Apoptosis in interleukin-3-dependent haemopoietic cells

Ormerod, M.G.; Collins, Mary; Rodríguez-Tarduchy, Gemma; Robertson, David

doi:10.1016/0022-1759(92)90305-d

Cited by 195 publications

(85 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Sequential cell staining with Hoechst 33342 and PI was used by Sun et al,(62) and Ormerod et al,(63) to discriminate live, apoptotic and dead cells, the latter having a permeable plasma membrane.…”

Section: Note Added In Proofsmentioning

confidence: 99%

Features of apoptotic cells measured by flow cytometry

et al. 1992

View full text Add to dashboard Cite

The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL‐60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only Sphase HL‐60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: (a) Activation of an endonuclease in apoptotic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA‐specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity oaf the apoptotic process. (b) Plasma membrane integrity, which is lost in necrotic but not in apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early‐ and late‐apoptotic cells. (c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. (d) The ATP‐dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. (e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. (f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. (g) The decrease in forward light shatter, paralleled either by no change (HL‐60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. (h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at love pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. (i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis. The data presented indicate that flow cytometry can be applied in basic research on molecular and biochemical mechanisms of apoptosis, as well as in the clinic, where the ability to monitor early signs of apoptosis in samples from patients' tumors may be predictive of the outcome of some treatment protocols. © 1992 Wiley‐Liss, Inc.

show abstract

Section: Note Added In Proofsmentioning

confidence: 99%

Features of apoptotic cells measured by flow cytometry

et al. 1992

View full text Add to dashboard Cite

show abstract

“…Cells were treated with H 2 O 2 or TNF-␣ (with or without E 2 ) or completely serum withdrawn for 72 h to induce apoptosis. DNA content was analyzed by flow cytometry and sub-G 1 population was considered as apoptotic (Ormerod et al, 1992).…”

Section: Treatment With Bad Oligonucleotidesmentioning

confidence: 99%

Estradiol Abrogates Apoptosis in Breast Cancer Cells through Inactivation of BAD: Ras-dependent Nongenomic Pathways Requiring Signaling through ERK and Akt

Fernando

Wimalasena

2004

MBoC

113

View full text Add to dashboard Cite

Estrogens such as 17-β estradiol (E2) play a critical role in sporadic breast cancer progression and decrease apoptosis in breast cancer cells. Our studies using estrogen receptor-positive MCF7 cells show that E2 abrogates apoptosis possibly through phosphorylation/inactivation of the proapoptotic protein BAD, which was rapidly phosphorylated at S112 and S136. Inhibition of BAD protein expression with specific antisense oligonucleotides reduced the effectiveness of tumor necrosis factor-α, H2O2, and serum starvation in causing apoptosis. Furthermore, the ability of E2 to prevent tumor necrosis factor-α-induced apoptosis was blocked by overexpression of the BAD S112A/S136A mutant but not the wild-type BAD. BAD S112A/S136A, which lacks phosphorylation sites for p90RSK1 and Akt, was not phosphorylated in response to E2 in vitro. E2 treatment rapidly activated phosphatidylinositol 3-kinase (PI-3K)/Akt and p90RSK1 to an extent similar to insulin-like growth factor-1 treatment. In agreement with p90RSK1 activation, E2 also rapidly activated extracellular signal-regulated kinase, and this activity was down-regulated by chemical and biological inhibition of PI-3K suggestive of cross talk between signaling pathways responding to E2. Dominant negative Ras blocked E2-induced BAD phosphorylation and the Raf-activator RasV12T35S induced BAD phosphorylation as well as enhanced E2-induced phosphorylation at S112. Chemical inhibition of PI-3K and mitogen-activated protein kinase kinase 1 inhibited E2-induced BAD phosphorylation at S112 and S136 and expression of dominant negative Ras-induced apoptosis in proliferating cells. Together, these data demonstrate a new nongenomic mechanism by which E2 prevents apoptosis.

show abstract

“…The 337H mutation is believed to be a silent mutation as it results in white colonies in the FASAY (a yeast based functional assay in which wild-type p53 results in white yeast colonies and mutant p53 results in red yeast colonies; (Flaman et al, 1995)). pC53-SN3 (an expression plasmid containing wildtype p53) and the mutant derivatives were transfected into Saos-2 cells and 72 h later the extent of apoptosis was assessed as the sub-G1 peak on a DNA histogram (Ormerod et al, 1992). In addition to staining DNA, p53 protein was also stained and therefore the percentage of p53 positive cells was known.…”

Section: Induction Of Apoptosismentioning

confidence: 99%

“…p53 protein levels were determined by measurement of green¯uorescence and DNA content was determined by measurement of red uorescence. The extent of apoptosis was measured as the sub-G 1 peak on a DNA histogram as described previously (Ormerod et al, 1992).…”

Section: Induction Of Apoptosismentioning

confidence: 99%