Chapter 9 The End of the (Cell) Line: Methods for the Study of Apoptosis in Vitro

McGahon, Anne J.; Martin, Séamus J.; Bissonnette, Reid P.; Mahboubi, Artin; Shi, Yufang; Mogil, Rona J.; Nishioka, Walter K.; Green, Douglas R.

doi:10.1016/s0091-679x(08)61929-9

Cited by 515 publications

(358 citation statements)

References 76 publications

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“…47 Cells were centrifuged at 1500 Â g for 5 min at room temperature and resuspended in 25 ml of complete medium, to which 2 ml of AcOr/EtBr solution was added. The cells were then visualized by fluorescence microscopy (Olympus IX51) and images were recorded on an Olympus DP70 camera, using an SWB filter.…”

Section: Treatment Of Cellsmentioning

confidence: 99%

The voltage-dependent anion channel-1 modulates apoptotic cell death

et al. 2005

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The role of the voltage-dependent anion channel (VDAC) in cell death was investigated using the expression of native and mutated murine VDAC1 in U-937 cells and VDAC inhibitors. Glutamate 72 in VDAC1, shown previously to bind dicyclohexylcarbodiimide (DCCD), which inhibits hexokinase isoform I (HK-I) binding to mitochondria, was mutated to glutamine. Binding of HK-I to mitochondria expressing E72Q-mVDAC1, as compared to native VDAC1, was decreased by B70% and rendered insensitive to DCCD. HK-I and ruthenium red (RuR) reduced the VDAC1 conductance but not that of E72Q-mVDAC1. Overexpression of native or E72Q-mVDAC1 in U-937 cells induced apoptotic cell death (80%). RuR or overexpression of HK-I prevented this apoptosis in cells expressing native but not E72Q-mVDAC1. Thus, a single amino-acid mutation in VDAC prevented HK-I-or RuR-mediated protection against apoptosis, suggesting the direct VDAC regulation of the mitochondria-mediated apoptotic pathway and that the protective effects of RuR and HK-I rely on their binding to VDAC.

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Section: Treatment Of Cellsmentioning

confidence: 99%

The voltage-dependent anion channel-1 modulates apoptotic cell death

et al. 2005

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“…Subsequently, cells were assessed for apoptotic morphology by supravital fluorescence microscopy. Cells were scored as apoptotic according to established morphologic criteria (McGahon et al, 1995).…”

Section: Quantitation Of Apoptotic Cellsmentioning

confidence: 99%

p53 protein regulates the effects of amifostine on apoptosis, cell cycle progression, and cytoprotection

et al. 2003

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To determine the role of p53 protein on the cellular effects of amifostine, we used molecularly engineered HCT116 colon cancer cells in which the p53 gene was inactivated by targeted homologous recombination or p53 protein was degraded by high-level expression of papillomavirus E6 protein. Amifostine induced a G1 arrest and protected against paclitaxel toxicity in p53-proficient but not in p53-deficient cells. In the absence of p53 protein, amifostine enhanced the cytotoxicity of paclitaxel. In addition, treatment of HCT116 cells with amifostine alone resulted in apoptotic cell death. Compared with p53-deficient cells, p53-proficient cells exhibited low-level resistance to amifostine-induced apoptosis. Amifostine induced the expression of p53 protein in p53-proficient cells and the expression of p21 protein in both p53-proficient and -deficient cells. These findings indicate that amifostine-induced G1 arrest and cytoprotection are mediated via a pathway that is dependent on p53 protein and that amifostine-induced expression of p21 protein is not sufficient to sustain a G1 arrest or to mediate cytoprotection. In addition, these findings identify p53 protein as a mechanism of resistance to amifostine-induced apoptosis.

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“…Percentage of cell death was calculated by changes in the light scattering properties due to reduction in cell size and increase in granularity (McGahon et al, 1995a). DNA fragmentation was quanti®ed by cell cycle analysis of total DNA content as described elsewhere (Nicoletti et al, 1991), with slight modi®cations.…”

Section: Determination Of Apoptosismentioning

confidence: 99%

“…The percentage of hypodiploid nuclei correlates with the extent of apoptosis in the sample. DNA breaks was measured by the TUNEL assay as previously described (McGahon et al, 1995a).…”

Section: Determination Of Apoptosismentioning

confidence: 99%