Adenovirus-mediated p21((WAF1/SDII/CIP1)) gene transfer induces apoptosis of human cervical cancer cell lines.
(57/5854)
p21((WAF1/SDII/CIP1)) (p21) arrests cell growth by inhibiting cyclin-depend kinases. To explore the potential of using p21 for the gene therapy of cervical cancer, we infected human papillomavirus (HPV)-positive cervical cancer cells (HeLa, SiHa, and Z172) and HPV-negative cervical cancer cells (C33A) with recombinant adenovirus encoding p21 cDNA. The results revealed that effective inhibition of cell growth could be achieved by sense p21 adenovirus but not antisense p21 adenovirus infection and occurred through apoptosis as measured by DNA fragmentation and chromatin condensation. Apoptosis was also observed in xenografts of human cervical cancer cells infected with sense p21 adenovirus, as confirmed by in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL). The apoptosis was not prevented by overexpression of the bcl-2 transgene. To sum up, the apoptotic effect suggests that p21 should be a tumoricidal agent instead of a tumoristatic agent in preventing cervical cancers. In addition, our report substantiates the combination of the high efficiency of adenovirus vector-mediated gene delivery and the apoptotic effect of p21. (+info)
CD95 ligand (Fas-L/APO-1L) and tumor necrosis factor-related apoptosis-inducing ligand mediate ischemia-induced apoptosis in neurons.
(58/5854)
Programmed cell death plays an important role in the neuronal degeneration after cerebral ischemia, but the underlying mechanisms are not fully understood. Here we examined, in vivo and in vitro, whether ischemia-induced neuronal death involves death-inducing ligand/receptor systems such as CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). After reversible middle cerebral artery occlusion in adult rats, both CD95 ligand and TRAIL were expressed in the apoptotic areas of the postischemic brain. Further recombinant CD95 ligand and TRAIL proteins induced apoptosis in primary neurons and neuron-like cells in vitro. The immunosuppressant FK506, which most effectively protects against ischemic neurodegeneration, prevented postischemic expression of these death-inducing ligands both in vivo and in vitro. FK506 also abolished phosphorylation, but not expression, of the c-Jun transcription factor involved in the transcriptional control of CD95 ligand. Most importantly, in lpr mice expressing dysfunctional CD95, reversible middle cerebral artery occlusion resulted in infarct volumes significantly smaller than those found in wild-type animals. These results suggest an involvement of CD95 ligand and TRAIL in the pathophysiology of postischemic neurodegeneration and offer alternative strategies for the treatment of cardiovascular brain disease. (+info)
Excimer laser-induced hydroxyl radical formation and keratocyte death in vitro.
(59/5854)
PURPOSE: To characterize the type of reactive oxygen species (ROS) produced by excimer photoablation of aqueous solutions and to show the effects of ROS and antioxidants on corneal stromal cells in vitro. METHODS: Electron spin-resonance spectroscopy was performed using the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) for the detection of the superoxide anion and the hydroxyl radical in an acellular DMPO solution irradiated with the excimer laser. Hydroxyl radicals were produced by the Fenton reaction in vitro by the mixture of hydrogen peroxide and ferrous iron (Fe2+), and the effects on cultured corneal fibroblasts were observed by fluorescent microscopy using the cell death marker, propidium iodide (PI) and TdT-mediated dUTP nick-end labeling (TUNEL). RESULTS: Excimer photoablation of a 1% DMPO solution produced a species-specific spin-trapping adduct for the hydroxyl radical ('OH), but not for the superoxide anion or other unidentified free radical. The signals were inhibited dose dependently by the hydroxyl radical scavenger dimethylsulfoxide (DMSO) and an L-ascorbic acid analogue, EPCK-1. The production of *OH in the supernatant of cultured rabbit corneal fibroblasts by the Fenton reaction caused an increase in PI (+) and TUNEL (+) cells by 90 minutes, which was significantly inhibited by the addition of DMSO. CONCLUSIONS: Hydroxyl radicals may be partly responsible for stromal fibroblast cell apoptosis after excimer photoablation. (+info)
Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid-beta precursor protein and amyloidogenic A beta peptide formation.
(60/5854)
The amyloid-beta precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated amyloid-beta (A beta) peptide formation. The predominant site of caspase-mediated proteolysis is within the cytoplasmic tail of APP, and cleavage at this site occurs in hippocampal neurons in vivo following acute excitotoxic or ischemic brain injury. Caspase-3 is the predominant caspase involved in APP cleavage, consistent with its marked elevation in dying neurons of Alzheimer's disease brains and colocalization of its APP cleavage product with A beta in senile plaques. Caspases thus appear to play a dual role in proteolytic processing of APP and the resulting propensity for A beta peptide formation, as well as in the ultimate apoptotic death of neurons in Alzheimer's disease. (+info)
Characterization and comparison of the mode of cell death, apoptosis versus necrosis, induced by 7beta-hydroxycholesterol and 7-ketocholesterol in the cells of the vascular wall.
(61/5854)
Oxidized low density lipoproteins (LDLs) play a central role in atherosclerosis, and their toxicity is due, at least in part, to the formation of oxysterols that have been shown to induce apoptosis in various cell types. As 7beta-hydroxycholesterol and 7-ketocholesterol are the major oxysterols found in oxidized LDLs, we have investigated and compared the mode of cell death, apoptosis versus necrosis, that they induce in the cells of the vascular wall, ie, endothelial cells, smooth muscle cells, and fibroblasts. To this end, human vascular endothelial cells from umbilical cord veins (HUVECs), human artery smooth muscle cells, A7R5 rat smooth muscle cells, MRC5 human fibroblasts, and human fibroblasts isolated from umbilical cord veins were taken at confluence and incubated for 48 hours with 7beta-hydroxycholesterol or 7-ketocholesterol (concentration range, 5 to 80 microg/mL). In all cells, both 7beta-hydroxycholesterol and 7-ketocholesterol exhibited toxic effects characterized by a loss of cell adhesion and an increased permeability to propidium iodide. In oxysterol-treated endothelial and smooth muscle cells, typical features of apoptosis were revealed: condensed and/or fragmented nuclei were detected by fluorescence microscopy after staining with Hoechst 33342, oligonucleosomal DNA fragments were visualized in situ in the cell nuclei by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method, and internucleosomal DNA fragmentation was found on agarose gel. In contrast, in oxysterol-treated fibroblasts, fragmented and/or condensed nuclei were never revealed, and no DNA fragmentation was observed either by the TUNEL method or by DNA analysis on agarose gel, indicating that these oxysterols induced necrosis in these cells but not apoptosis. In addition, acetylated Asp-Glu-Val-L-aspartic acid aldehyde (an inhibitor of Asp-Glu-Val-L-aspartic acid-sensitive caspases) prevented 7beta-hydroxycholesterol- and 7-ketocholesterol-induced cell death in HUVECs and smooth muscle cells but not in fibroblasts. Thus, 7beta-hydroxycholesterol and 7-ketocholesterol have dual cytotoxic effects on the cells of the vascular wall by their ability to induce apoptosis in endothelial and smooth muscle cells and necrosis in fibroblasts. (+info)
Induction of necrosis but not apoptosis after anoxia and reoxygenation in isolated adult cardiomyocytes of rat.
(62/5854)
OBJECTIVES: Apoptosis is one feature of myocardial damage after ischemia-reperfusion, but the causes for its induction are unclear. The present study was undertaken to investigate whether apoptosis in cardiomyocytes is directly initiated by their sub-lethal injury that results from ischemia-reperfusion. METHODS: Ischemia was simulated on isolated ventricular cardiomyocytes of adult rats by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected by (1) DNA laddering of genomic DNA, (2) TUNEL positive cells (terminal deoxynucleotidyl transferase-mediated-UTP nick end labelling) and (3) annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide. Necrotic cells were identified by (1) staining with both annexinV-FITC and propidium iodide, (2) unspecific DNA degradation and (3) enzyme release. RESULTS: Simulated ischemia caused a > 75% loss of high-energy phosphates within 2 h, which was reversible upon reoxygenation at pH 7.4. Even after 18 h of simulated ischemia, creatine phosphate contents recovered to 55.2 +/- 7.3% of control within 1 h. Apoptosis could be induced by UV irradiation (80 J/m2), H2O2 and the NO-donor N2-acetyl-S-nitroso-D,L-penicillinaminamide. In contrast to this, simulated ischemia and reoxygenation could not induce apoptosis in the cells, but with prolonged ischemia more cells became necrotic. After 18 hours of simulated ischemia and 4 h of reoxygenation 41.2 +/- 10.2% myocytes were necrotic (vs. 6.3 +/- 4.4% of control) and only 1.7 +/- 0.5% (vs. 8.7 +/- 4.6% of control) were apoptotic. The percentage of necrotic cells correlated with an increase in lactate dehydrogenase release from 9.9 +/- 0.6% (of total activity) of normoxic controls to 37.9 +/- 5.1% after 18 h of simulated ischemia and 12 h of reoxygenation. CONCLUSIONS: Simulated ischemia-reoxygenation causes necrosis of isolated cardiomyocytes but is not sufficient for induction of apoptosis. (+info)
Adenovirus-mediated p53 gene transfer in advanced non-small-cell lung cancer.
(63/5854)
BACKGROUND: Preclinical studies in animal models have demonstrated tumor regression following intratumoral administration of an adenovirus vector containing wild-type p53 complementary DNA (Ad-p53). Therefore, in a phase I clinical trial, we administered Ad-p53 to 28 patients with non-small-cell lung cancer (NSCLC) whose cancers had progressed on conventional treatments. METHODS: Patients received up to six, monthly intratumoral injections of Ad-p53 by use of computed tomography-guided percutaneous fine-needle injection (23 patients) or bronchoscopy (five patients). The doses ranged from 10(6) plaque-forming units (PFU) to 10(11) PFU. RESULTS: Polymerase chain reaction (PCR) analysis showed the presence of adenovirus vector DNA in 18 (86%) of 21 patients with evaluable posttreatment biopsy specimens; vector-specific p53 messenger RNA was detected by means of reverse transcription-PCR analysis in 12 (46%) of 26 patients. Apoptosis (programmed cell death) was demonstrated by increased terminal deoxynucleotide transferase-mediated biotin uridine triphosphate nick-end labeling (TUNEL) staining in posttreatment biopsy specimens from 11 patients. Vector-related toxicity was minimal (National Cancer Institute's Common Toxicity Criteria: grade 3 = one patient; grade 4 = no patients) in 84 courses of treatment, despite repeated injections (up to six) in 23 patients. Therapeutic activity in 25 evaluable patients included partial responses in two patients (8%) and disease stabilization (range, 2-14 months) in 16 patients (64%); the remaining seven patients (28%) exhibited disease progression. CONCLUSIONS: Repeated intratumoral injections of Ad-p53 appear to be well tolerated, result in transgene expression of wild-type p53, and seem to mediate antitumor activity in a subset of patients with advanced NSCLC. (+info)
Developmental activation of the capability to undergo checkpoint-induced apoptosis in the early zebrafish embryo.
(64/5854)
In this study, we demonstrate the developmental activation, in the zebrafish embryo, of a surveillance mechanism which triggers apoptosis to remove damaged cells. We determine the time course of activation of this mechanism by exposing embryos to camptothecin, an agent which specifically inhibits topoisomerase I within the DNA replication complex and which, as a consequence of this inhibition, also produces strand breaks in the genomic DNA. In response to an early (pre-gastrula) treatment with camptothecin, apoptosis is induced at a time corresponding approximately to mid-gastrula stage in controls. This apoptotic response to a block of DNA replication can also be induced by early (pre-MBT) treatment with the DNA synthesis inhibitors hydroxyurea and aphidicolin. After camptothecin treatment, a high proportion of cells in two of the embryo's three mitotic domains (the enveloping and deep cell layers), but not in the remaining domain (the yolk syncytial layer), undergoes apoptosis in a cell-autonomous fashion. The first step in this response is an arrest of the proliferation of all deep- and enveloping-layer cells. These cells continue to increase in nuclear volume and to synthesize DNA. Eventually they become apoptotic, by a stereotypic pathway which involves cell membrane blebbing, "margination" and fragmentation of nuclei, and cleavage of the genomic DNA to produce a nucleosomal ladder. Fragmentation of nuclei can be blocked by the caspase-1,4,5 inhibitor Ac-YVAD-CHO, but not by the caspase-2,3,7[, 1] inhibitor Ac-DEVD-CHO. This suggests a functional requirement for caspase-4 or caspase-5 in the apoptotic response to camptothecin. Recently, Xenopus has been shown to display a developmental activation of the capability for stress- or damaged-induced apoptosis at early gastrula stage. En masse, our experiments suggest that the apoptotic responses in zebrafish and Xenopus are fundamentally similar. Thus, as for mammals, embryos of the lower vertebrates exhibit the activation of surveillance mechanisms, early in development, to produce the selective apoptosis of damaged cells. (+info)