Gabriel Pons

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.

A zinc-finger transcription factor induced by TGF-β promotes apoptotic cell death in epithelial Mv1Lu cells

Transforming growth factor‐β (TGF‐β) superfamily members constitute a group of multifunctional factors that are able to stimulate apoptotic cell death in a variety of cells. In this report, we show that a zinc‐finger transcription factor (TIEG) is an immediate early gene transcriptionally induced by TGF‐β in the epithelial Mv1Lu cell line. We also demonstrate that, mimicking TGF‐β effects, ectopic overexpression of TIEG is sufficient to trigger the apoptotic cell program in these cells, which is preceded by a decrease of Bcl‐2 protein levels. Finally, apoptotic events elicited by TIEG overexpression can be effectively prevented by ectopic co‐expression of Bcl‐2. On the basis of these results we suggest that induction of TIEG expression has a role in the pro‐apoptotic properties of TGF‐β.

Mitoxantrone, a topoisomerase II inhibitor, induces apoptosis of B‐chronic lymphocytic leukaemia cells

B‐chronic lymphocytic leukaemia (B‐CLL) is characterized by the accumulation of long‐lived CD5+ B lymphocytes. The effect of mitoxantrone, a topoisomerase II inhibitor, on B‐CLL cells was studied. Treatment of B‐CLL cells for 48 h with mitoxantrone (0.5 μg/ml) induced a decrease in cell viability as determined by MTT assay. The IC50 calculated for the cells of three patients was 0.7 μg/ml for two of them and 1.4 μg/ml for the third. In all three patients the maximum effect was observed with 2 μg/ml. An additive cytotoxic effect was observed when mitoxantrone (0.5 μg/ml) was combined with fludarabine (5 μg/ml). Mitoxantrone induced DNA fragmentation and the proteolytic cleavage of poly(ADP‐ribose) polymerase (PARP), a marker of the activation of caspases, in all the patients studied, demonstrating that the cytotoxic effect of mitoxantrone was due to induction of apoptosis. These results suggest that mitoxantrone, and possibly other topoisomerase II inhibitors, may be used in the chemotherapy of B‐CLL, and that combination of mitoxantrone with fludarabine or other drugs could improve the effectiveness of the treatment.

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.

Effect of vanadate on phosphoryl transfer enzymes involved in glucose metabolism

Different types of enzymes from yeast and from rabbit muscle which catalyze phosphoryl transfer reactions involved in glucose metabolism differ in their sensitivity to vanadate. Phospho glucomutase and phosphoglycerate mutase are inhibited at the μM range. 2,3-Bisphosphoglycerate phosphatase is completely inhibited by 0.5 mM vanadate. 2,3-Bisphosphoglycerate synthase, hexokinase, phosphoglycerate kinase and fructose-1,6-P2 phosphatase are partially inhibited by mM vanadate. Phosphofructokinase and pyruvate kinase are not affected. The glycolytic enzymes which mechanism does not involves phosphoryl transfer step are not affected by vanadate.

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.

Phylogeny and ontogeny of the phosphoglycerate mutases—IV. Distribution of glycerate-2,3-P2 dependent and independent phosphoglycerate mutases in algae, fungi, plants and animals

1. The distribution of the glycerate-2,3-P2 dependent and independent phosphoglycerate mutases (PGM) has been studied in more than eighty species. 2. PGM activity in the extracts has been measured in the presence and in the absence of glycerate-2,3-P2, at pH 7.5 and at pH 8.5. 3. All samples with glycerate-2,3-P2 dependent PGM possess higher activity at pH 7.5 than at pH 8.5. In contrast, samples with glycerate-2,3-P2 independent PGM possess lower activity at pH 7.5 than at pH 8.5. 4. In algae and fungi both glycerate-2,3-P2 dependent and independent PGM have been found. 5. In plants only glycerate-2,3-P2 independent PGM has been detected. 6. In animals both types of PGM are present. Independent PGM activity is present in sponges, coelenterates, myriapods, arachnids and echinoderms. Glycerate-2,3-P2 dependent PGM is present in platyhelminths, mollusks, annelids, crustaceans, insects and vertebrates.