Use of flow cytometry techniques in studying mechanisms of apoptosis in leukemic cells

Gorman, Adrienne M.; Samali, Afshin; McGowan, Adrian J.; Cotter, Thomas G.

doi:10.1002/(sici)1097-0320(19971001)29:2<97::aid-cyto1>3.0.co;2-j

Cited by 45 publications

(26 citation statements)

References 38 publications

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“…After 4-MU treatment, the cells showed a clear G 1 arrest (84.1% in G 1 , 10.8% in S, and 5.1% in G 2 ). Interestingly, in addition to the G 1 peak, another sharp peak appeared after 4-MU treatment, which is similar to the extra peak that has been associated with apoptosis in other cell types (25). Furthermore, in AoSMCs treated with 4-MU ϩ HMW-HA, the extra peak disappeared, even though these cells continued to be blocked in G 1 (84.2% in G 1 , 9.0% in S, and 6.9% in G 2 ).…”

Section: Resultssupporting

confidence: 61%

Glycosaminoglycans and Glucose Prevent Apoptosis in 4-Methylumbelliferone-treated Human Aortic Smooth Muscle Cells

Vigetti

Rizzi

Moretto

et al. 2011

Journal of Biological Chemistry

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Smooth muscle cells (SMCs) have a pivotal role in cardiovascular diseases and are responsible for hyaluronan (HA) deposition in thickening vessel walls. HA regulates SMC proliferation, migration, and inflammation, which accelerates neointima formation. We used the HA synthesis inhibitor 4-methylumbelliferone (4-MU) to reduce HA production in human aortic SMCs and found a significant increase of apoptotic cells. Interestingly, the exogenous addition of HA together with 4-MU reduced apoptosis. A similar anti-apoptotic effect was observed also by adding other glycosaminoglycans and glucose to 4-MU-treated cells. Furthermore, the anti-apoptotic effect of HA was mediated by Toll-like receptor 4, CD44, and PI3K but not by ERK1/2. Hyaluronan (HA)3 is one of the most abundant glycosaminoglycans (GAGs) in extracellular matrices (ECMs) and is composed of linear, unsulfated repetitions of D-glucuronic acid and N-acetylglucosamine. In mammals, two specific HA synthases (HAS1 and -2) produce high molecular weight HA (HMW-HA), in the range of millions of Da, whereas the other isoenzyme (HAS3) synthesizes HA of lower molecular mass, in the range of several thousands of Da (1). The size of HA depends also on specific degrading enzymes (i.e. hyaluronidases) that can produce bioactive HA oligosaccharides. Therefore, in vivo, HA chains can greatly vary in lengths and can differently regulate cell behavior through interactions with several receptors, including CD44, RHAMM (receptor for HA-mediated motility), lymphatic vessel endothelial receptor 1 (Lyve-1), HA receptor for endocytosis (HARE), and Toll-like Receptor 4 (TLR4) (2).In cardiovascular pathologies, HA accumulates during neointima formation and alters smooth muscle cell (SMC) behavior (3). In some pathological conditions, contractile SMCs dedifferentiate to form a synthetic phenotype characterized by a high production of ECM components, including HA and versican, by synthesis of ECM-modifying metalloproteinases (4) and by increased rates of proliferation and migration. Therefore, SMCs acquire the capability to invade the vascular tunica intima, thereby contributing to vessel wall thickening. HMW-HA is involved in the modulation of SMC migration and proliferation through interaction with CD44 (5-7), which can mediate a signaling cascade inside the cell that activates different pathways, including PI3K, AKT, and ERK1/2 (8).We demonstrated previously that human aortic SMC (AoSMC) migration is strictly dependent on HA-CD44 signaling and recently reported that the HA synthesis inhibitor 4-methylumbelliferone (4-MU) reduced proatherosclerotic properties of AoSMCs by decreasing cell migration and proliferation and by inhibiting monocyte binding to the HA-rich ECM that contributes to inflammation (9, 10). Moreover, we found that the simultaneous addition of HMW-HA to 4-MUtreated AoSMCs restored cell proliferation to the levels of controls. Therefore, the aim of this study was to investigate at the molecular level the effects of HMW-HA after 4-MU treatment of AoSMCs and the pathway...

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Section: Resultssupporting

confidence: 61%

Glycosaminoglycans and Glucose Prevent Apoptosis in 4-Methylumbelliferone-treated Human Aortic Smooth Muscle Cells

Vigetti

Rizzi

Moretto

et al. 2011

Journal of Biological Chemistry

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“…Furthermore, as we needed a method to distinguish between early and late PCD, we developed a modified ISEL technique for FCM (10,11). ISEL is a variant of the TUNEL technique, but TUNEL is used much more frequently in research (16,17). DNA-Polymerase-I used in ISEL was tested and it was able to detect DNA fragments generated by endonuclease activity in Jurkat and human BM CD34ϩ cells during PCD (18 -21).…”

Section: Discussionmentioning

confidence: 99%

The dynamic process of apoptosis analyzed by flow cytometry using Annexin‐V/propidium iodide and a modified in situ end labeling technique

Span¹,

Pennings

Vierwinden

et al. 2001

Cytometry

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BackgroundTo study the apoptotic process in time, we used the following flow cytometric (FCM) techniques: phosphatidylserine (PS) translocation by Annexin‐V (AnV), DNA fragmentation by in situ end labeling (ISEL), and propidium iodide (PI) staining. Because PS translocation is assumed to be an early feature of programmed cell death (PCD), we questioned if AnV positivity implies inevitable cell death.MethodsApoptosis was induced in Jurkat cells by γ‐irradiation, incubation with camptothecin (CPT), or cytosine β‐D‐arabinofuranoside (Ara‐C). At different time intervals, PCD was quantified by AnV/PI and ISEL. To analyze the influence of cell handling procedures on PCD, we applied these three FCM techniques on CD34+ bone marrow (BM) stem cells after selection and after a freeze‐thaw procedure. Various AnV/PI− CD34+ fractions were cultured in a single‐cell single‐well (SCSW) assay.ResultsJurkat cells under three different detrimental conditions showed essentially the same pattern of apoptosis in time. Initially developed AnV+/PI− cells subsequently (within 1 h) showed ISEL positivity, after which they turned into AnV+/PI++ cells with even higher levels of ISEL positivity (80–90%). Eventually, they lost some of their PI and ISEL positivity and formed the AnV+/PI+ fraction. Cell handling of CD34+ cells caused high and variable AnV+/PI− fractions (overall range 23–62%). Within total AnV+ and AnV+/PI− populations, only a minority of CD34+ cells showed ISEL positivity (range 4–8% and 0.8–6%, respectively). Different fractions of AnV+/PI− CD34+ cells did have clonogenic capacity.ConclusionsPCD of cell suspensions in vitro can be followed accurately in time by these three FCM techniques. PS translocation is followed rapidly (within 1 h) by oligo‐nucleosomal DNA fragmentation, after which cell (and nuclear) membrane leakage occurs. Detection of PS asymmetry by AnV‐fluorescein isothiocyanate (FITC) is not always associated with (inevitable) apoptosis, as can be concluded from the proliferative capacity of AnV+ /PI− CD34+ cells in the SCSW assay. Cytometry 47:24–31, 2002. © 2001 Wiley‐Liss, Inc.

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“…Mitochondrial membrane depolarization, peroxide and superoxide levels were measured as previously described (Gorman et al, 1997a;Costa-Pereira and Cotter, 1999). Briefly, cells (2.5 ϫ 10 5 ) were harvested and incubated with 5 µg ml Ϫ1 JC-1 (∆ψm measurement), for 15 min; or with 5 µM DCFH-DA (peroxide level) for 1 h at 37ЊC; or with 5 µM DHE (superoxide anion) for 30 min at 37ЊC.…”

Section: Detection Of Intracellular Roi and Measurement Of Mitochondrmentioning

confidence: 99%

Camptothecin sensitizes androgen-independent prostate cancer cells to anti-Fas-induced apoptosis

Costa-Pereira

Cotter

1999

Br J Cancer

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Despite expressing both Fas and Fas ligand, DU145 and LNCaP prostate cancer cells were resistant to anti-Fas-induced cell death. Resistance to Fas-mediated cytotoxicity could be overcome in DU145, but not in LNCaP, cells by pretreating cells with sublethal doses of cytotoxic drugs, such as camptothecin. Activated caspases were shown to be required for this cytotoxicity. Indeed, poly(ADP-Ribose) polymerase was shown to be proteolytically cleaved in cells treated with camptothecin plus anti-Fas, but not in cells treated with anti-Fas only. Moreover, pretreatment of cells with ZVAD completely blocked camptothecin-mediated Fas-induced apoptosis. Sensitization of cells to Fas-induced cell death did not involve up-regulation of Fas or FasL, and it was independent of alterations in the cell cycle. Reactive oxygen intermediates (ROI) have been shown to be important mediators of drug-induced apoptosis. Here, we demonstrate that treatment of DU145 cells with camptothecin, anti-Fas, or both, did not alter the intracellular levels of peroxide or superoxide anion. © 1999 Cancer Research Campaign

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Use of flow cytometry techniques in studying mechanisms of apoptosis in leukemic cells

Cited by 45 publications

References 38 publications

Glycosaminoglycans and Glucose Prevent Apoptosis in 4-Methylumbelliferone-treated Human Aortic Smooth Muscle Cells

Glycosaminoglycans and Glucose Prevent Apoptosis in 4-Methylumbelliferone-treated Human Aortic Smooth Muscle Cells

The dynamic process of apoptosis analyzed by flow cytometry using Annexin‐V/propidium iodide and a modified in situ end labeling technique

Camptothecin sensitizes androgen-independent prostate cancer cells to anti-Fas-induced apoptosis

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