UNIPROT:P10415 - FACTA Search

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Query: UNIPROT:P10415 (Bcl-2)
33,771 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

All-trans retinoic acid (ATRA) increases the sensitivity of AML blast cells to cytosine arabinoside (Ara-C) or daunorubicin (DNR) when ATRA is given after drug. We have proposed that down-regulation of bcl-2 is part of the mechanism by which ATRA regulates drug sensitivity. To test this hypothesis cDNA encoding bcl-2 was transfected into cells of the continuous lines OCI/AML-2 and OCI/AML-5. Four transfectant lines were isolated; three contained transfected bcl-2 in the sense orientation (AML5-BCL2sa, AML5-BCL2sb and 2-bcl2) and one with anti-sense bcl-2(AML5-bcl2as). The presence of the transfected gene was demonstrated by Northern blot; translation of the sense transfected genes into protein was demonstrated by Western blotting. Lines with sense-oriented transfected bcl-2 were significantly less sensitive to Ara-C or H2O2 than the parental lines; the cells with anti-sense transfected genes were more sensitive than their parent but the difference did not reach statistical significance. The effect of ATRA on bcl-2 expression was compared in sense-transfected cells and their parents; by Northern blotting it was shown that the endogenous but not the transfected genes were down-regulated after ATRA exposure. The capacity of cells with transfected genes to respond to ATRA was tested by obtaining Ara-C survival curves for ATRA-treated cells. Compared to controls not exposed to ATRA, the transfected cells showed little or statistically insignificant changes in Ara-C sensitivity after ATRA treatment. We conclude that data from the transfectants provides evidence that expression of bcl-2 is a determinant of sensitivity to Ara-C and H2O2; and that the effect of ATRA on sensitivity requires the presence of bcl-2 genes in association with regulatory elements.
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PMID:Direct evidence for the participation of bcl-2 in the regulation by retinoic acid of the Ara-C sensitivity of leukemic stem cells. 756 7

We have previously shown that blasts from acute myeloid leukaemia (AML) patients which grow autonomously in culture have high bcl-2 expression which in turn has been linked to a poor clinical response to chemotherapy. The bcl-2 protein promotes cell survival by preventing the onset of apoptosis or programmed cell death following growth-factor deprivation. Bcl-2 has also been shown to be responsible for chemo-resistance in human leukaemic cell lines. Here we have investigated the role of bcl-2 expression in mediating resistance to apoptosis induced by cytosine arabinoside in vitro. The blasts from 17 AML patients exhibiting autonomous growth in a blast cell colony assay and expressing high levels of bcl-2 protein were studied. Incubation of the blasts with antisense oligonucleotides directed against bcl-2 mRNA resulted in a significant decrease in expression of the bcl-2 protein in seven of the 17 samples. In these seven cases the decreased expression of bcl-2 was accompanied by increased apoptosis and the susceptibility of the blasts to apoptosis induced by Ara-C was increased in the presence of bcl-2 antisense. As a high level of bcl-2 defines a group of AML patients who exhibit a poor response to chemotherapy, the demonstration that chemosensitivity of a significant proportion of these patients can be increased by bcl-2 antisense suggests this approach may have clinical potential.
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PMID:Inhibition of bcl-2 with antisense oligonucleotides induces apoptosis and increases the sensitivity of AML blasts to Ara-C. 784 7

Ara-C has been shown to induce apoptosis of human acute myelogenous leukemia HL-60 cells. The DNA repair enzyme poly(ADP-ribose) polymerase (PARP) is known to be degraded during apoptosis. PARP as a substrate is cleaved by the Yama protease, encoded by the CPP32beta/Yama gene. Yama belongs to the interleukin 1beta converting enzyme/ced-3 family of cysteine proteases that are activated as a cascade, producing proteolytic cleavage of specific substrates that results in the morphological and biochemical features of apoptosis. In the present studies, we determined the effect of high intracellular levels of the antiapoptosis Bcl-2 or Bcl-xL protein on Yama protease activation and PARP degradation during Ara-C-induced apoptosis. For this, we utilized HL-60/Bcl-2, HL-60/Bcl-xL, or control HL-60/neo cells, which were created by transfection of the cDNA of the bcl-2, bcl-xL, or the neomycin-resistant genes, respectively. As compared to HL-60/neo, HL-60/Bcl-2 and HL-60/Bcl-xL cells have 5-fold greater expression of Bcl-2 and Bcl-xL, respectively. However, these cell lines have similar levels of p32Yama and PARP. Treatment with 10 or 100 microM Ara-C for 4 h produced DNA fragmentation and morphological features of apoptosis in HL-60/neo cells. This was associated with the cleavage and activation of p32Yama and PARP degradation but not with the induction of Yama mRNA. In contrast, in HL-60/Bcl-2 and HL-60/ Bcl-xL cells, Ara-C-induced p32Yama activation by its cleavage, PARP degradation and apoptosis were significantly inhibited. High Bcl-2 and Bcl-xL levels in these cells also inhibited Yama protease activity, PARP degradation, and apoptosis due to clinically relevant concentrations of etoposide and mitoxantrone. These results suggest that the activation of the Yama protease and PARP degradation are involved in Ara-C-, etoposide-, or mitoxantrone-induced apoptosis. In addition, they suggest that Bcl-2 and Bcl-xL antagonize drug-induced apoptosis by a mechanism that interferes in the activity of a key cysteine protease that is involved in the execution of apoptosis.
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PMID:Overexpression of Bcl-2 or Bcl-xL inhibits Ara-C-induced CPP32/Yama protease activity and apoptosis of human acute myelogenous leukemia HL-60 cells. 884 Sep 93

We examined the effects of high intracellular levels of Bcl-2 on the metabolism and DNA incorporation of high-dose Ara-C (HIDAC) as well as on Ara-C-induced DNA strand breaks and apoptosis of human AML HL-60 cells. HL-60/Bcl-2 and HL-60/neo cells were created by retrovirally transfecting the human AML HL-60 cells with the pZip-bcl-2 and pZip-neo plasmids, respectively. As compared to HL-60/neo, HL-60/Bcl-2 cells contained significantly higher (approximately 10-fold) p26Bcl-2, but equivalent levels of Bax and undetectable levels of Bcl-xL. HIDAC (10 or 100 microM for 4 h) produced the kilobase size and internucleosomal DNA fragmentation associated with apoptosis in HL-60/neo but not in HL-60/Bcl-2 cells. Significantly greater loss of survival (by MTT assay) and flowcytometric and morphologically recognizable apoptosis were observed in HL-60/neo cells. HIDAC did not affect Bcl-2 levels in either cell type. The intracellular accumulation of Ara-CTP relative to dCTP, Ara-C DNA incorporation and Ara-C-induced early DNA damage in the form of strand breaks (detected by alkaline elution assay) were not significantly different between HL-60/Bcl-2 and HL-60/neo cells. In addition, HIDAC treatment caused similar DNA synthesis inhibition in the two cell types. These results indicate that high intracellular levels of Bcl-2 operate distally to inhibit the final apototic cell death pathway by preventing the conversion of HIDAC-induced early DNA damage into lethal DNA fragmentation associated with apoptosis.
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PMID:Intracellular metabolism of Ara-C and resulting DNA fragmentation and apoptosis of human AML HL-60 cells possessing disparate levels of Bcl-2 protein. 889 76

The response to therapy of leukemic cells is largely determined by their capacity of proliferation and apoptosis in presence of the administered drugs. We describe here the main markers used in flow cytometry (FCM) and involved in the assessment of cell cycle parameters: single labeling by Propidium Iodide (PI) and double labeling anti-Bromodeoxyuridine (BrdUrd)/PI which, both in vitro and in vivo, gives cell percentages in the different cell cycle phases. The markers of cell cycle progression can be divided into proliferation markers such as PCNA (proliferating cell nuclear antigen) or Ki-67 and cell cycle progression markers. The latter, which are the core of the cell cycle machinery, are molecules recently characterized (Cyclins, CDKs (cell dependent kinases), CDIs (cyclin-dependent kinase inhibitors)) and their cell expression can be analyzed using FCM. FCM is also one of the best means to detect and quantitate apoptotic cells. Several techniques are described: Nuclear labeling using Hoechst 33342: mitochondrial labeling using DiOC6(3): detection of DNA fragmentation using 1) labeling of fixed and permeabilized cells with a DNA marker or 2) labeling of the free 3' DNA ends using incorporation of labeled deoxynucleotides; detection in apoptotic cells (Bcl-2, Fas, phospholipids...). At last, we analyzed flow cytometry methods to study the cell resistance to Ara-C and anthracyclins. In combination with cell kinetic studies and detection of apoptotic cells, they should increase the efficiency of the acute leukemia treatment.
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PMID:Flow cytometry study of cell cycle, apoptosis and drug resistance in acute leukemia. 903 Sep 62

High-dose Ara-C (HIDAC) induces the cleavage and activity of caspase-3 (CPP32beta/Yama/apopain), resulting in the morphological and biochemical features of apoptosis. High levels of the antiapoptotic Bcl-x(L) or Bcl-2, relative to the proapoptotic Bax, have been shown to inhibit HIDAC-induced cleavage and activity of caspase-3 and apoptosis of the human acute myeloid leukemia HL-60 cells. In a previous report, we demonstrated this inhibition, using the control HL-60 (HL-60/neo) cells and their counterparts, HL-60/Bcl-x(L), which have enforced overexpression of Bcl-x(L) and a significantly lower ratio of free to bound Bax. Results of the present studies demonstrate that, in the initiation phase of apoptosis of HL-60/neo cells due to HIDAC (10 or 100 microM for 4 h), cytochrome c is released from the mitochondria to the cytosol, followed by the loss of mitochondrial membrane potential (deltapsi m) and an increase in the reactive oxygen species; these events precede and trigger the cleavage and activity of caspase-3. These HIDAC-induced early mitochondrial and cytosolic perturbations, which represent the initiation phase of HIDAC-induced apoptosis, were inhibited in HL-60/Bcl-x(L) cells. HIDAC treatment for 4 h also modestly increased the intracellular levels of free Bax relative to Bax bound to Bcl-2 and Bcl-x(L) in HL-60/neo but not in HL-60/Bcl-x(L) cells. In HL-60/neo cells, HIDAC-induced progressive accumulation of cytochrome c in the cytosol, the decrease in deltapsi m, and the increase in reactive oxygen species were not inhibited by coculture with the tetrapeptide inhibitors of caspases that have been previously shown to inhibit Ara-C-induced cleavage and activity of caspase-3 and apoptosis. These findings indicate that Bcl-x(L) inhibits HIDAC-induced preapoptotic mitochondrial perturbations, which prevent the accumulation of cytochrome c in the cytosol, thereby preserving caspase-3 in the inactive zymogen state and checking the molecular cascade of apoptosis.
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PMID:Overexpression of Bcl-X(L) inhibits Ara-C-induced mitochondrial loss of cytochrome c and other perturbations that activate the molecular cascade of apoptosis. 924 35

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that induces apoptosis in various cell systems by binding to the TNF receptor (TNFR). To study TNF-alpha-induced apoptosis, we isolated and characterized a novel TNF-alpha-resistant variant, U937/TNF clone UA, from human monocytic leukemia U937 cells. The UA cells resist apoptosis induced by TNF-alpha and anti-Fas antibody but not by anticancer drugs, such as VP-16 and Ara-C. Somatic cell hybridization between U937 and UA showed that apoptosis resistance to TNF-alpha in UA was genetically recessive. The hybridization analysis also showed that UA and another recessive mutant clone, UC, belong to different complementation groups in TNF-alpha-induced apoptosis signaling. In UA cells, TNF-alpha-induced disruption of mitochondrial membrane potential and CPP32 activation were abrogated. Expression of TNFR, Fas, and Bcl-2 family proteins was not changed in UA cells. These results suggest that the apoptosis resistant UA cells could have a functional defect in apoptosis signaling from the TNFR to mitochondria and interleukin-1beta converting enzyme (ICE) family protease activation. UA cells could be used to study signaling linkage between cell death-inducing receptor and mitochondria.
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PMID:Genetically recessive mutant of human monocytic leukemia U937 resistant to tumor necrosis factor-alpha-induced apoptosis. 942 4

Bcr-Abl expression in leukemic cells is known to exert a potent effect against apoptosis due to antileukemic drugs, but its mechanism has not been elucidated. Recent reports have indicated that a variety of apoptotic stimuli cause the preapoptotic mitochondrial release of cytochrome c (cyt c) into cytosol, which mediates the cleavage and activity of caspase-3 involved in the execution of apoptosis. Whether Bcr-Abl exerts its antiapoptotic effect upstream to the cleavage and activation of caspase-3 or acts downstream by blocking the ensuing degradation of substrates resulting in apoptosis, has been the focus of the present studies. In these, we used (1) the human acute myelogenous leukemia (AML) HL-60 cells that are stably transfected with the bcr-abl gene (HL-60/Bcr-Abl) and express p185 Bcr-Abl; and (2) the chronic myelogenous leukemia (CML)-blast crisis K562 cells, which have endogenous expression of p210 Bcr-Abl. Exposure of the control AML HL-60 cells to high-dose Ara-C (HIDAC), etoposide, or sphingoid bases (including C2 ceramide, sphingosine, or sphinganine) caused the accumulation of cyt c in the cytosol, loss of mitochondrial membrane potential (MMP), and increase in the reactive oxygen species (ROS). These preapoptotic events were associated with the cleavage and activity of caspase-3, resulting in the degradation of poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) and DNA fragmentation factor (DFF), internucleosomal DNA fragmentation, and morphologic features of apoptosis. In contrast, in HL-60/Bcr-Abl and K562 cells, these apoptotic stimuli failed to cause the cytosolic accumulation of cyt c and other associated mitochondrial perturbations, as well as the failure to induce the activation of caspase-3 and apoptosis. While the control HL-60 cells showed high levels of Bcl-2 and barely detectable Bcl-xL, HL-60/Bcr-Abl cells expressed high levels of Bcl-xL and undetectable levels of Bcl-2, a pattern of expression similar to the one in K562 cells. Bax and caspase-3 expressions were not significantly different between HL-60/Bcr-Abl or K562 versus HL-60 cells. These findings indicate that Bcr-Abl expression blocks apoptosis due to diverse apoptotic stimuli upstream by preventing the cytosolic accumulation of cyt c and other preapoptotic mitochondrial perturbations, thereby inhibiting the activation of caspase-3 and execution of apoptosis.
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PMID:Bcr-Abl exerts its antiapoptotic effect against diverse apoptotic stimuli through blockage of mitochondrial release of cytochrome C and activation of caspase-3. 947 36

The myeloid leukemic cell line HL-60 was studied by using DNA gel electrophoresis, flow cytomery, McAb C-myc, McAb Bcl-2 and CFU-L. From zero to 36 h, the apoptosis rates of 8 different phases and other indexes were observed. The results showed that with the prolonged time of drug incubation, apoptosis of HL-60 cells increased progressively. This effect can be enhanced obviously by rh-IL-3 and rh-GM-CSF. At the same time, the killed rate of leukemic cells by Ara-C induction was increased. C-myc expression was decreased and Bcl-2 expression did not display apparent change. Interestingly, the normal hemopoietic cells were not affected by these two kinds of cytokine. The theoretical basis was provided for concurrent use of rh-IL-3, rh-GM-CSF and cytotoxic drugs whose purpose is to elevate remission rate during the phase of induced remission of leukemia.
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PMID:Effect of concurrent use of rh-IL-3 and rh-GM-CSF on apoptosis of HL-60 cells induced by Ara-C. 963 77

The expression of Bcl-2 family members was examined in normal and leukemic hematopoietic cells. Immature hematopoietic progenitor cells (CD34+/33-/13-) did not express Bcl-2 but Bcl-XL, the majority of CD34 cells expressed Bcl-2, Bcl-XL and BAD, and normal promyelocytes (CD34-/33+) lacked expression of both Bcl-2 and Bcl-XL, while leukemic CD34+progenitors and promyelocytes expressed these anti-apoptotic proteins. In AML, Bcl-2 expression was higher on CD34+ than on all AML cells, however, expression of Bcl-2 or Bcl-XL did not predict achievement of complete remission. Surprisingly, low Bcl-2 content was associated with poor survival in a group of patients with poor prognosis cytogenetics. The anti-apoptotic BAD protein was found to be expressed in AML, but was phosphorylated in 41/42 samples. Phosphorylation was found at both sites, Ser 112 and Ser 136. During induction chemotherapy, Bcl-2 levels of CD34 cells increased significantly. In the context of evidence for small numbers of leukemic CD34+ cells expressing very high levels of Bcl-2 prior to therapy, this finding is interpreted as a survival advantage of Bcl-2 overexpressing progenitors and rapid elimination of cells with low Bcl-2. Bcl-2 and Bcl-XL were both expressed in minimal residual disease cells. Downregulation of Bcl-2 mRNA and protein was observed by ATRA and the combination of Ara-C, followed by ATRA, resulted in markedly increased cytotoxicity in HL-60 cells, as compared to Ara-C alone or ATRA followed by Ara-C. Implications of these findings for the development of new therapeutic strategies for AML are discussed.
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PMID:Expression of Bcl-2-related genes in normal and AML progenitors: changes induced by chemotherapy and retinoic acid. 1055 66

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