Constitutive cytoplasmic localization of p21Waf1/Cip1 affects the apoptotic process in monocytic leukaemia

In the present study, we analysed the expression and localization of p21Waf1/Cip1 in normal and malignant haematopoietic cells. We demonstrate that in normal monocytic cells, protein kinase C (PKC)-induced p21 gene activation, which is nuclear factor-κB (NF-κB) independent, results in predominantly cytoplasmic localized p21 protein. In acute monocytic leukaemia (M4, M5), monocytic blasts (N=12) show constitutive cytoplasmic p21 expression in 75% of the cases, while in myeloid leukaemic blasts (N=10), low nuclear and cytoplasmic localization of p21 could be detected, which is also PKC dependent. Constitutive p21 expression in monocytic leukaemia might have important antiapoptotic functions. This is supported by the finding that in U937 cells overexpressing p21, VP16-induced apoptosis is significantly reduced (20.0±0.9 vs 55.8±3.8%, P<0.01, N=5), reflected by a reduced phosphorylation of p38 and JNK. Similarly, AML blasts with high cytoplasmic p21 were less sensitive to VP16-induced apoptosis as compared to AML cases with low or undetectable p21 expression (42.25 vs 12.3%, P<0.01). Moreover, complex formation between p21 and ASK1 could be demonstrated in AML cells, by means of coimmunoprecipitation. In summary, these results indicate that p21 has an antiapoptotic role in monocytic leukaemia, and that p21 expression is regulated in a PKC-dependent and NF-κB independent manner.

Inhibition of Caspase 3 Abrogates Lipopolysaccharide-Induced Nitric Oxide Production by Preventing Activation of NF-κB and c-Jun NH(2)-Terminal Kinase/Stress-Activated Protein Kinase in RAW 264.7 Murine Macrophage Cells

The effect of caspase inhibitors on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 267.4 murine macrophage cells was investigated. Pretreatment of RAW cells with a broad caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), resulted in a striking reduction in LPS-induced NO production. Z-VAD-FMK inhibited LPS-induced NF-κB activation. Furthermore, it blocked phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) but not that of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases. Similarly, a caspase 3-specific inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone, inhibited NO production, NF-κB activation, and JNK/SAPK phosphorylation in LPS-stimulated RAW cells. The attenuated NO production was due to inhibition of the expression of an inducible-type NO synthase (iNOS). The overexpression of the dominant negative mutant of JNK/SAPK and the addition of a JNK/SAPK inhibitor blocked iNOS expression but did not block LPS-induced caspase 3 activation. It was therefore suggested that the inhibition of caspase 3 might abrogate LPS-induced NO production by preventing the activation of NF-κB and JNK/SAPK. The caspase family, especially caspase 3, is likely to play an important role in the signal transduction for iNOS-mediated NO production in LPS-stimulated mouse macrophages

Effects of overexpression of the SH2-containing inositol phosphatase SHIP on proliferation and apoptosis of erythroid AS-E2 cells

Previous studies have demonstrated that SH2-containing inositol phosphatase (SHIP) is involved in the control of B cell, myeloid cell and macrophage activation and proliferation. The goal of the present study was to examine the role of SHIP during proliferation and apoptosis in cells of the erythroid lineage. Wild-type and catalytically inactive SHIP proteins were overexpressed in the erythropoietin (EPO)-dependent cell line AS-E2. Stable overexpression of catalytically inactive SHIP decreased proliferation and resulted in prolonged activation of the extracellular signal-regulated protein kinases ERK1/2 and protein kinase B (PKB), while wild-type SHIP did not affect EPO-mediated proliferation or phosphorylation of ERK and PKB. When AS-E2 cells were EPO deprived a significant increase in apoptosis was observed in clones overexpressing wild type. Mutational analysis showed that this increase in apoptosis was independent of the enzymatic activity of SHIP. The enhanced apoptosis due to overexpression of SHIP was associated with an increase in caspase-3 and -9 activity, without a distinct effect on caspase-8 activity or mitochondrial depolarization. Moreover, in cells overexpressing SHIP apoptosis could be reduced by a caspase-3 inhibitor. These data demonstrate that in the erythroid cell line AS-E2 overexpression of catalytically inactive SHIP reduced proliferation, while overexpression of wild-type SHIP had no effect. Furthermore, overexpression of SHIP enhanced apoptosis during growth factor deprivation by inducing specific caspase cascades, which are regulated independently of the 5-phosphatase activity of SHIP