptotic cells were detected in tubuli and only 20 % in glomeruli of the renal biopsies of HUS patients. Furthermore, most apoptotic cells were detected in those patients that had received peritoneal dialysis suggesting that there is a relationship between severity of the disease and amount of apoptotic cells. The finding of apoptotic cells suggest that apoptosis plays a role in HUS.

Adenovirus-mediated E2F-1 gene transfer efficiently induces apoptosis in melanoma cells.

E2F-1 is a transcription factor that stimulates cellular proliferation and cell cycle progression from G(1) to S-phase. Somewhat paradoxically, E2F-1 also has the properties of a tumor suppressor. Overexpression of E2F-1 has been shown to induce apoptosis in some cancer cells. In the current study, the effect of adenovirus-mediated E2F-1 gene transfer on human melanoma cell growth was investigated. Two human melanoma cell lines, SK-MEL-28 (wild-type p53) and SK-MEL-2 (mutant p53), were treated by mock infection, infection with a control vector expressing the beta-galactosidase gene (Ad5CMV-LacZ), or infection with a vector expressing E2F-1 (Ad5CMV-E2F-1) at a multiplicity of infection of 100. Cell proliferation and viability were determined by WST-1 assay and trypan blue exclusion, respectively. Apoptosis was assessed by cell flow cytometry and confirmed by cell morphology, in situ terminal deoxynucleotidyl nick end labeling assay, and poly(ADP-ribose) polymerase cleavage assay. Marked overexpression of E2F-1 was evident in both cell lines 24 hours after infection with Ad5CMVE2F-1 by Western blot analysis. E2F-1 overexpression resulted in growth inhibition and rapid loss of cell viability. Overexpression of E2F-1 also resulted in premature S-phase entry and G(2) arrest at 24 hours followed by apoptotic cell death at 48 hours. After Ad5CMVE2F-1 infection, expression of Bax and Bak was unchanged, whereas Mcl-1 levels decreased markedly. In SK-MEL-28 cells, Bcl-XL levels also declined after E2F-1 expression. Bcl-2 was undetectable in SK-MEL-28 cells but was increased in SK-MEL-2 cells in response to E2F-1 overexpression. Adenovirus-mediated E2F-1 gene transfer efficiently induces widespread apoptosis in human melanoma cells. E2F-1 overexpression induced apoptosis in cell lines containing wild-type and mutant p53, suggesting that this effect does not require wild-type p53 function. Anti-apoptotic proteins of the Bcl-2 family, notably Mcl-1 and Bcl-XL, may be involved in mediating the response to E2F-1. These data suggest that adenovirus-mediated E2F-1 gene therapy may be effective in the treatment of melanoma.

Induction of Target Cell Apoptosis by Channel Catfish Cytotoxic Cells

This study examines cytotoxic mechanisms used by channel catfish peripheral blood-derived effector cells. Transmission electron microscopy (TEM), coupled with [3H]thymidine DNA fragmentation (JAM) and terminal deoxynucleotidyl nick-end labeling (TUNEL) assays, provided the first evidence that catfish peripheral blood cytotoxic effectors killed allogeneic targets via an apoptotic pathway. TEM demonstrated that the effector cell population present within peripheral blood leukocytes (PBLs) was composed of agranular lymphocytes that formed conjugates with, and induced apoptosis in, allogeneic target cells. Both JAM and TUNEL assays showed that PBLs induced target cell DNA fragmentation within 1 h of coculture. In addition, fixed effectors did not induce target cell necrosis or apoptosis, and target cell lysis was completely inhibited by chelation of free Ca2+ by EGTA. These results suggest that catfish peripheral blood-derived effector cells utilize a secretory mechanism rather than a ligand-based mechanism to trigger apoptosis.

Morphological changes in prostatic adenomas after transurethral microwave thermotherapy.

To study the extension and type of tissue damage in prostatic adenomas after transurethral microwave thermotherapy (TUMT). Seven patients with benign prostatic hyperplasia and scheduled for open adenectomy underwent TUMT before surgery. TUMT was performed 2 h before operation in one patient, 24 h before in five and one week before in the remaining patient. The excised adenomas were examined by routine light microscopy. In addition, the terminal deoxynucleotidyl nick-end labelling (TUNEL) technique was used to search for DNA-strand breaks in areas with morphological alterations suggestive of apoptosis. Due to technical problems, TUMT was inadequate for one patient, i.e. the effect given was extremely low. In six cases, histopathological changes were found in limited areas extending from the urethra for 20-25 mm into the prostatic tissue. The predominant histopathological findings were areas of necrosis surrounded by cells with apoptotic features. The latter proved to be TUNEL-positive, i.e. they contained nuclei with DNA fragmentation of the apoptotic type. In the case with inadequate TUMT, there was no heat-induced tissue damage. The area of tissue damage seen after TUMT was relatively small compared with the volume of the prostates. The main histopathological finding was massive necrosis, but cells undergoing apoptosis were also identified. Obviously, temperatures lower than those leading to necrosis induced apoptosis, which is a discrete type of cell death not associated with oedema or inflammatory reaction.