Terminal Deoxynucleotidyltransferase-mediated Deoxyuridine Triphosphate Nick End Labeling Research Articles

Overexpression of circRNA circUCK2 Attenuates Cell Apoptosis in Cerebral Ischemia-Reperfusion Injury via miR-125b-5p/GDF11 Signaling

Circular RNAs (circRNAs) are expressed at high levels in the brain and are involved in various central nervous system diseases. However, the potential role of circRNAs in ischemic stroke-associated neuronal injury remains largely unknown. Herein, we uncovered the function and underlying mechanism of the circRNA UCK2 (circUCK2) in ischemia stroke. The oxygen-glucose deprivation model in HT-22 cells was used to mimic ischemia stroke in vitro. Neuronal viability and apoptosis were determined by Cell Counting Kit-8 (CCK-8) assays and TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling) staining, respectively. Middle cerebral artery occlusion was conducted to evaluate the function of circUCK2 in mice. The levels of circUCK2 were significantly decreased in brain tissues from a mouse model of focal cerebral ischemia and reperfusion. Upregulated circUCK2 levels significantly decreased infarct volumes, attenuated neuronal injury, and improved neurological deficits. circUCK2 reduced oxygen glucose deprivation (OGD)-induced cell apoptosis by regulating transforming growth factor β (TGF-β)/mothers against decapentaplegic homolog 3 (Smad3) signaling. Furthermore, circUCK2 functioned as an endogenous miR-125b-5p sponge to inhibit miR-125b-5p activity, resulting in an increase in growth differentiation factor 11 (GDF11) expression and a subsequent amelioration of neuronal injury. Consequently, these findings showed that the circUCK2/miR-125b-5p/GDF11 axis is an essential signaling pathway during ischemia stroke. Thus, the circRNA circUCK2 may serve as a potential target for novel treatment in patients with ischemic stroke.

Exosome-Shuttled circSHOC2 from IPASs Regulates Neuronal Autophagy and Ameliorates Ischemic Brain Injury via the miR-7670-3p/SIRT1 Axis

The aim of the present study was to investigate the neuroprotective roles and mechanisms of the circular RNA circSHOC2 in ischemic-preconditioned astrocyte-derived exosomes (IPAS-EXOs) against ischemic stroke. We established an ischemia model based on oxygen glucose deprivation (OGD) in vitro and isolated resultant exosomes from astrocytes. Neuronal viability and apoptosis were determined by Cell Counting Kit-8 (CCK-8) assays and TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling) staining, respectively. Autophagy-related proteins were analyzed by western blotting. We found that exosomes derived from IPAS-preconditioned medium (IPAS-CM) exerted neuroprotection. Furthermore, circSHOC2 expression was significantly upregulated in exosomes released from IPAS-CM. Overexpression of circSHOC2 in neurons yielded the same protective effects as those from IPAS-EXOs in vitro, and similar results were also observed in the middle cerebral artery occlusion (MCAO) mouse model. Mechanistically, circSHOC2 reduced neuronal apoptosis via regulating autophagy. Furthermore, circSHOC2 was found to sponge miR-7670-3p, which regulated SIRT1 expression. Transfection with an miR-7670-3p small interfering RNA (siRNA) (siRNA-7670-3p) and incubation with circSHOC2 extracellular vesicles attenuated ischemia-induced neuronal apoptosis in vivo and in vitro, while silencing of SIRT1 reversed the protective effects of exosomal circSHOC2 on hypoxic cerebral neurons. Taken together, our findings indicate that circSHOC2 in IPAS-EXOs suppressed neuronal apoptosis and ameliorated neuronal damage by regulating autophagy and acting on the miR-7670-3p/SIRT1 axis, which might contribute to a therapeutic strategy for ischemic stroke treatment.

ScAAV2-Mediated C3 Transferase Gene Therapy in a Rat Model with Retinal Ischemia/Reperfusion Injuries

Glaucoma is characterized by retinal ganglion cell (RGC) death and axonal loss. Therefore, neuroprotection is important in treating glaucoma. In this study, we explored whether exoenzyme C3 transferase (C3)-based gene therapy could protect retinas in an ischemia/reperfusion (I/R) injury rat model. Self-complementary adeno-associated virus 2 (scAAV2) vectors encoding either C3 protein (scAAV2-C3) or enhanced green fluorescence protein (scAAV2-EGFP) were intravitreally delivered into both eyes of rats, and I/R models (acute ocular hypertension) were made in one eye of each rat at day 7 after the injection. The rats were divided into six groups: scAAV2-C3, scAAV2-C3 with I/R, scAAV2-EGFP, scAAV2-EGFP with I/R, blank control, and blank control with I/R. TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling), immunohistochemistry of cleaved caspase-3, NeuN and Brn-3a, and H&E staining were used to detect apoptotic cells and other changes in the retina. The results showed that scAAV2-C3 significantly reduced the number of apoptotic RGCs and decreased cell loss in the ganglion cell layer after I/R injury, and the I/R-injured retinas treated with scAAV2-C3 were the thickest in all I/R groups. These results suggest that scAAV2-mediated C3 gene therapy is able to protect the rat retina from I/R injury and has potential in the treatment of glaucoma in the future.

Aptamer-Functionalized Drug Nanocarrier Improves Hepatocellular Carcinoma toward Normal by Targeting Neoplastic Hepatocytes

Site-specific delivery of chemotherapeutics specifically to neoplastic hepatocytes without affecting normal hepatocytes should be a focus for potential therapeutic management of hepatocellular carcinoma (HCC). The aptamer TLS 9a with phosphorothioate backbone modifications (L5) has not been explored so far for preferential delivery of therapeutics in neoplastic hepatocytes to induce apoptosis. Thus, the objective of the present investigation was to compare the therapeutic potential of L5-functionalized drug nanocarrier (PTX-NPL5) with those of the other experimental drug nanocarriers functionalized by previously reported HCC cell-targeting aptamers and non-aptamer ligands, such as galactosamine and apotransferrin. A myriad of well-defined investigations such as cell cycle analysis, TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling) assay, and studies related to apoptosis, histopathology, and immunoblotting substantiated that PTX-NPL5 had the highest potency among the different ligand-attached experimental formulations in inducing selective apoptosis in neoplastic hepatocytes via a mitochondrial-dependent apoptotic pathway. PTX-NPL5 did not produce any notable toxic effects in healthy hepatocytes, thus unveiling a new and a safer option in targeted therapy for HCC. Molecular modeling study identified two cell-surface biomarker proteins (tumor-associated glycoprotein 72 [TAG-72] and heat shock protein 70 [HSP70]) responsible for ligand-receptor interaction of L5 and preferential internalization of PTX-NPL5 via clathrin-mediated endocytosis in neoplastic hepatocytes. The potential of PTX-NPL5 has provided enough impetus for its rapid translation from the pre-clinical to clinical domain to establish itself as a targeted therapeutic to significantly prolong survival in HCC.

Noninvasive Whole-Body Imaging of Phosphatidylethanolamine as a Cell Death Marker Using 99mTc-Duramycin During TNF-Induced SIRS.

Systemic inflammatory response syndrome (SIRS) is an inflammatory state affecting the whole body. It is associated with the presence of pro- and antiinflammatory cytokines in serum, including tumor necrosis factor (TNF). TNF has multiple effects and leads to cytokine production, leukocyte infiltration, and blood pressure reduction and coagulation, thereby contributing to tissue damage and organ failure. A sterile mouse model of sepsis, TNF-induced SIRS, was used to visualize the temporal and spatial distribution of damage in susceptible tissues during SIRS. For this, a radiopharmaceutical agent, 99mTc-duramycin, that binds to exposed phosphatidylethanolamine on dying cells was longitudinally visualized using SPECT/CT imaging. Methods: C57BL/6J mice were challenged with intravenous injections of murine TNF or vehicle, and necrostatin-1 was used to interfere with cell death. Two hours after vehicle or TNF treatment, mice received 99mTc-duramycin intravenously (35.44 ± 3.80 MBq). Static whole-body 99mTc-duramycin SPECT/CT imaging was performed 2, 4, and 6 h after tracer injection. Tracer uptake in different organs was quantified by volume-of-interest analysis using PMOD software and expressed as SUVmean After the last scan, ex vivo biodistribution was performed to validate the SPECT imaging data. Lastly, terminal deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed to correlate the obtained results to cell death. Results: An increased 99mTc-duramycin uptake was detected in mice injected with TNF, when compared with control mice, in lungs (0.55 ± 0.1 vs. 0.34 ± 0.05), intestine (0.75 ± 0.13 vs. 0.56 ± 0.1), and liver (1.03 ± 0.14 vs. 0.64 ± 0.04) 4 h after TNF and remained significantly elevated until 8 h after TNF. The imaging results were consistent with ex vivo γ-counting results. Significantly increased levels of tissue damage were detected via TUNEL staining in the lungs and intestine of mice injected with TNF. Interestingly, necrostatin-1 pretreatment conferred protection against lethal SIRS and reduced the 99mTc-duramycin uptake in the lungs 8 h after TNF (SUV, 0.32 ± 0.1 vs. 0.51 ± 0.15). Conclusion: This study demonstrated that noninvasive 99mTc-duramycin SPECT imaging can be used to characterize temporal and spatial kinetics of injury and cell death in susceptible tissues during TNF-induced SIRS, making it useful for global, whole-body assessment of tissue damage during diseases associated with inflammation and injury.

Neuroprotection of agomelatine against cerebral ischemia/reperfusion injury through an antiapoptotic pathway in rat

Agomelatine is an agonist of the melatonergic MT1/MT2 receptors and an antagonist of the serotonergic 5-HT receptors. Its actions mimic melatonin in antioxidative and anti-inflammation. However, the protective mechanism of agomelatine in ischemic/reperfusion (I/R) injury has not been investigated. In this study, cerebral I/R injury rats were induced by middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion. The rats were randomly divided into 6 groups (12 rats per group): sham-operated; vehicle-treated I/R; 20 mg/kg, 40 mg/kg, and 80 mg/kg agomelatine-treated I/R; and 10 mg/kg melatonin-treated I/R. Agomelatine and melatonin were intraperitoneally administrated to the rats 1 h before MCAO induction. After reperfusion for 24 h, the brain samples were harvested for evaluating the infarct volume, histological changes, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining as well as cleaved caspase-3, Bax, Bcl-XL, nuclear factor erythroid-2-related factor (Nrf2), and heme oxygenase (HO-1) levels. Agomelatine treatment significantly decreased apoptosis, with decreases in Bax and cleaved caspase-3, and increased Bcl-XL, along with a decrease in apoptotic neuronal cells. Moreover, agomelatine was also found to markedly increase the expression of HO-1, the antioxidative enzymes, and the activity of superoxide dismutase (SOD) mediated by Nrf2 pathway. Agomelatine treatment protects the brain from cerebral I/R injury by suppressing apoptosis and agomelatine has antioxidant properties. Hence, there exists the possibility of developing agomelatine as a potential candidate for treating ischemic stroke.

Antitumor Properties of Taxol in Combination With Cyclooxygenase-1 and Cyclooxygenase-2 Selective Inhibitors on Ovarian Tumor Growth In Vivo

The present study was designed to investigate whether taxol in combination with cyclooxygenase (COX) inhibitors could be superior on inhibitory effect of ovarian cancer growth than taxol alone as drug therapy of mice implanted with human ovarian carcinoma cell line SKOV-3. The animals were treated with 100 mg/ kg celecoxib (a COX-2 selective inhibitor) alone or in combination with 3 mg/kg SC-560 (a COX-1 selective inhibitor) by gavage twice a day, 20mg/kg taxol alone by intraperitoneal (IP) once a week or in combination with celecoxib, or SC-560/celecoxib/taxol for 3 weeks. To test the mechanism of the combination treatment, the index of cell proliferation, expression of cyclin D1, and microvessel density (MVD) in tumor tissues were determined by immunohistochemistry and the index of apoptotic cells by the terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. Mean tumor volume in the SC-560/celecoxib/taxol group was first significantly lower than control at day 14 (p < 0.05). In the SC-560/celecoxib/taxol group, the index of cell proliferation and apoptosis and quantification of cyclin D1-postive cells were 6.93%, 69.62%, and 19.14%, respectively, which are statistically significant compared with those of the control group (29.85%, p < 0.001; 32.81% and 36.99%, both p < 0.05). Statistical significance on MVD was observed between the SC-560/celecoxib/taxol (39.57 +/- 4.98) and the control (73.2 +/- 1.96) group (p < 0.001). Our results suggest that the combined antitumor efficacy of taxol and COX inhibitors may be superior to taxol alone as drug therapy against ovarian cancer in mice, and that synergism of the combination treatment in part may be mediated through accelerated apoptosis and suppression of cell proliferation and angiogenesis.

Combined Effects of Cyclooxygenase-1 and Cyclooxygenase-2 Selective Inhibitors on Ovarian Carcinoma in Vivo

The present study was designed to investigate the combined effects of cyclooxygenase (COX)-1 and COX-2 selective inhibitors on human ovarian SKOV-3 carcinoma cells xenograft-bearing mice. The animals were treated with 3 mg/kg SC-560 (a COX-1 selective inhibitor) alone, 25 mg/kg celecoxib (a COX-2 selective inhibitor) alone, or SC-560/celecoxib by gavage, twice a day for three weeks. To test the mechanism of inhibition of tumor growth by COX selective inhibitors, the index of proliferating cells in tumor tissues was determined by immunostaining and the index of apoptotic cells by the terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. The inhibitory rate on tumor growth in the combination group was 35.54% which is significant statistically compared with that of the control group (P < 0.05). In the combination group, the index of cell proliferation and apoptosis were 12.40% and 51.03% respectively, which are significant statistically compared with those of the control group (22.56%, 19.07%, all P < 0.05). These studies indicate that synergism between two COX inhibitors and inhibitor combination treatment has particular potential for chemoprevention of ovarian cancer growth.

Anti-Tumoral Effect of a Celecoxib Low Dose on a Model of Human Medullary Thyroid Cancer in Nude Mice

Medullary thyroid carcinoma (MTC) is a C cell neoplasm secreting calcitonin (CT). Surgery remains the only treatment as MTC is resistant to radio- and chemotherapies. Anti-tumoral effects of nonsteroidal anti-inflammatory drugs have been observed in various cancers. Thus, we tested the anti-tumoral action of an nonsteroidal anti-inflammatory drug, celecoxib, on MTC development. We studied the expression of prostaglandin (PG) metabolism enzymes in our in vitro (TT cells) and in vivo (TT tumors) models and in human MTCs by Western blot. We checked the effect of celecoxib on xenografted subcutaneous tumors in nude mice. Celecoxib was administrated in powder food during 9 weeks from day 1 after TT cell injection. At the end of the experiment plasma CT was measured by radioimmunoassay, the number of proliferating cells in tumor tissues was detected by Ki67 immunocytochemistry and apoptotic nuclei by caspase 3 ad Bcl-2 expression and terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. PGE(2) concentrations in TT cell medium were evaluated by an enzyme immunoassay kit. Our in vitro and in vivo models were validated: the status of PG metabolism enzymes was comparable in these models and in human MTCs. A very low dose of celecoxib, 120 ppm in food, inhibited tumor volume by 71% and reduced plasma CT level. Although no proapoptotic effect was detectable in tumors, a decrease of proliferating cells was revealed. The inducible PG synthesis enzyme, cyclooxygenase 2, was only detectable in rare stromal cells. The expression of the constitutive PG synthesis enzyme, cyclooxygenase 1, was diminished, while the level of the catabolism enzyme, 15-PG dehydrogenase, was decreased. In vitro, TT cells treated for 12 days with 25 muM celecoxib reproduced these changes, and PGE(2) secretion was not significantly modified by the treatment, in these conditions. Celecoxib has a good therapeutic potential for MTC to prevent metastasis growth, and its anti-tumoral effect is, at least in part, independent of PGE(2).

Extracellular ATP and ATPγS Suppress the Proliferation of Human Periodontal Ligament Cells by Different Mechanisms

Periodontitis, similar to many other known inflammatory diseases, is thought to increase adenosine triphosphate (ATP) levels in extracellular spaces. Extracellular ATP acts on specific receptors to modulate the proliferation of various cell types. Platelet-rich plasma (PRP) contains high levels of ectonucleotidase activity capable of degrading ATP. The aim of this study was to investigate the effects of ATP on the proliferation of human periodontal ligament (PDL) cells and how these effects are altered by ectonucleotidases. PDL cells were derived from healthy young volunteers. ATP content and DNA synthesis were quantified by a bioluminescence and an enzyme-linked immunosorbent assay, respectively. CD39 and p21(WAF1/cip1) expression was analyzed by Western blot. Apoptosis was evaluated by caspase-3/7 activity, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) activity, annexin-V-binding, and DNA fragmentation. CD39 and ectonucleotidase-like activity were found in PDL cells and serum, respectively. Because less CD39 is expressed in freshly plated cells, both exogenous ATP and ATPgammaS, a slowly hydrolysable analog, inhibited cell proliferation under low serum conditions. ATP upregulated p21(WAF1/cip1), an inhibitor of cell-cycle progression, whereas ATPgammaS induced caspase-dependent apoptosis. Either upregulation of CD39 or added serum rescued cells from the cytostatic actions of exogenous ATP. In PDL cells expressing low CD39 levels, both ATP and ATPgammaS inhibited proliferation but by different mechanisms. ATP-induced growth arrest suggests that periodontal tissue regeneration is often suppressed at the site of injury. Furthermore, added ectonucleotidases protected PDL cells from ATP's cytostatic actions, suggesting that ectonucleotidase-rich PRP augments the regenerative actions of its constituent growth factors by protecting against exogenous ATP at clinical sites.

Chronic Treatment with Mood Stabilizers Lithium and Valproate Prevents Excitotoxicity by Inhibiting Oxidative Stress in Rat Cerebral Cortical Cells

Recent studies indicate that chronic treatment with the mood-stabilizing drugs lithium and valproate produces a neuroprotective effect against excitotoxicity. In this study, we aimed to determine whether inhibiting oxidative damage plays a role in a neuroprotective effect of lithium and valproate against excitotoxicity. Intracellular free calcium concentration was measured with the fluorescent calcium ion indicator fluo-3. Malondialdehyde, an end product derived from peroxidation of polyunsaturated fatty acid, and protein carbonyls were used to assess oxidative damage to lipid and protein. Excitotoxicity was assayed by measuring cell viability with the MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide] method and by measuring deoxyribonucleic acid (DNA) fragmentation with TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling) staining. We found that chronic treatment with lithium and valproate at their therapeutically relevant concentrations significantly inhibited the glutamate-induced increase of intracellular free calcium concentration, lipid peroxidation, protein oxidation, DNA fragmentation, and cell death in primary cultured rat cerebral cortical cells. This treatment had no effect on basal intracellular free calcium concentration, lipid peroxidation, protein oxidation, DNA fragmentation, and cell death. Our results suggest that chronic treatment with lithium and valproate inhibits oxidative damage to lipid and protein and in turn produces a neuroprotective effect against excitotoxicity.

2‐Chloroethyl‐3‐sarcosinamide‐1‐nitrosourea (SarCNU) inhibits prostate carcinoma cell growth via p53‐dependent and p53‐independent pathways

Prostate carcinoma is the most commonly occurring malignancy in men. Although 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU), an analog of the chloroethylnitrosoureas, has been used in the treatment of advanced solid tumors, the molecular mechanisms underlying the antineoplastic activity of this agent are not well understood. In the current study, the authors sought to investigate the effects of SarCNU on prostate carcinoma cell growth in vivo and in vitro. Male SCID mice underwent subcutaneous implantation (on both flanks) of human CWR-22 and CWR-22R prostate carcinoma xenografts. Mice were treated with either vehicle or 60 or 80 mg SarCNU per kg body weight for 5 days, with tumor growth being assessed every 3 days. Animals were sacrificed 21 days after the final injection, and tumors subsequently were collected, weighed, and processed for analysis. Immunohistochemical analyses were performed to obtain data on the localization of p53 and p21Cip1/Waf1. Cell counting, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assays, cell cycle analyses, Western blotting, and in vitro kinase assays were performed to determine the effects of SarCNU on growth, apoptosis, cell cycle arrest, cell cycle-regulated protein levels, and Cdc-2 activity, respectively. SarCNU reduced tumor incidence and inhibited the growth of CWR-22 and CWR-22R xenografts. In addition, treatment with this agent led to increases in p21Cip1/Waf1 levels and p53 phosphorylation at Ser15. In vitro administration of SarCNU to cells with wild-type p53 (LNCaP and primary CWR-22 cells) and cells with mutant p53 (PC-3 cells) resulted in G2/M arrest and the reduction of cellular Cdc-2 activity. Up-regulation of p53 levels, p53 phosphorylation at Ser15, and p21Cip1/Waf1 levels in primary CWR-22 and LNCaP cells, as well as up-regulation of Cdc-2 phosphorylation at Tyr15 in PC-3 cells, was detected. SarCNU induced G2/M arrest in prostate carcinoma cells via p53-dependent up-regulation of p21Cip1/Waf1 and p53-independent phosphorylation of Cdc-2 at Tyr15. These findings suggest a potential role for SarCNU in the treatment of prostate malignancies.

Paradoxical action of fulvestrant in estradiol-induced regression of tamoxifen-stimulated breast cancer.

Long-term tamoxifen treatment of breast cancer can result in tamoxifen-stimulated breast cancer, in which estrogen inhibits tumor growth after tamoxifen withdrawal. We investigated the molecular mechanism(s) of estradiol-induced tumor regression by using an in vivo model of tamoxifen-stimulated human breast cancer. Growth of parental estradiol-stimulated MCF-7E2 and long-term tamoxifen-stimulated MCF-7TAMLT xenografts in athymic mice was measured during treatment with vehicle, estradiol, estradiol plus tamoxifen, tamoxifen alone, estradiol plus fulvestrant, or fulvestrant alone. Apoptosis was detected by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Protein expression was assessed by western blot analysis. mRNA expression was assessed by real-time reverse transcription-polymerase chain reaction. All statistical tests were two-sided. MCF-7E2 tumor growth was stimulated by estradiol (cross-sectional area at week 13 = 1.06 cm2, 95% confidence interval [CI] = 0.82 to 1.30 cm2; P<.001) compared with control (0.06 cm2, 95%CI = -0.02 to 0.14 cm2), but tumor growth was inhibited by tamoxifen or fulvestrant. MCF-7TAMLT tumor growth was stimulated by tamoxifen) cross-sectional area at week 10 = 0.60 cm2, 95% CI = 0.50 to 0.70 cm2; P<.001) compared with control (0.02 cm2, 95% CI = 0.00 to 0.04 cm2). For MCF-7TAMLT tumors that were initially 0.35 cm2, estradiol-induced regression to 0.18 cm2 (95% CI = 0.15 to 0.21 cm2; P<.001), and tamoxifen or estradiol plus fulvestrant enhanced tumor growth to 1.00 cm2 (95% CI = 0.88 to 1.22 cm2). Estradiol increased the number of apoptotic cells in tumors by 23% (95% CI = 20% to 26%; P<.001) compared with all other treatments, decreased estrogen receptor alpha(ERalpha) protein expression, increased the expression of Fas mRNA and protein, decreased the expression of HER2/neu mRNA and protein and nuclear factor kappaB (NF-kappaB) protein but did not affect Fas ligand protein expression compared with control. Paradoxically, fulvestrant reversed this effect and stimulated MCF-7TAMLT tumor growth apparently through ERalpha-mediated regulation of Fas, HER2/neu, and NF-kappaB. Physiologic levels of estradiol induced regression of tamoxifen-stimulated breast cancer tumors, apparently by inducing the death receptor Fas and suppressing the antiapoptotic/prosurvival factors NF-kappaB and HER2/neu.

Apoptosis of auditory neurons following central process injury

Although apoptotic changes in auditory neurons induced by injury to peripheral processes (dendrites) have been intensively studied, apoptotic changes in auditory neurons induced by injury to central processes (axons of spiral ganglion cells, SGCs) have not been reported previously, probably due to lack of an experimental model. The present study reports for the first time the appearance, extent, and time course of SGC apoptosis following injury to the central processes. Apoptosis was studied in a rat model that consisted of compression of the auditory nerve in the cerebellopontine (CP) angle cistern with intraoperative recordings of auditory nerve compound action potentials (CAPs) to ensure highly reproducible results. Rats were killed between day 0 and day 14 after compression and apoptosis of SGCs was evaluated quantitatively as well as qualitatively by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, anti-activated caspase-3 immunostaining, Hoechst 33342 staining, and electron microscopy. The average number of TUNEL-positive apoptotic SGCs in each cochlear turn increased from day 1 to day 5 and then decreased gradually to an undetectable level on day 14 after compression. The average proportion of apoptotic SGCs identified in any cochlear turn on any day was always lower than 10%. The results of our present study should be useful in determining the therapeutic time window for rescuing auditory neurons undergoing apoptosis due to injury during surgery in the CP angle.

Involvement of the Fas system in liver allograft rejection

Recent studies suggest that apoptosis is an important mechanism of cell death in the rejection of liver allografts and that this process is mediated via Fas. The aim of this study was to analyze the expression of the Fas system during the liver allograft rejection and its evolution after treatment. We evaluated 14 patients with liver allograft rejection before and after treatment. Fas immunostaining was performed by the labeled streptavidin-biotin peroxidase method using a 200-fold dilution of a monoclonal antibody. Assessment of apoptosis was determined by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) technique on deparaffined liver samples. Serum levels of soluble Fas antigen (sFas) were detected by an enzyme immunoassay procedure. Twelve liver transplant patients without allograft rejection were analyzed as a control group. The number of hepatocytes expressing Fas antigen, the percentage of apoptotic hepatocytes, and the sFas levels were higher in patients with liver allograft rejection than in controls (27.9+/-23.1% vs 1.4+/-1.2%, p < 0.001; 2.2+/-0.9% vs 1.0+/-0.1%, p = 0.02; 24.2+/-39.6 vs 2.8+/-4.0 IU/ml, p = 0.03, respectively). There was a correlation between the levels of sFas, AST (r = 0.86, p < 0.001), ALT (r = 0.78, p = 0.02), and gamma-globulin levels (r = 0.86, p < 0.001). After the rejection treatment we found a significant decrease in the Fas antigen expression (18.6+/-13.3%, p < 0.05), TUNEL index (0.2+/-0.4, p < 0.05), and levels of sFas (9.9+/-30.25 IU/ml, p = 0.005). 1) The demonstration of hepatocytes with Fas antigen expression and the labeling of the nuclei by the TUNEL assay suggest that apoptosis mediated by the Fas system plays a role in the pathogenesis of liver allograft rejection. 2) The Fas expression and the sFas levels decreased in patients with treatment response.

Involvement of the Fas system in liver allograft rejection

OBJECTIVE: Recent studies suggest that apoptosis is an important mechanism of cell death in the rejection of liver allografts and that this process is mediated via Fas. The aim of this study was to analyze the expression of the Fas system during the liver allograft rejection and its evolution after treatment. METHODS: We evaluated 14 patients with liver allograft rejection before and after treatment. Fas immunostaining was performed by the labeled streptavidin-biotin peroxidase method using a 200-fold dilution of a monoclonal antibody. Assessment of apoptosis was determined by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) technique on deparaffined liver samples. Serum levels of soluble Fas antigen (sFas) were detected by an enzyme immunoassay procedure. Twelve liver transplant patients without allograft rejection were analyzed as a control group. RESULTS: The number of hepatocytes expressing Fas antigen, the percentage of apoptotic hepatocytes, and the sFas levels were higher in patients with liver allograft rejection than in controls (27.9 ± 23.1% vs 1.4 ± 1.2%, p < 0.001; 2.2 ± 0.9% vs 1.0 ± 0.1%, p = 0.02; 24.2 ± 39.6 vs 2.8 ± 4.0 IU/ml, p = 0.03, respectively). There was a correlation between the levels of sFas, AST ( r = 0.86, p < 0.001), ALT ( r = 0.78, p = 0.02), and γ-globulin levels ( r = 0.86, p < 0.001). After the rejection treatment we found a significant decrease in the Fas antigen expression (18.6 ± 13.3%, p < 0.05), TUNEL index (0.2 ± 0.4, p < 0.05), and levels of sFas (9.9 ± 30.25 IU/ml, p = 0.005). CONCLUSIONS: 1) The demonstration of hepatocytes with Fas antigen expression and the labeling of the nuclei by the TUNEL assay suggest that apoptosis mediated by the Fas system plays a role in the pathogenesis of liver allograft rejection. 2) The Fas expression and the sFas levels decreased in patients with treatment response.

Apoptosis and the expression of Fas and Fas-ligand antigen after orthotopic liver transplantation

OBJECTIVE:Recent studies suggest that apoptosis is an important mechanism of cell death in the rejection of liver allografts and that this process is mediated via Fas. The aim of this study was to analyze the expression of the Fas system during the liver allograft rejection and its evolution after treatment.METHODS:We evaluated 14 patients with liver allograft rejection before and after treatment. Fas immunostaining was performed by the labeled streptavidin-biotin peroxidase method using a 200-fold dilution of a monoclonal antibody. Assessment of apoptosis was determined by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) technique on deparaffined liver samples. Serum levels of soluble Fas antigen (sFas) were detected by an enzyme immunoassay procedure. Twelve liver transplant patients without allograft rejection were analyzed as a control group.RESULTS:The number of hepatocytes expressing Fas antigen, the percentage of apoptotic hepatocytes, and the sFas levels were higher in patients with liver allograft rejection than in controls (27.9 ± 23.1% vs 1.4 ± 1.2%, p < 0.001; 2.2 ± 0.9% vs 1.0 ± 0.1%, p = 0.02; 24.2 ± 39.6 vs 2.8 ± 4.0 IU/ml, p = 0.03, respectively). There was a correlation between the levels of sFas, AST (r = 0.86, p < 0.001), ALT (r = 0.78, p = 0.02), and γ-globulin levels (r = 0.86, p < 0.001). After the rejection treatment we found a significant decrease in the Fas antigen expression (18.6 ± 13.3%, p < 0.05), TUNEL index (0.2 ± 0.4, p < 0.05), and levels of sFas (9.9 ± 30.25 IU/ml, p = 0.005).CONCLUSIONS:1) The demonstration of hepatocytes with Fas antigen expression and the labeling of the nuclei by the TUNEL assay suggest that apoptosis mediated by the Fas system plays a role in the pathogenesis of liver allograft rejection. 2) The Fas expression and the sFas levels decreased in patients with treatment response.

Novel cell imaging techniques show induction of apoptosis and proliferation in mesothelial cells by asbestos.

We developed in situ dual-fluorescence detection techniques for measuring apoptosis and proliferation simultaneously in single dishes of cells. The deoxyribonucleic acid (DNA)-specific labeling method, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), first was used in conjunction with a 4',6-diamidino-2-phenylindole (DAPI) counterstain to detect and measure morphologic characteristics of apoptotic rat pleural mesothelial (RPM) cells isolated from Fischer 344 rats and exposed to 300 microM hydrogen peroxide (H2O2). For this purpose, 100 TUNEL-positive nuclei were measured while being viewed with DAPI counterstaining for area, perimeter, longest diameter, and average diameter, using imaging software and an image-collection apparatus. We then exposed cells to a range of concentrations of crocidolite asbestos and putative apoptotic and mitogenic agents. Exposure to crocidolite asbestos (5 microg/cm2) caused a striking dose-dependent apoptotic response at 24 h, 48 h, and 72 h. The nonfibrous crocidolite analogue riebeckite failed to induce apoptosis. At 24 h, tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml) caused an increase in apoptotic nuclei. A second method, utilizing an antibody to 5'-bromodeoxyridine (BrdU) and oxazole yellow homodimer (YOYO), showed a dose-dependent increase in proliferation occurring in cells exposed to asbestos (5 microg/cm2) at 48 h and 72 h. In addition, increased numbers of rat pleural mesothelial (RPM) cells exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA), TNF-alpha, and epidermal growth factor (EGF) exhibited incorporation of BrdU at these time points, although total numbers of cells per unit area were unchanged. Results indicate a dynamic balance between apoptosis and increased DNA synthesis after exposure of mesothelial cells to asbestos.