Ana M. Cosialls

AICAR induces apoptosis independently of AMPK and p53 through up-regulation of the BH3-only proteins BIM and NOXA in chronic lymphocytic leukemia cells

5-Aminoimidazole-4-carboxamide riboside or acadesine (AICAR) induces apoptosis in chronic lymphocytic leukemia (CLL) cells. A clinical study of AICAR is currently being performed in patients with this disease. Here, we have analyzed the mechanisms involved in AICAR-induced apoptosis in CLL cells in which it activates its only well-known molecular target, adenosine monophosphate-activated protein kinase (AMPK). However, AMPK activation with phenformin or A-769662 failed to induce apoptosis in CLL cells and AICAR also potently induced apoptosis in B lymphocytes from Ampkα1(-/-) mice, demonstrating an AMPK-independent mechanism of cell death. Importantly, AICAR induced apoptosis irrespective of the tumor suppressor TP53 or ataxia telangiectasia mutated (ATM) status via induction of the mitochondrial pathway. Apoptosis was preceded by an increase in mRNA and protein levels of proapoptotic BCL-2 family proteins of the BH3-only subgroup, including BIM, NOXA, and PUMA in CLL cells. Strikingly, B lymphocytes from Noxa(-/-) or Bim(-/-) mice were partially protected from the cytotoxic effects of AICAR. Consistently, B cells from Noxa(-/-)/Bim(-/-) mice resisted induction of apoptosis by AICAR as potently as B lymphocytes overexpressing transgenic BCL-2. These findings support the notion that AICAR is an interesting alternative therapeutic option for CLL patients with impaired p53 function and resistance to conventional chemotherapy.

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.

Multiplex ligation‐dependent probe amplification for detection of genomic alterations in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) is the commonest form of leukaemia in adults in Western countries. We performed multiplex ligation‐dependent probe amplification (MLPA) analysis in 50 CLL patients to identify multiple genomic CLL‐specific targets, including genes located at 13q14, 17p13 (TP53), 11q23 (ATM) and chromosome 12, and compared the results with those obtained with fluorescence in situ hybridization (FISH). There was a good correlation between MLPA and FISH results, as most alterations (89%) were detected by both techniques. Only three cases with a low percentage (<25%) of cells carrying the alterations were not detected by MLPA. On the other hand, as MLPA uses multiple probes it identified intragenic or small alterations undetected by FISH in three cases. MLPA also detected alterations in 8q24 (MYC) and 6q25–26. In summary, unlike interphase FISH, MLPA enabled the simultaneous analysis of many samples with automated data processing at a low cost. Therefore, the combination of robust multiplexing and high throughput makes MLPA a useful technique for the analysis of genomic alterations in CLL.

Akt inhibitors induce apoptosis in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is now divisible into subsets with distinct clinical behaviors. The biology underlying these differences appears to include signaling pathways through the B-cell receptor and other receptors. The phosphatidylinositol-3-kinase/Akt pathway is important, raising the possibility that inhibitors could have therapeutic potential. Encouragingly, two inhibitors were found to induce preferential apoptosis of CLL cells, irrespective of TP53 status, with increases in NOXA and PUMA protein levels and a decrease in MCL-1. Background The phosphatidylinositol-3-kinase/Akt pathway has been described to be critical in the survival of chronic lymphocytic leukemia cells. In this study we analyzed the effect of two selective chemical inhibitors of Akt (Akti-1/2 and A-443654) on the survival of chronic lymphocytic leukemia cells. Design and Methods Using cytometry we studied the cytotoxic effects of Akt inhibitors on peripheral B and T lymphocytes from patients with chronic lymphocytic leukemia and from healthy donors. We studied the changes induced by Akti-1/2 and A-443654 at the mRNA level by performing reverse transcriptase multiplex ligation–dependent probe amplification. We also studied the changes induced by both Akt inhibitors in some BCL-2 protein family members on chronic lymphocytic leukemia cells by western blotting. Moreover, we analyzed the cytotoxic effect of Akt inhibitors in patients’ cells with deleted/mutated TP53. Results Both inhibitors induced apoptosis in chronic lymphocytic leukemia cells in a dose-dependent manner. Moreover, B cells from patients with chronic lymphocytic leukemia were more sensitive to Akt inhibitors than T cells from leukemic patients, and B or T cells from healthy donors. Survival factors for chronic lymphocytic leukemia cells, such as interleukin-4 and stromal cell-derived factor-1α, were not able to block the apoptosis induced by either Akt inhibitor. Akti-1/2 did not induce any change in the mRNA expression profile of genes involved in apoptosis, while A-443654 induced some changes, including an increase in NOXA and PUMA mRNA levels, suggesting the existence of additional targets for A-443654. Both inhibitors induced an increase in PUMA and NOXA protein levels, and a decrease in MCL-1 protein level. Moreover, Akti-1/2 and A-443654 induced apoptosis irrespective of TP53 status. Conclusions These results demonstrate that Akt inhibitors induce apoptosis of chronic lymphocytic leukemia cells and might be a new therapeutic option for the treatment of chronic lymphocytic leukemia.

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.

The potential anticancer agent PK11195 induces apoptosis irrespective of p53 and ATM status in chronic lymphocytic leukemia cells

Background and Objectives The potential anticancer agent 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a translocator protein (18KDa) (TSPO) ligand, facilitates the induction of cell death by a variety of cytotoxic and chemotherapeutic agents. Primary chronic lymphocytic leukemia (CLL) cells overexpress TSPO. The aim of this study was to examine the effects of PK11195 on CLL cells. Design and Methods Using cytometric analysis, we studied the cytotoxic effects of PK11195 on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Western blot and cytometric analyses were used to study the mitochondrial effects of PK11195 on CLL cells. Moreover, we analyzed the cytotoxic effect of PK11195 in patients’ cells with mutated p53 or ATM. Results PK11195 induces apoptosis and had additive effects with chemotherapeutic drugs in primary CLL cells. Other TSPO ligands such as RO 5-4864 and FGIN-1-27 also induce apoptosis in CLL cells. PK11195 induces mitochondrial depolarization and cytochrome c release upstream of caspase activation, and dithiocyana-tostilbene-2,2- disulfonic acid (DIDS), a voltage-dependent anion channel (VDAC) inhibitor, inhibits PK11195-induced apoptosis, demonstrating a direct involvement of mitochondria. CLL cells and normal B cells are more sensitive than T cells to PK11195-induced apoptosis. Interestingly, PK11195 induced apoptosis in CLL cells irrespective of their p53 or ATM status. Interpretation and Conclusions These results suggest that PK11195 alone or in combination with chemotherapeutic drugs might be a new therapeutic option for the treatment of CLL.

Analysis of apoptosis regulatory genes altered by histone deacetylase inhibitors in chronic lymphocytic leukemia cells.

Histone deacetylases (HDACs) play a key role in the regulation of acetylation status not only of histones but also of many other non-histone proteins involved in cell cycle regulation, differentiation or apoptosis. Therefore, histone deacetylase inhibitors (HDACi) have emerged as promising anticancer agents. Herein, we report the characterization of apoptosis in B-cell chronic lymphocytic leukemia (CLL) induced by two HDACi, Kendine 92 and SAHA. Both inhibitors induce dose-, time- and caspase-dependent apoptosis through the mitochondrial pathway. Interestingly, Kendine 92 and SAHA show a selective cytotoxicity for B lymphocytes and induce apoptosis in CLL cells with mutated or deleted TP53 as effectively as in tumor cells harboring wild-type TP53. The pattern of apoptosis-related gene and protein expression profile has been characterized. It has shown to be irrespective of TP53 status and highly similar between SAHA and Kendine 92 exposure. The balance between the increased BAD, BNIP3L, BNIP3, BIM, PUMA and AIF mRNA expression levels, and decreased expression of BCL-W, BCL-2, BFL-1, XIAP and FLIP indicates global changes in the apoptosis mRNA expression profile consistent with the apoptotic outcome. Protein expression analysis shows increased levels of NOXA, BIM and PUMA proteins upon Kendine 92 and SAHA treatment. Our results highlight the capability of these molecules to induce apoptosis not only in a selective manner but also in those cells frequently resistant to standard treatments. Thus, Kendine 92 is a novel HDACi with anticancer efficacy for non-proliferating CLL cells.

AICAR induces Bax/Bak-dependent apoptosis through upregulation of the BH3-only proteins Bim and Noxa in mouse embryonic fibroblasts

Abstract5-Aminoimidazole-4-carboxamide (AICA) riboside (AICAR) is a nucleoside analogue that is phosphorylated to 5-amino-4-imidazolecarboxamide ribotide (ZMP), which acts as an AMP mimetic and activates AMP-activated protein kinase (AMPK). It has been recently described that AICAR triggers apoptosis in chronic lymphocytic leukemia (CLL) cells, and its mechanism of action is independent of AMPK as well as p53. AICAR-mediated upregulation of the BH3-only proteins BIM and NOXA correlates with apoptosis induction in CLL cells. Here we propose mouse embryonic fibroblasts (MEFs) as a useful model to analyze the mechanism of AICAR-induced apoptosis. ZMP formation was required for AICAR-induced apoptosis, though direct Ampk activation with A-769662 failed to induce apoptosis in MEFs. AICAR potently induced apoptosis in Ampkα1−/−/α2−/− MEFs, demonstrating an Ampk-independent mechanism of cell death activation. In addition, AICAR acts independently of p53, as MEFs lacking p53 also underwent apoptosis normally. Notably, MEFs lacking Bax and Bak were completely resistant to AICAR-induced apoptosis, confirming the involvement of the mitochondrial pathway in its mechanism of action. Apoptosis was preceded by ZMP-dependent but Ampk-independent modulation of the mRNA levels of different Bcl-2 family members, including Noxa, Bim and Bcl-2. Bim protein levels were accumulated upon AICAR treatment of MEFs, suggesting its role in the apoptotic process. Strikingly, MEFs lacking both Bim and Noxa displayed high resistance to AICAR. These findings support the notion that MEFs are a useful system to further dissect the mechanism of AICAR-induced apoptosis.

Transcriptional modulation of apoptosis regulators by roscovitine and related compounds.

Chemical inhibitors of cyclin-dependent kinase (CDK), like roscovitine, are promising drugs in the context of new cancer therapies. Roscovitine and related compounds, like seliciclib and olomoucine, are effective inducers of apoptosis in many proliferating cells in culture. These compounds are known to activate the intrinsic or mitochondrial pathway of apoptosis. In order to better characterize this intrinsic pathway, a transcriptional analysis was performed using the reverse transcriptase-multiplex ligation-dependent probe amplification procedure (RT-MLPA). In five cell lines, we detected an early and marked reduction of most transcripts, which is consistent with the disruption of transcription that results from the inhibition of CDK7 and CDK9. However, the mRNA of p53-upregulated modulator of apoptosis(PUMA) gene escaped from this transcription inhibition in neuroblastoma cells with a functional p53 protein. The increase of PUMA mRNA was not found in roscovitine-treated cell lines defective in p53, which underwent apoptosis like their p53 proficient counterparts. In addition, in SH-SY5Y cells, sublethal and lethal concentrations of roscovitine produced equivalent increases of PUMA mRNA and protein. In conclusion, the increased expression of PUMA was not associated with apoptosis induction. On the contrary, mRNA and protein depletion of MCL-1 gene correlated the best with cell demise. Moreover, NOXA protein suffered a far minor decrease than MCL-1. Because of the selective neutralization of NOXA by MCL-1, we hypothesize that the disruption of this balance is a critical event in apoptosis induction by roscovitine and related compounds.