An ELISA for detection of apoptosis

Salgame, Padmini; Varadhachary, Arun S.; Primiano, Laura L.; Fincke, John E.; Muller, Sylviane; Monestier, Marc

doi:10.1093/nar/25.3.680

Cited by 117 publications

(70 citation statements)

References 7 publications

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“…DNA fragmentation was detected using a terminal dUTP nick-end labeling apoptosis detection kit (Upstate Biotechnology) according to the manufacturer's instruction. To quantify cell death, DNA fragmentation was assayed using a cell death ELISA kit as described (45).…”

Section: Methodsmentioning

confidence: 99%

Selective, Reversible Caspase-3 Inhibitor Is Neuroprotective and Reveals Distinct Pathways of Cell Death after Neonatal Hypoxic-ischemic Brain Injury

Han¹,

Xu²,

Choi³

et al. 2002

Journal of Biological Chemistry

170

135

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Hypoxia-ischemia (H-I) in the developing brain results in brain injury with prominent features of both apoptosis and necrosis. A peptide-based pan-caspase inhibitor is neuroprotective against neonatal H-I brain injury, suggesting a central role of caspases in brain injury. Because previously studied peptide-based caspase inhibitors are not potent and are only partially selective, the exact contribution of specific caspases and other proteases to injury after H-I is not clear. In this study, we explored the neuroprotective effects of a small, reversible caspase-3 inhibitor M826. M826 selectively and potently inhibited both caspase-3 enzymatic activity and apoptosis in cultured cells in vitro. In a rat model of neonatal H-I, M826 blocked caspase-3 activation and cleavage of its substrates, which begins 6 h and peaks 24 h after H-I. Although M826 significantly reduced DNA fragmentation and brain tissue loss, it did not prevent calpain activation in the cortex. This activation, which is associated with excitotoxic/necrotic cell injury, occurred within 30 min to 2 h after H-I even in the presence of M826. Similar to calpain activation, we found evidence of caspase-2 processing within 30 min to 2 h after H-I that was not affected by M826. Caspase-2 processing appeared to be secondary to calpain-mediated cleavage and was not associated with caspase-2 activation. These data suggest that caspase-3 specifically contributes to delayed cell death and brain injury after neonatal H-I and that calpain activation is associated with and likely a marker for the early component of excitotoxic/necrotic brain injury previously demonstrated in this model. Hypoxic-ischemic (H-I)1 encephalopathy in the prenatal and perinatal period is a major cause of morbidity and mortality and often results in cognitive impairment, seizures, and motor impairment leading to cerebral palsy (1, 2). Many studies of neonatal H-I brain injury have utilized the well characterized Levine model in which unilateral carotid ligation is followed by exposure to hypoxia in postnatal day (P) 7 rats (3-5). This model of H-I results in a reproducible pattern of hemispheric injury ipsilateral, but not contralateral, to the carotid ligation (5-7). There are prominent features of both apoptosis and necrosis when this model is performed in neonatal rats and mice (1, 8 -11). Inhibition of caspases utilizing a pan-caspase inhibitor partially protects against brain injury after neonatal H-I injury in this model (12), and similar inhibitors have been shown to partially protect against ischemic injury in adult models (13-16). Previously utilized peptide-based caspase inhibitors (e.g. Boc-D-fmk, z-VAD-fmk, z-DEVD-fmk) required relatively large doses in vivo for their protective effects, and at high concentrations, their effects are more likely to be less selective. Thus, although these studies suggest a role for caspases, the specific caspases and other proteases, which contribute to brain injury after neonatal H-I, have not been clarified.Caspases are a family of cysteine asp...

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Section: Methodsmentioning

confidence: 99%

Selective, Reversible Caspase-3 Inhibitor Is Neuroprotective and Reveals Distinct Pathways of Cell Death after Neonatal Hypoxic-ischemic Brain Injury

Han¹,

Xu²,

Choi³

et al. 2002

Journal of Biological Chemistry

170

135

View full text Add to dashboard Cite

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“…Effects on the apoptotic fraction of even greater magnitude were observed in UMSCC-11B cells with the combination treatment (p65 siRNA and TSA, 43%; NaBu, 32.6%) versus control oligonucleotide (9.0%) or p65 siRNA (12.4%) alone (histograms not shown). As another independent indicator of apoptosis, we did a DNA fragmentation assay validated against standard DNA laddering assay for apoptosis to confirm the presence of apoptotic nucleosomal DNA fragmentation induced by p65 siRNA and HDIs (28,29). The level of DNA fragmentation induced by the HDIs in combination with p65 siRNA significantly exceeded that observed with the agents alone (P < 0.05; Fig.…”

Section: Limited Inhibitory Effects Of Hdis In Umscc Cells With Basalmentioning

confidence: 97%

“…After 24 h, cells were harvested, and the extent of apoptosis was determined by quantitation of nucleosomes released into the cytoplasm using the Cell Death Detection ELISA Plus kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer's directions. The assay has been standardized against DNA laddering, a hallmark of apoptosis (28,29).…”

Section: Inhibitors and Antibodiesmentioning

confidence: 99%

Nuclear factor-κB p65 small interfering RNA or proteasome inhibitor bortezomib sensitizes head and neck squamous cell carcinomas to classic histone deacetylase inhibitors and novel histone deacetylase inhibitor PXD101

Duan

Friedman

Nottingham

et al. 2007

Molecular Cancer Therapeutics

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Histone deacetylase inhibitors (HDI) can inhibit proliferation and enhance apoptosis in a wide range of malignancies. However, HDIs show relatively modest activity in head and neck squamous cell carcinomas (HNSCC), in which we have shown the activation of nuclear factor-KB (NF-KB; NF-KB1/RelA or p50/p65), a transcription factor that promotes expression of proliferative and antiapoptotic genes. In this study, we examined if HDIs enhance activation of NF-KB and target genes and if genetic or pharmacologic inhibition of NF-KB can sensitize HNSCC to HDIs. Limited activity of classic HDIs trichostatin A and sodium butyrate was associated with enhanced activation of NF-KB reporter activity in a panel of six HNSCC cell lines. HDIs enhanced NF-KB p50/p65 DNA binding and acetylation of the RelA p65 subunit. Transfection of small interfering RNAs targeting p65 strongly inhibited NF-KB expression and activation, induced cell cycle arrest and cell death, and further sensitized HNSCC cells when combined with HDIs. The p65 small interfering RNA inhibited HDI-enhanced expression of several NF-KB -inducible genes implicated in oncogenesis of HNSCC, such as p21, cyclin D1, and BCL-XL. Bortezomib, an inhibitor of proteasome-dependent NF-KB activation, also increased sensitization to trichostatin A, sodium butyrate, and a novel HDI, PXD101, in vitro, and to the antitumor effects of PXD101 in bortezomib-resistant UMSCC-11A xenografts. However, gastrointestinal toxicity, weight loss, and mortality of the combination were dose limiting and required parenteral fluid administration. We conclude that HDI-enhanced NF-KB activation is one of the major mechanisms of resistance of HNSCC to HDIs.

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“…B: CA-OV-3 and SK-OV-3 ovarian tumor cells (5 Â 10 5 ) were treated with 10 À6 M CD437 or 4-HPR (10 À5 M) or TNF-a (50 ng/ml). An apoptosis ELISA was performed on day 5 (Salgame et al, 1997). DMSO treated was used as controls.…”

Section: Cd437 Induced Apoptosis Is Meditated In Part By Retinoid Recmentioning

confidence: 99%

Synthetic retinoids as inducers of apoptosis in ovarian carcinoma cell lines

Holmes¹,

Soprano²,

Soprano³

2004

Journal Cellular Physiology

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Apoptosis is also known as programmed cell death. Apoptosis plays an essential role in maintaining normal tissue and cell physiology in multicellular organisms. Clearance of aberrant or pre-cancerous cells occurs through the induction of apoptosis. It has been reported that many tumors and tumor cell lines have dysfunctional apoptosis signaling, causing these tumors to escape immune monitoring and internal cellular control mechanisms. One potential cause of this dysfunctional apoptosis is the tumor suppressor p53, an important regulator of growth arrest and apoptosis that is mutated in over 50% of all cancers. Retinoids have great potential in the areas of cancer therapy and chemoprevention. While some tumor cells are sensitive to the growth inhibitory effects of natural retinoids such as all-trans-retinoic acid (ATRA), many ovarian tumor cells are not. 6-[3-(1-Admantyl)]-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and fenretinide N-[4-hydroxyphenyl] retinamide (4-HPR) are conformationally restricted synthetic retinoids that induce growth arrest and apoptosis in both ATRA-sensitive and ATRA-resistant ovarian tumor cell lines. Recently, we have identified the molecular pathways of apoptosis induced by treatment of ovarian carcinoma cells with mutated p53 by CD437 and 4-HPR.

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An ELISA for detection of apoptosis

Cited by 117 publications

References 7 publications

Selective, Reversible Caspase-3 Inhibitor Is Neuroprotective and Reveals Distinct Pathways of Cell Death after Neonatal Hypoxic-ischemic Brain Injury

Selective, Reversible Caspase-3 Inhibitor Is Neuroprotective and Reveals Distinct Pathways of Cell Death after Neonatal Hypoxic-ischemic Brain Injury

Nuclear factor-κB p65 small interfering RNA or proteasome inhibitor bortezomib sensitizes head and neck squamous cell carcinomas to classic histone deacetylase inhibitors and novel histone deacetylase inhibitor PXD101

Synthetic retinoids as inducers of apoptosis in ovarian carcinoma cell lines

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