Isolation of Intact Organelles by Differential Centrifugation of Digitonin-Treated Hepatocytes Using a Table Eppendorf Centrifuge

Bronfman, Miguel; Loyola, Gloria; Koenig, Cecilia S.

doi:10.1006/abio.1997.2453

Cited by 27 publications

(23 citation statements)

References 6 publications

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“…After shearing the DNA, the extracts were centrifuged at 18,000 Â g for 20 minutes (all done at 4jC), and the supernatant was retained. To separate cytosolic proteins from mitochondrial proteins, at least 5 Â 10 6 cells were first incubated in ''sucrose buffer'' [250 mmol/L sucrose, 20 mmol/L HEPES, 10 mmol/L KCl, 1.5 mmol/L MgCl 2 , 1 mmol/L EDTA, 1 mmol/L DTT (pH 7.4), and protease inhibitor cocktail as above] containing 0.1 mg/mL of digitonin for 3 minutes at 37jC to solubilize the plasma membrane and then centrifuged at 12,000 Â g for 5 minutes at 4jC (33). The supernatant was removed and diluted with a 50% volume of cell lysis buffer (''cytosolic'' fraction).…”

Section: Methodsmentioning

confidence: 99%

Nitric Oxide–Induced Apoptosis in Lymphoblastoid and Fibroblast Cells Dependent on the Phosphorylation and Activation of p53

McLaughlin

Demple²

2005

Cancer Research

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When nitric oxide (NO) is produced at micromolar concentrations, as during inflammation, exposure to surrounding cells is potentially cytotoxic. The NO-dependent signaling pathways that initiate cell death are thought to involve the tumor suppressor protein p53, but the degree to which this factor contributes to NO-induced cell death is less clear. Various reports either confirm or negate a role for p53 depending on the cell type and NO donor used. In this study, we have used several pairs of cell lines whose only differences are the presence or absence of p53, and we have treated these cell lines with the same NO donor, spermineNONOate (SPER/ NO). Treatment with SPER/NO induced such apoptotic markers as DNA fragmentation, nuclear condensation, poly-(ADP-ribose) polymerase cleavage, cytochrome c release, and Annexin V staining. p53 was required for at least 50% of SPER/ NO-induced apoptotic cell death in human lymphoblastoid cells and for almost all in primary and E1A-tranformed mouse embryonic fibroblasts, which highlights the possible importance of DNA damage for apoptotic signaling in fibroblasts. In contrast, p53 did not play a significant role in NO-induced necrosis. NO treatment also induced the phosphorylation of p53 at Ser 15 ; pretreatment with phosphoinositide-3 kinase (PI3K) family inhibitors, wortmannin, LY294002, and caffeine, blocked such phosphorylation, but the p38 mitogen-activated protein kinase inhibitor, SB203580, did not. Pretreatment with the PI3K family inhibitors also led to a switch from NOinduced apoptosis to necrosis, which implicates a PI3K-related kinase such as ataxia telangiectasia mutated (ATM) or ATR (ATM and Rad3 related) in p53-dependent NO-induced apoptosis. (Cancer Res 2005; 65(14): 6097-104)

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Section: Methodsmentioning

confidence: 99%

Nitric Oxide–Induced Apoptosis in Lymphoblastoid and Fibroblast Cells Dependent on the Phosphorylation and Activation of p53

McLaughlin

Demple²

2005

Cancer Research

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“…Subcellular fractions were prepared by the method of Bronfman et al (1998). At the time of harvest, the media was removed from the cells, and each plate was washed 3 times with PBS.…”

Section: Subcellular Fractionationmentioning

confidence: 99%

Survivin splice variants regulate the balance between proliferation and cell death

et al. 2005

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Survivin is an inhibitor of apoptosis protein that also plays critical roles in regulating the cell cycle and mitosis. Its prominent expression in essentially all human malignancies, and low or absent expression in most normal tissues, suggests that it would be an ideal target for cancerdirected therapy. Impeding development of safe and effective survivin antagonists for clinical use is a lack of understanding of the molecular mechanisms by which survivin differentially affects apoptosis and cell division, in normal and malignant cells. We show that the diverse functional roles of survivin can be explained, in part, by its heterodimerization with survivin splice variants in tumor cells. Survivin and survivin-DEx3 interact within the mitochondria where they may inhibit mitochondrialdependent apoptosis. If the expression of all survivin forms is eliminated by siRNA transfections, cells undergo both apoptosis and defective cell division. Overall, we provide new insights suggesting that targeting specific survivin isoforms, rather than survivin alone, may selectively and effectively destroy tumor cells. These findings are likely to have a significant impact in the design of biologic agents for clinical therapy.

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“…As a control of cytochrome c release, the cells were exposed to staurosporine (100 µg/mL) [33]. Subcellular fractionation of the proteins was performed according to previously described protocols [8,10] with slight modifications. Briefly, the cells were trypsinized, centrifuged (800 × g for 5 min), lysed in a digitonin buffer (1 mg/mL digitonin, 20 mM Hepes, pH 7.4, 250 mM sucrose) supplemented with protease inhibitors for 1 min, and microcentrifuged at 11 000 × g for 2 min.…”

Section: Analysis Of Cytochrome C Translocationmentioning

confidence: 99%

The bovine viral diarrhea virus (BVDV) NS3 protein, when expressed alone in mammalian cells, induces apoptosis which correlates with caspase-8 and caspase-9 activation

2005

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-The bovine viral diarrhea virus (BVDV) strains exist as two biotypes, cytopathic (cp) and noncytopathic (ncp), according to their effects on tissue culture cells. It has been previously reported that cell death associated to cp BVDV in vitro is mediated by apoptosis. Here, experiments were conducted to determine the involvement of the NS3 protein in the induction of apoptosis. The NS3-and NS3∆50 (deleted from the NH2-terminal 50 amino acids)-cDNA encoding sequences of BVDV NADL cp reference strain were cloned into adenoviral vectors (AdV) from which the BVDV gene of interest could be expressed from a tetracycline-responsive promoter. A549tTA cells infected in vitro with NS3 or NS3∆50-expressing AdV showed cytopathic changes characterized by cell rounding and detachment, and nucleus chromatin condensation. DNA fragmentation assays, cytochrome c release, and activation of cellular caspases performed on these infected cells clearly correlated with the observed cytopathic changes with apoptosis. The BVDV NS3∆50-induced apoptotic process was inhibited by caspase-8-and -9-specific peptide inhibitors (Z-IETD-FMK and Z-LEHD-FMK). Furthermore, apoptosis was inhibited in cells expressing the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase (HSV2-R1) or hsp70, two proteins which are known to inhibit apoptosis associated with caspase-8 activation and cytochrome c release-dependent caspase-9 activation, respectively. Given that HSV2-R1, a specific inhibitor of the caspase-8 activation pathway, efficiently suppressed apoptosis and also prevented caspase-9 activation, the overall results indicate that the BVDV NS3/NS3∆50 induces apoptosis initiated by caspase-8 activation and subsequent cytochrome c release-dependent caspase-9 activation. bovine viral diarrhea virus / NS3 protein / apoptosis / caspases

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Isolation of Intact Organelles by Differential Centrifugation of Digitonin-Treated Hepatocytes Using a Table Eppendorf Centrifuge

Cited by 27 publications

References 6 publications

Nitric Oxide–Induced Apoptosis in Lymphoblastoid and Fibroblast Cells Dependent on the Phosphorylation and Activation of p53

Nitric Oxide–Induced Apoptosis in Lymphoblastoid and Fibroblast Cells Dependent on the Phosphorylation and Activation of p53

Survivin splice variants regulate the balance between proliferation and cell death

The bovine viral diarrhea virus (BVDV) NS3 protein, when expressed alone in mammalian cells, induces apoptosis which correlates with caspase-8 and caspase-9 activation

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