Montserrat Barragán

Aspirin induces apoptosis through mitochondrial cytochrome c release

Aspirin and other non‐steroidal anti‐inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin‐induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT‐4, Raji and HL‐60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin‐induced cytochrome c release was not affected by the caspase inhibitor Z‐VAD·fmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin‐induced apoptosis involves caspase activation through cytochrome c release.

Regulation of the proapoptotic BH3-only protein BIM by glucocorticoids, survival signals and proteasome in chronic lymphocytic leukemia cells.

Glucocorticoids induce apoptosis in chronic lymphocytic leukemia (CLL) cells through a caspase-dependent mechanism. However, their mechanism of action remains unknown. We have studied the regulation of the proapoptotic BH3-only Bcl-2 interacting mediator of cell death (BIM) in CLL cells. We demonstrate that glucocorticoids upregulate BIM at protein and mRNA levels. We have investigated the ability of different survival signals, such as 12-O-tetradecanoylphorbol 13-acetate (TPA), stromal cell-derived factor-1α (SDF-1α), interleukin 4 (IL-4) and B-cell receptor (BCR) activation, to influence the levels of BIM and its induction by glucocorticoids. TPA downregulates BIMEL by extracellular signal-regulated kinase (ERK)-mediated BIM phosphorylation and further proteasome-mediated degradation. However, SDF-1α and BCR activation induce transient BIM phosphorylation, without protein degradation. Proteasome inhibitors do not modify the levels of BIM with respect to untreated cells. However, they induce apoptosis and inhibit TPA-induced BIMEL degradation, leading to its accumulation. In conclusion, the results implicate BIM in glucocorticoid-induced apoptosis in CLL cells. BIMEL phosphorylation through the ERK pathway targets the protein for proteasomal degradation.

Regulation of Akt/PKB by phosphatidylinositol 3-kinase-dependent and -independent pathways in B-cell chronic lymphocytic leukemia cells: role of protein kinase Cβ

Apoptosis of B cell chronic lymphocytic leukemia (B‐CLL) cells is regulated by the PI‐3K‐Akt pathway. In the present work, we have analyzed the mechanisms of Akt phosphorylation in B‐CLL cells. Freshly isolated cells present basal Akt phosphorylation, which is PI‐3K‐dependent, as incubation with the PI‐3K inhibitor LY294002 decreased Ser‐473 and Thr‐308 phosphorylation in most samples analyzed (seven out of 10). In three out of 10 cases, inhibition of protein kinase C (PKC) inhibited basal Akt phosphorylation. Stromal cell‐derived factor‐1α, IL‐4, and B cell receptor activation induced PI‐3K‐dependent Akt phosphorylation. PMA induced the phosphorylation of Akt at Ser‐473 and Thr‐308 and the phosphorylation of Akt substrates, independently of PI‐3K in B‐CLL cells. In contrast, PKC‐mediated phosphorylation of Akt was PI‐3K‐dependent in normal B cells. Finally, a specific inhibitor of PKCβ blocked the phosphorylation and activation of Akt by PMA in B‐CLL cells. Taken together, these results suggest a model in which Akt could be activated by two different pathways (PI‐3K and PKCβ) in B‐CLL cells.

Protein Kinases in the Regulation of Apoptosis in B-cell Chronic Lymphocytic Leukemia

The involvement of several protein kinase pathways in the regulation of apoptosis and cell survival has been analyzed in a wide range of models. This article reviews current understanding of the protein kinases involved in the control of apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells. Protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) and nuclear factor-kappa B (NF-κB) play important roles in the survival of these leukemic cells. These survival pathways affect proteins involved in the control of apoptosis by altering their expression or function. The elucidation of the signal transduction network involved in the survival of B-CLL cells could provide novel pharmacological targets for the therapy of B-CLL.

Aspirin induces apoptosis in human leukemia cells independently of NF-κB and MAPKs through alteration of the Mcl-1/Noxa balance

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in most cell types. In this study we examined the mechanism of aspirin-induced apoptosis in human leukemia cells. We analyzed the role of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) pathways. Furthermore, we studied the changes induced by aspirin in some genes involved in the control of apoptosis at mRNA level, by performing reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA), and at protein level by Western blot. Our results show that aspirin induced apoptosis in leukemia Jurkat T cells independently of NF-κB. Although aspirin induced p38 MAPK and c-Jun N-terminal kinase activation, selective inhibitors of these kinases did not inhibit aspirin-induced apoptosis. We studied the regulation of Bcl-2 family members in aspirin-induced apoptosis. Aspirin increased the mRNA levels of some pro-apoptotic members, such as BIM, NOXA, BMF or PUMA, but their protein levels did not change. In contrast, aspirin decreased the protein levels of Mcl-1. Interestingly, in the presence of aspirin the protein levels of Noxa remained high. This alteration of the Mcl-1/Noxa balance was also found in other leukemia cell lines and primary chronic lymphocytic leukemia cells (CLL). Furthermore, in CLL cells aspirin induced an increase in the protein levels of Noxa. Knockdown of Noxa or Puma significantly attenuated aspirin-induced apoptosis. These results indicate that aspirin induces apoptosis through alteration of the Mcl-1/Noxa balance.