Flow cytometry and biochemical analysis of DNA degradation characteristic of two types of cell death

Afanas'ev, V. N.; Korol, B.A.; Mantsygin, Yurii A.; Nelipovich, P.A.; Pechatnikov, V A; Umansky, Samuil R.

doi:10.1016/0014-5793(86)80115-6

Cited by 93 publications

(38 citation statements)

References 11 publications

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“…48 sion of Myc or of Myc and Bcl-2 had no significant effect on cell cycle distribution in proliferating cells when compared with Rat la cells ( Fig. 6a to c This peak represents cells with <2c DNA content; specifically, these are cells with fragmented DNA (1,30). These cells constituted 47.0% ± 0.2% of total Rat lalmyclneo cells (Table 1) and demonstrate apoptosis in the adherent-cell population.…”

Section: Methodsmentioning

confidence: 91%

Myc-mediated apoptosis is blocked by ectopic expression of Bcl-2.

1993

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The product of the c-myc proto-oncogene is an important positive regulator of cell growth and proliferation. Recently, c-Myc has also been demonstrated to be a potent inducer of apoptosis when expressed in the absence of serum or growth factors. To further examine Myc-induced apoptosis, we coexpressed the proto-oncogene bcl2, which has been shown to block apoptosis in other systems, with c-myc in serum-deprived Rat 1a fibroblasts. Here we report that ectopic expression of bcl2 specifically blocks apoptosis induced by constitutive c-myc expression. Constitutive c-myc expression in serum-deprived Rat 1a cells caused a > 15-fold increase in the number of dead cells, accompanied by DNA fragmentation. However, coexpression of bcl2 with c-myc in these cells led to a 10-fold increase in the number of live cells and a significant decrease in DNA fragmentation. Thus, Bcl-2 effectively inhibits Myc-induced apoptosis in serum-deprived Rat 1a fibroblasts without blocking entry into the cell cycle. These results imply that apoptosis serves as a protective mechanism to prevent tumorigenicity elicited by deregulated Myc expression. This protective mechanism is abrogated, however, by Bcl-2 and therefore may explain the synergism between Myc and Bcl-2 observed in certain tumor cells.

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Section: Methodsmentioning

confidence: 91%

Myc-mediated apoptosis is blocked by ectopic expression of Bcl-2.

1993

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“…These results strongly suggest that a particular binding mechanism is not a requirement for the resolution of the A, region in thymocyte apoptosis. Although intercalating dyes have been utilized most extensively as probes of chromatin structure (7,ll) and have been previously used to distinguish apoptosis (1,6,20,27), the changes in chromatin conformation associated with apoptosis can apparently result in reduced overall fluorescence with nuclear dyes possessing a variety of primary binding mechanisms.…”

Section: Discussionmentioning

confidence: 99%

“…Cell cycle analysis was carried out using an Ortho Diagnostics Cytofluorograph 50-H fluorescence acti- 1. Flow cytometric cell cycle histograms of fixed mouse thymocytes stained with propidium iodide (PI) at 50 pg/ml, acridine orange (AO) at 10 Fgirnl, or 7-aminoactinornycin D (7-AAD) at 5 p.g/ml after no prior treatment (unincubated) or incubation for 8 h with or without 0.1 JLM dexarnethasone (Dex) or following a 5 gray (5 Gy) dose vated cell sorter (FACS)/2150 computer system using a cell cycle analysis doublet discrimination protocol.…”

Section: Flow Cytometrymentioning

confidence: 99%

“…Apoptotic (A,) Region The formation of a distinct, quantifiable cell cycle region below G,/G, i n mouse thymocytes after incubation with glucocorticoids or prior exposure to ionizing radiation has been previously shown to represent cells undergoing apoptosis-associated DNA degradation (1,6,20,27). The region was designated the A, region, and a percentage of events in this region with respect to the entire cell cycle could be easily calculated.…”

Section: Criterion For Evaluating the Presence Of Anmentioning

confidence: 99%

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Comparative evaluation of several DNA binding dyes in the detection of apoptosis‐associated chromatin degradation by flow cytometry

1992

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Mouse thymocytes readily undergo apoptosis-associated DNA degradation upon exposure to glucocorticoids or ionizing radiation. It has been previously shown that flow cytometric cell cycle analysis of propidium iodide-stained apoptotic thymocytes results in the appearance of a distinct cell cycle region (the A, region) below the GdGl region. Cells in this region were shown to be undergoing apoptosis, and determination of apoptosis by flow cytometric analysis was proposed as a superior method for evaluating thymocyte apoptosis. In this study, a variety of DNA binding dyes with diverse primary binding mechanisms were evaluated for their ability to detect glucocorticoid and ionizing radiation-induced apoptosis in mouse thymocytes. Apoptotic thymocytes stained with DNA binding dyes from the phenanthridinium, acridine, actinomycin, chromomycinone, anthracycline, and bisbenzimidazole groups all demonstrated clearly defined A, , regions with percentages comparable to those obtained for propidium iodide. These results indicate that the appearance of the A, region is not dependent on a particular dye binding characteristic and may be the consequence of extensive changes in chromatin structure resulting in a significant degree of dye exclusion.Key terms: Programmed cell death, phenanthridinium, acridine, chromomycinone, anthracycline, bisbenzimidazole Apoptosis, or programmed cell death, is a well-documented phenomenon in many cellular systems (31). Apoptosis is characterized by a number of structural changes in the cell, including the degradation of nuclear chromatin into internucleosomal fragments, presumably by the activation of an endogenous endonuclease (32). Glucocorticoid-(5,32) and ionizing radiation-(28) induced apoptosis in mouse thymocytes has been particularly well characterized in this regard. Internucleosomal DNA fragmentation has formed the basis for the majority of assay systems used to detect apoptosis, including electrophoresis of internucleosoma1 DNA fragments (32) and the separation of small internucleosomal DNA fragments by high-speed centrifugation (33). As previously described, assay systems of this type are by their very nature inadequate since they fail to evaluate apoptosis on a cell by cell basis (27).In a previous paper we described a method by which glucocorticoid-induced apoptosis could be detected in individual, intact mouse thymocytes by reduced fluorescence of the DNA binding dye propidium iodide in the apoptotic subpopulation (27). The apoptotic subpopulation manifested itself as a discrete peak displaying reduced fluorescence with respect to the G$G, cell cycle region. Reduced DNA binding dye fluorescence in apoptotic cells has also been observed using acridine orange (6,12,20), mithramycidethidium bromide (11, and the Hoechst dyes (13,171 in several systems. The apparent ability of DNA binding dyes to distinguish apoptotic cells based on reduced fluorescence by an as yet unclear mechanism has the potential to be an important means of studying programmed cell death,

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“…DNA degradation is a widely used hallmark of apoptosis and, thus, apoptotic cells can be measured by flow cytometry by determining the proportion of cells that contain less than 2C DNA (Afanasyev et al, 1986(Afanasyev et al, , 1993. Figure 1 shows that about 34% of dead non-adherent cells contain less than 2C DNA and are presumed to be apoptotic based on this criterion.…”

Section: Resultsmentioning

confidence: 99%

Prevention of rat neonatal cardiomyocyte apoptosis induced by simulated in vitro ischemia and reperfusion

et al. 1997

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Apoptosis, or programmed cell death, is an active metabolic response to physiological signals or exposure to cytotoxic agents. Recent evidence has shown that the cell death response can be modified by agents presumed to be unrelated to the initial signal, but capable of interfering with the molecular mechanisms of the apoptotic pathway progression. Here we show the results of investigations on the use of a phospholipid-based pharmaceutical preparation for suppression of myocardial damage. First, we show that serum or serum/glucose deprivation, in vitro ischemia with subsequent simulated reperfusion, inhibition of protein synthesis, and treatment with ceramide, staurosporine, adriamycin, cis-platinum and menadione induce apoptotic death in a primary culture of rat neonatal cardiomyocytes. Then we demonstrate that a mixture of specific phospholipids, which has been originally purified from soy flour on the basis of its antiapoptotic activity, prevents cardiomyocyte death induced by serum or serum/glucose deprivation, by ischemia with subsequent simulated reperfusion, and by ceramide, but not by other cytotoxic treatments. This suggests that ceramide, a lipid secondary messenger which triggers apoptosis induced by some cytotoxic agents, may be involved in the process of signaling ischemia/reperfusion induced apoptotic death of cardiomyocytes. These results further demonstrate that an active pharmaceutical preparation for the suppression of cardiomyocyte death can be formulated based upon a novel strategy of apoptosis modification.

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Flow cytometry and biochemical analysis of DNA degradation characteristic of two types of cell death

Cited by 93 publications

References 11 publications

Myc-mediated apoptosis is blocked by ectopic expression of Bcl-2.

Myc-mediated apoptosis is blocked by ectopic expression of Bcl-2.

Comparative evaluation of several DNA binding dyes in the detection of apoptosis‐associated chromatin degradation by flow cytometry

Prevention of rat neonatal cardiomyocyte apoptosis induced by simulated in vitro ischemia and reperfusion

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