Apoptosis of human myeloid leukemia cells induced by an inhibitor of protein phosphatases (okadaic acid) is prevented by Bcl-2 and Bcl-XL

Abstract

2. 13 On the other hand, Haldar et al 14 described that treatment this report, we found that OA induced apoptosis in three Keywords: Bcl-2; Bcl-XL; apoptosis; okadaic acid; myeloid myeloid leukemia cell lines, which was accompanied by down-regulation of Bcl-2, Bcl-X L and Bax. Furthermore, we also demonstrated that overexpression of either Bcl-X L or BclIntroduction 2 blocked OA-induced apoptosis in K562 cells.Phosphorylation and dephosphorylation of cellular proteins are implicated in many biologically important processes such Materials and methods as cell growth and differentiation. Protein phosphorylation has been recently implicated in pathways that lead to apoptosis. Cell cultureApoptosis is induced in U937 myeloid leukemia cells by cellpermeable analogues of ceramide. 1 One of the candidate K562, KU812 and HL-60 myeloid cell lines were maintained ceramide-regulated targets is ceramide-activated protein phosin RPMI 1640 medium (Seromed; Biochrom KG, Berlin, phatase (CAPP), which belongs to the phosphatase class 2A Germany) supplemented with 10% heat-inactivated fetal calf (PP2A) family of serine/threonine protein phosphatases. 2 Furserum (Flow Laboratories, Irvine, UK), non-essential aminothermore, ceramide-induced apoptosis is inhibited by okadaic acids, 2 mM glutamine, 100 U/ml penicillin and 100 g/ml acid (OA), an inhibitor of serine/threonine protein phosphastreptomycine. Viability and total cell counts were determined tases, being more potent on the PP2A type phosphatases. [2][3][4] at various times by trypan blue exclusion and counting of at Okadaic acid also inhibits glucocorticoid-induced apoptosis least 200 cells from each individual culture. Cells (5-9 × 10 5 in murine T cell hybridomas 5 and apoptosis induced in B cell cells/ml) were cultured in the presence of 15 nM okadaic acid lymphoma and T cell leukemia lines by either heat treatment (Boehringer Mannheim, Mannheim, Germany) for various or ionizing radiation. 6 Apoptosis of lymphoid tumor cells is times. In some experiments, cells were treated with 5 nM accompanied by dephosphorylation of a limited number of calyculin A (Boehringer Mannheim). specific proteins and OA is able to prevent apoptosis and protein dephosphorylation in these cells. 6 On the contrary, this inhibitor of protein phosphatases has been described to Apoptotic cell death analysis induce apoptosis in different mammalian cells, 7 mouse fibroblasts, 8 human breast tumor cells, 9 HeLa cells, 10 and K562, Morphological characteristics of apoptosis were examined HL-60 and U937 myeloid leukemic cells. 11,12 Thus OA has under a light microscope following 48 h of treatment with OA. opposite effects on apoptosis depending on the cell system For the DNA fragmentation analysis, cells were treated as studied.described previously. 17 Briefly, cell pellets were resuspended in lysis buffer containing 0.5% SDS and centrifuged. Supernatants were incubated with 0.5 mg/ml proteinase K,

Olanzapine Attenuates the Okadaic Acid-Induced Spatial Memory Impairment and Hippocampal Cell Death in Rats

“…Furthermore, the prevention of Bcl-2 decrease has been associated with the protective effects of olanzapine on methamphetamine-induced neurotoxicity (He et al, 2004). Thus our own previous data, together with the findings by other investigators that OA-induced apoptosis is associated with downregulation of Bcl-2 and can be prevented by upregulation of Bcl-2 (Benito et al, 1997;Nuydens et al, 2000;Cabado et al, 2004), suggest that Bcl-2 plays an important role in the neuroprotective effects of olanzapine on OA-induced neurodegeneration and apoptosis. Olanzapine may also attenuate OA-induced neurotoxicity by upregulating superoxide dismutase (Manna et al, 1998;Li et al, 1999;Matias et al, 1999), and perform protective effects on OA-induced apoptotic cell death by modulating the expression of pro-and antiapoptotic proteins such as Bax and Bcl-X L (Wei et al, 2003b;Cabado et al, 2004).…”

RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines

“…Transfection with 800 nM siRNA duplexes for bcl -2 gene induced a minimal apoptotic response in HL -60, U937, and THP -1 cell lines, which expressed high Bcl -2 levels, but not in the K562 cell line, which is Bcl -2-negative. 31,32 However, a significant induction of programmed cell death (P < .05) was observed after the combined transfection with 400 nM siRNA duplexes for c -raf and 400 nM siRNA duplexes for bcl -2 gene in HL -60, U937, and THP -1 cell lines.…”

“…Indeed, it was shown that the K562 cell line does not express Bcl -2, but instead expresses high levels of the antiapoptotic protein Bcl -xL from the same family of apoptosis inhibitors. 31,32 It is also possible that the Mcl -1 protein -belonging to the same Bcl -2 family of programmed cell death inhibitors and localized predominantly in the endoplasmic reticulum and nuclear envelope -having a cellular pattern of expression that overlaps with, but is not identical to, that of Bcl -2, 38 plays a significant role in the case of K562 line cells. Indeed, Mcl -1 was found to exert a function analogous to Bcl -2, targeting Raf -1 to mitochondria and reducing cell damageinduced release of mitochondrial cytochrome c -the increase in Mcl -1 proteins correlating with a reduced extent of apoptotic cell death induced by etoposide 39 and by fludarabine.…”

Myc Antagonizes Ras-mediated Growth Arrest in Leukemia Cells through the Inhibition of the Ras-ERK-p21Cip1 Pathway

“…As described in other models, this effect is likely due to c-Mycinduced apoptosis, as it was partly relieved by using high serum concentrations (not shown) and by enforced expression of Bcl2 (see below). To allow the study of Myc and Ras functional interaction by clonogenic assays, we first generated a K562/S subline with constitutive expression of Bcl-2 (not shown), as K562 cells do not express this antiapoptotic protein (67). We co-transfected K562/S-Bcl2 cells with Ras-G12V-T35S and c-Myc expression vectors in a ratio of 5:1 (Ras/Myc) and scored colonies after selection with hygromycin B, the resistance gene harbored in the c-Myc vector.…”