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The death receptor CD95 plays a pivotal role in immune surveillance and immune tolerance. Binding of CD95L to CD95 leads to recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates caspase-8 and caspase-10. Efficient formation of the CD95/FADD/caspase complex, known as the death-inducing signaling complex (DISC), culminates in the induction of apoptosis. We show that cells exposed to CD95L undergo a reorganization of the plasma membrane in which the Ca(2+) release-activated Ca(2+) channel Orai1 and the endoplasmic reticulum-resident activator stromal interaction molecule 1 colocalize with CD95 into a micrometer-sized cluster in which the channel elicits a polarized entry of calcium. Orai1 knockdown and expression of a dominant negative construct (Orai1E106A) reveal that on CD95 engagement, the Orai1-driven localized Ca(2+) influx is fundamental to recruiting the Ca(2+)-dependent protein kinase C (PKC) β2 to the DISC. PKCβ2 in turn transiently holds the complex in an inactive status, preventing caspase activation and transmission of the apoptotic signal. This study identifies a biological role of Ca(2+) and the Orai1 channel that drives a transient negative feedback loop, introducing a lag phase in the early steps of the CD95 signal. We suggest that these localized events provide a time of decision to prevent accidental cell death.

Objective: Triptolide has shown antitumor activity in a broad range of solid tumors and on leukemic cells in vitro., Materials and Methods: The THP1 cell line and primary acute myeloid leukemia (AML) cells were cultured with triptolide alone or in association with AraC or idarubicin in increasing concentrations. Apoptosis was measured by flow cytometry using DiOC6(3) for the cell line and fluorescein isothiocyanateAnnexin-V and CD45 labeling for fresh blast cells. Protein expression was measured by Western blot. Cell cycle distribution of apoptotic cells was measured by flow cytometry., Results: A synergistic effect was observed when triptolide was added to idarubicin or to AraC to induce apoptosis of THP-1 leukemic cells. The triptolide/AraC association was also investigated in vitro on primary blast cells from 25 AML patients. This combination induced significantly higher percentages of apoptosis vs treatment with each drug separately (p<0.005). The IkappaB and X-linked inhibitor of apoptosis protein contents, which were altered by triptolide in idarubicin-treated cells, were not modified in AraC-treated cells. The association of AraC with triptolide increased the number of cells blocked in the S phase and most underwent apoptosis., Conclusion: These results suggest that, by modifying the cell cycle kinetics, AraC sensitizes AML cells to apoptosis induced by low concentration triptolide. The in vitro proapoptotic effect of triptolide associated with the antiproliferative activity of AraC warrants further clinical investigation for treatment of AML patients, especially elderly patients for whom low-dose AraC treatment could be improved by the addition of triptolide.

Proteasome inhibitors are a novel class of compounds that might increase sensitivity to chemotherapy for acute myeloid leukemia (AML). We quantified apoptosis in THP-1 cells incubated with idarubicin (IDA) alone or together with a low concentration of MG132 or bortezomib. The combination of both drugs yielded a percentage of apoptotic cells that was significantly higher than the additive effect of both drugs administered separately (p < 0.01). Isobologram analysis showed that both MG132 and bortezomib interacted synergistically with IDA to induce apoptosis of THP1 cells. Western blot analysis of Bax and Bim show an acumulation of these pro-apoptotic proteins in THP1 treated cells. This increase in Bim preceded the induction of apoptosis and participated in idarubicin-induced apoptosis. Proteasome inhibition also potentiated IDA-induced apoptosis in primary blast cells from 22 AML patients while no such effect was found on normal lymphocytes, PHA-stimulated lymphocytes, normal cord blood CD34+ cells or bone marrow normal myeloid cells. These data show that MG132 and bortezomib specifically sensitize leukemic cells to IDA through an increase in BIM and Bax pro-apoptotic Bcl-2 family proteins.

Background: Imatinib mesylate, a BCR-ABL tyrosine kinase inhibitor, induces apoptosis in chronic myeloid leukemia cells. Resistance to imatinib is currently the most important concern of this treatment. One of the main mechanisms of this resistance is overexpression of BCR-ABL., Methods: In the current study, the authors investigated the correlation between BCR-ABL overexpression and apoptosis in BaF/BCR-ABL and LAMA84 cell lines resistant to imatinib suddenly deprived of the inhibitor, and compared with their sensitive counterpart., Results: Removal of imatinib from culture medium led to a decrease in Bcr-Abl protein expression by Day 5, which was sustained for > or = 3 weeks of imatinib deprivation. Apoptosis was observed after 3 days of imatinib deprivation in resistant lines accompanied by caspase activation, loss of membrane asymmetry (annexin V staining), and alteration of mitochondrial potential (dihexyloxacarbocyanine iodide [DiOC6]). Transient activation of the STAT5/Bcl-xL pathway and Akt kinase activity preceded these responses., Conclusions: Thus, imatinib removal led to apoptosis of BCR-ABL-overexpressing leukemic cells, a phenomenon that could be exploited to sensitize imatinib-resistant cells to the cytotoxic effect of other drugs.

It is often postulated that trans-3,4',5-trihydroxystilbene (resveratrol, RES) exhibits cell growth regulatory and chemopreventive activities. However, mechanisms by which this polyphenol inhibits tumor cell growth, and its therapeutic potential are poorly understood. Using various human leukemia cells, we have first defined the anti-tumoral doses of this compound. RES inhibited the proliferation and induced the apoptosis of all tested lymphoid and myeloid leukemia cells with IC(50) = 5-43 microM. Prior to apoptosis, RES-induced caspase activity in a dose-dependent manner and cell cycle arrest in G(2)/M-phase, correlating with a significant accumulation of cyclins A and B. Leukemia cell death with RES required both caspase-dependent and -independent proteases, as it was significantly inhibited by simultaneous addition of Z-VAD-FMK and leupeptin to these cultures. While RES did not affect non-activated normal lymphocytes, this agent decreased the growth and induced the apoptosis of cycling normal human peripheral blood lymphocytes at lower concentrations (IC(50) <8 microM) than those required for most leukemia cells. RES also induced the apoptosis of early normal human CD34(+) cells and decreased the number of colonies generated by these precursor cells in a dose-dependent manner (IC(50) = 60 microM). Together, the data point to the complexity of RES-mediated signaling pathways and revealed the high anti-proliferative and proapoptotic activities of RES in normal cycling hemopoietic cells.

The death receptor CD95 plays a pivotal role in immune surveillance and immune tolerance. Binding of CD95L to CD95 leads to recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates caspase-8 and caspase-10. Efficient formation of the CD95/FADD/caspase complex, known as the death-inducing signaling complex (DISC), culminates in the induction of apoptosis. We show that cells exposed to CD95L undergo a reorganization of the plasma membrane in which the Ca2+ release-activated Ca2+ channel Orai1 and the endoplasmic reticulum-resident activator stromal interaction molecule 1 colocalize with CD95 into a micrometer-sized cluster in which the channel elicits a polarized entry of calcium. Orai1 knockdown and expression of a dominant negative construct (Orai1E106A) reveal that on CD95 engagement, the Orai1-driven localized Ca2+ influx is fundamental to recruiting the Ca2+-dependent protein kinase C (PKC) β2 to the DISC. PKCβ2 in turn transiently holds the complex in an inactive status, preventing caspase activation and transmission of the apoptotic signal. This study identifies a biological role of Ca2+ and the Orai1 channel that drives a transient negative feedback loop, introducing a lag phase in the early steps of the CD95 signal. We suggest that these localized events provide a time of decision to prevent accidental cell death. [ABSTRACT FROM AUTHOR]

Apoptosis was studied in parental and mdr-1 expressing U937, HL60 and K562 myeloid leukemic cell lines using mdr unrelated inducers of apoptosis such as Ara-C, cycloheximide, serum deprivation, ceramide, monensin and UV irradiation. Apoptosis was efficiently induced by all these treatments in U937 and HL60 cells while K562 cells exhibited an apoptosis-resistant phenotype except with UV and monensin. The pattern of apoptosis resistance in mdr-1 expressing U937 (U937-DR) and HL60 (HL60-DR100) was similar to that presented by K562. This apoptosis-resistant phenotype of mdr cells was not overcome by concentrations of verapamil inhibiting the P-gp 170 pump. The acquisition of this phenotype was posterior to the mdr-1 expressing phenotype since a HL60-DR5 variant, selected at the beginning of the induction of resistance, presented a low level of mdr-1 expression without resistance to apoptosis. The variations observed in the Fas (CD95) expression between sensitive and resistant cells were not sufficient to account for apoptosis resistance. However, a high expression in Abl antigen was found in all the apoptosis-resistant cells. RT – PCR and Western blot analysis showed that this increase in Abl antigen content was accompanied by the expression in U937-DR and HL60-DR100 cells of a hybrid bcr/abl mRNA and a 210 kD Bcr/Abl protein which was constitutive in K562. This expression was due to the translocation of abl and the amplification of the bcr-abl translocated gene. These results are in agreement with the role of Bcr/Abl tyrosine protein kinase as an inhibitor of apoptosis independently of the mdr-1 expression. They also suggest that translocation of the abl gene in the bcr region is a highly probable rearrangement in the mdr-1 expressing myeloid cells and that Bcr/Abl tyrosine kinase effect on apoptosis needs the regulation of intracellular pH and is inactive against UV-induced apoptosis. [ABSTRACT FROM AUTHOR]