Screening and detection of apoptosis.
ABSTRACT Since programmed cell death was first described by the electron microscopic cellular changes demonstrating an organized form of cell death over 30 years ago, it has undergone a great deal of scrutiny as a potential target for several diseases including cancer. The techniques for the study of apoptosis have evolved accordingly. Methodologies for the study of apoptosis were examined by a MEDLINE search of the English-language literature and are summarized in this review. This review discusses the various ways to study apoptosis with specific assays, reagents, and molecules. The particular advantages and disadvantages of each method are reviewed.
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ABSTRACT: In this study, we reported the cloning and over expression of a gene coding for human collagen peptide (CP6) in Escherichia coli and investigated the protective effects of CP6 on UVA-irradiated human skin fibroblasts cells. The collagen peptide (CP6) was highly soluble and the expression level was approximately 10% of the total bacteria proteins. Also, we performed assays with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Annexin V-fluorescein isothiocyanate/propidine iodide (Annexin V-FITC/PI) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) methods to investigate the cytoprotective effects of CP6 on the proliferation of UVA-damaged human skin fibroblasts cells. Radiation dosages (5 J/cm 2) significantly decreased the proliferation activities of human skin fibroblasts cells (HSF). Compared with UVA irradiated group, in the given concentration, CP6 could improve the activities of cell's proliferation (P<0.05) and decrease the apoptosis rate of cell significantly (P<0.01). UVA could damage the human skin fibroblasts cell in vitro. The CP6 had protective effects on HSF irradiated by UVA, and the mechanism of this effect might be associated with its anti-oxidative effect and enhancing cell's proliferation.
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ABSTRACT: Several pathologies are associated with elevated levels of serum ferritin, for which growth inhibitory properties have been reported, however, the underlying mechanisms are still poorly defined. Previously we have described cytotoxic properties of isoferritins released from primary hepatocytes in vitro, which induce apoptosis in an iron and oxidative stress dependent mode. Here we show that this ferritin species stimulates endosome clustering and giant endosome formation in primary hepatocytes accompanied by enhanced lysosomal membrane permeability (LMP). In parallel, protein modification by lipid peroxidation derived 4-hydroxynonenal (HNE) is strongly promoted by ferritin, the HNE-modified proteins (HNE-P) showing remarkable aggregation. Emphasizing the prooxidant context, GSH is rapidly depleted and the GSH/GSSG ratio substantially declining in ferritin treated cells. Furthermore, ferritin triggers a transient upregulation of macroautophagy which is abolished by iron chelation and apparently supports HNE-P clearance. Macroautophagy inhibition by 3-methyladenine strongly amplifies ferritin cytotoxicity in a time and concentration dependent mode suggesting an important role of macroautophagy on cellular responses to ferritin endocytosis. Moreover, pointing at an involvement of lysosomal proteolysis, ferritin cytotoxicity and lysosome fragility are aggravated by the protease inhibitor leupeptin. In contrast, EGF which suppresses ferritin induced cell death attenuates ferritin mediated LMP. In conclusion, we propose that HNE-P accumulation, lysosome dysfunction and macroautophagy stimulated by ferritin endocytosis provoke lysosomal "metastability" in primary hepatocytes which permits cell survival as long as in- and extrinsic determinants (e.g. antioxidant availability, damage repair, EGF signaling) keep the degree of lysosomal destabilization below cell death inducing thresholds. Copyright © 2014. Published by Elsevier Inc.Free Radical Biology and Medicine 12/2014; 80. DOI:10.1016/j.freeradbiomed.2014.12.007 · 5.71 Impact Factor
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ABSTRACT: Nonylphenol (NP) is considered an important environmental toxicant, which may disrupt male reproductive system. The aim of this study was to investigate 4-n-nonylphenol (4-n-NP) induced apoptosis and its related mechanism in mouse Sertoli cell line, TM4 cells. Our results showed that NP treatment (0.1, 1, 10, 20 and 30μM) decreased cell viability and induced apoptosis in the cells, accompanied by alteration of Bcl-2 family mRNA expression, activation of caspases-3, release of Ca(2+), and increase of reactive oxygen species (ROS) generation. Subsequently, it was found that the levels of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) in the cells were markedly decreased, and maleic dialdehyde (MDA) content was increased by NP treatment. Then activation of the mitogen-activated protein kinases (MAPKs) pathways and inhibition of Akt pathway were simultaneously detected in NP challenged TM4 cells. Taken together, it was concluded that NP induced cytotoxicity and apoptosis in TM4 cells, and the apoptosis may be mediated via MAPKs and Akt pathways in addition to Ca(2+) release and ROS generation. Copyright © 2015. Published by Elsevier B.V.Environmental Toxicology and Pharmacology 02/2015; 39(2):815-824. DOI:10.1016/j.etap.2015.02.004 · 1.86 Impact Factor