l-663536 and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

The block of hematopoietic differentiation program in acute myeloid leukemia cells can be overcome by differentiating agent like retinoic acid, but it has several side effects. A study of other differentiation signaling pathways is therefore useful to predict potential targets of anti-leukemic therapy. We demonstrated previously that the co-treatment of HL-60 cells with Tumor necrosis factor-alpha (TNF-alpha) (1 ng/mL) and inhibitor of 5-lipoxygenase MK886 (5 microm) potentiated both monocytic differentiation and apoptosis. In this study, we detected enhanced activation of three main types of mitogen-activated protein kinases (MAPKs) (p38, c-Jun amino-terminal kinase [JNK], extracellular signal-regulated kinase [ERK]), so we assessed their role in differentiation using appropriate pharmacologic inhibitors. The inhibition of pro-apoptotic MAPKs (p38 and JNK) suppressed the effect of MK886 + TNF-alpha co-treatment. On the other hand, down-regulation of pro-survival ERK pathway led to increased differentiation. Those effects were accompanied by increased activation of caspases in cells treated by MK886 + TNF-alpha. Pan-caspase inhibitor ZVAD-fmk significantly decreased both number of apoptotic and differentiated cells. The same effect was observed after inhibition of caspase 9, but not caspase 3 and 8. To conclude, we evidenced that the activation of apoptotic processes and pathways supporting apoptosis (p38 and JNK MAPKs) is required for the monocytic differentiation of HL-60 cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cell Differentiation; Cysteine Proteinase Inhibitors; Enzyme Activation; HL-60 Cells; Humans; Indoles; Leukemia, Promyelocytic, Acute; Lipoxygenase Inhibitors; MAP Kinase Signaling System; Monocytes; NF-kappa B; Tumor Necrosis Factor-alpha

Inhibitors of 5-lipoxygenase-activating protein (FLAP) have been found to induce apoptosis. The current study examined the expression of FLAP and bcl family proteins and the induction of apoptosis in interleukin-3-dependent control and bcl-xL-overexpressing FL5.12 cell lines after treatment with MK886, a specific FLAP inhibitor. FL5.12 cells contained a substantial amount of FLAP protein and mRNA but surprisingly had no measurable 5-lipoxygenase protein or 5-, 12-, or 15-lipoxygenase activity. The basal level of FLAP protein in cells overexpressing bcl-xL was 70% less than in controls. FLAP disappeared 4 h after withdrawal of interleukin-3 in bcl-xL cells but not in control cells, which underwent apoptosis. A dose- and time-response study revealed that 5 nmol of MK886/10(6) cells was sufficient to induce apoptosis both in control and bcl-xL cells, respectively, but to different degrees. bcl-xL and bcl-2 proteins, but not bax or FLAP, were decreased by 4 h after 5 nmol of MK886/10(6) cells in both cell lines, although the higher levels of bcl-xL in overexpressors took longer to disappear. This early loss of bcl-xL and bcl-2 was not attributable to generalized proteolysis, as shown by Coomassie Blue staining and by the maintenance of bax. Caspase-3 was activated 2 h after MK886 treatment in control cells but not in bcl-xL cells. Inhibition of caspase-3 decreased MK886-induced apoptosis by 50% in control cells. Inhibition of this caspase after MK886 treatment was unable to prevent the loss of bcl-xL in control cells but did provide partial protection for the loss of the transfected form, but not the endogenous form, in overexpressing cells. These data indicate that MK886 induces extensive apoptosis that is partially caspase-3 dependent and may be related to a rapid loss of bcl-xL. Although caspase-3 inhibitors had no effect on the loss of bcl-xL, other caspases or protease systems may still be involved. The absence of 5-lipoxygenase in cells containing FLAP, the lower level of FLAP in bcl-xL cells, the apoptosis-inducing activity of MK886, and the rapid loss of bcl-xL and bcl-2 proteins after treatment with MK886 strongly indicate that FLAP has activities unrelated to lipoxygenase and suggest a possible functional or regulatory link between these proteins, which share similar subcellular localizations.

Topics: 5-Lipoxygenase-Activating Proteins; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Arachidonate 5-Lipoxygenase; B-Lymphocytes; bcl-X Protein; Carrier Proteins; Caspase 3; Caspase Inhibitors; Caspases; Enzyme Activation; Hematopoietic Stem Cells; Indoles; Interleukin-3; Lipoxygenase Inhibitors; Masoprocol; Membrane Proteins; Mice; Proto-Oncogene Proteins c-bcl-2; Signal Transduction