Glucocorticoid resistance in chronic lymphocytic leukaemia is associated with a failure of upregulated Bim/Bcl-2 complexes to activate Bax and Bak

Glucocorticoids (GCs) represent an important component of modern treatment regimens for fludarabine-refractory or TP53-defective chronic lymphocytic leukemia (CLL). However, GC therapy is not effective in all patients. The molecular mechanisms responsible for GC-induced apoptosis and resistance were therefore investigated in primary malignant cells obtained from a cohort of 46 patients with CLL. Dexamethasone-induced apoptosis was unaffected by p53 dysfunction and more pronounced in cases with unmutated IGHV genes. Cross-resistance was observed between dexamethasone and other GCs but not fludarabine, indicating non-identical resistance mechanisms. GC treatment resulted in the upregulation of Bim mRNA and protein, but to comparable levels in both GC-resistant and sensitive cells. Pre-incubation with Bim siRNAs reduced GC-induced upregulation of Bim protein and conferred resistance to GC-induced apoptosis in previously GC-sensitive cells. GC-induced upregulation of Bim was associated with the activation of Bax and Bak in GC-sensitive but not -resistant CLL samples. Co-immunoprecipitation experiments showed that Bim does not interact directly with Bax or Bak, but is almost exclusively bound to Bcl-2 regardless of GC treatment. Taken together, these findings suggest that the GC-induced killing of CLL cells results from the indirect activation of Bax and Bak by upregulated Bim/Bcl-2 complexes, and that GC resistance results from the failure of such activation to occur.     

The BH3-only protein Puma is both necessary and sufficient for neuronal apoptosis induced by DNA damage in sympathetic neurons

DNA damage activates apoptosis in several neuronal populations and is an important component of neuropathological conditions. While it is well established that neuronal apoptosis, induced by DNA damage, is dependent on the key cell death regulators p53 and Bax, it is unknown which proteins link the p53 signal to Bax. Using rat sympathetic neurons as an in vitro model of neuronal apoptosis, we show that cytosine arabinoside is a DNA damaging drug that induces the expression of the BH3-only pro-apoptotic genes Noxa, Puma and Bim. Increased expression occurred after p53 activation, measured by its phosphorylation at serine 15, but prior to the conformational change of Bax at the mitochondria, cytochrome c (cyt c) release and apoptosis. Hence Noxa, Puma and Bim could potentially link p53 to Bax. We directly tested this hypothesis by the use of nullizygous mice. We show that Puma, but not Bim or Noxa, is a crucial mediator of DNA damage-induced neuronal apoptosis. Despite the powerful pro-apoptotic effects of overexpressed Puma in Bax-expressing neurons, Bax nullizygous neurons were resistant to Puma-induced death. Therefore, Puma provides the critical link between p53 and Bax, and is both necessary and sufficient to mediate DNA damage-induced apoptosis of sympathetic neurons.     

沙眼衣原体感染HeLa229细胞Bim表达及抑制凋亡研究

研究沙眼衣原体(D血清型)感染的HeLa229细胞中Bim蛋白质的表达及凋亡诱导剂作用后的凋亡情况。Western-blot检测沙眼衣原体感染和未感染的HeLa229细胞Bim蛋白质的表达水平。凋亡诱导剂etopo- side作用HeLa229细胞后,经Hoechst33258染色用荧光显微镜观察核浓缩和凋亡小体;流式细胞仪检测凋亡率。HeLa229细胞在未感染及感染沙眼衣原体6 h后可检测到Bim的表达;在感染24、48 h后均未检测到Bim的表达。经etoposide作用后,未感染的HeLa229细胞观察到明显的核浓缩和凋亡小体;流式细胞仪检测的凋亡率为90.64%。感染24 h的HeLa229细胞,未观察到核浓缩和凋亡小体;流式细胞仪检测的凋亡率为11.50%,与未感染的HeLa229细胞诱导后的凋亡率比较有统计学意义(P<0.05)。沙眼衣原体感染HeLa229细胞后可降低Bim蛋白质的表达;并能抑制etoposide诱导的细胞凋亡。     

Glucagon-like Peptide-1 (GLP-1) Diminishes Neuronal Degeneration and Death Caused by NGF Deprivation by Suppressing Bim Induction

Glucagon-like peptide-1 (GLP-1) is a glucoincretin hormone most intensively studied for its actions on insulin secreting β-cells. GLP-1 and its receptor are also found in brain and accumulating evidence indicates that GLP-1 has neuroprotective actions. Here, we investigated whether GLP-1 protects neuronal cells from death evoked by nerve growth factor (NGF) withdrawal. Compromised trophic factor signaling may underlie neurodegenerative diseases ranging from Alzheimer disease to diabetic neuropathies. We report that GLP-1 provides sustained protection of cultured neuronal PC12 cells and sympathetic neurons from degeneration and death caused by NGF deprivation. Past work shows that NGF deprivation induces the pro-apoptotic protein Bim which contributes to neuron death. Here, we find that GLP-1 suppresses Bim induction promoted by NGF deprivation. Thus, GLP-1 may protect neurons, at least in part, by suppressing Bim induction. Our findings support the idea that drugs that mimic or elevate GLP-1 represent potential therapeutics for neurodegenerative diseases.     

The tubulin-depolymerising agent combretastatin-4 induces ectopic aster assembly and mitotic catastrophe in lung cancer cells H460

The relationship between microtubular dynamics, dismantling of pericentriolar components and induction of apoptosis was analysed after exposure of H460 non-small lung cancer cells to anti-mitotic drugs. The microtubule destabilising agent, combretastatin-A4 (CA-4) led to microtubular array disorganization, arrest in mitosis and abnormal metaphases, accompanied by the presence of numerous centrosome-independent “star-like” structures containing tubulin and aggregates of pericentrosomal matrix components like γ-tubulin, pericentrin and ninein, whereas the structural integrity of centrioles was not affected by treatment. On the contrary, in condition of prolonged exposure or high concentrations of CA-4 such aggregates never formed. Treatment with 7.5 nM CA-4, which produced a high frequency “star-like” aggregates, was accompanied by mitotic catastrophe commitment characterized by translocation of the proapoptotic Bim protein to mitochondria activation of caspases-3/9 and DNA fragmentation as a result of either prolonged metaphase arrest or attempt of cells to divide. Drug concentrations which fail to block cells at mitosis were also unable to activate apotosis. A detailed time-course analysis of cell cycle arrest and apoptosis indicated that after CA-4 washout the number of metaphases with “star-like” structures decreased as a function of time and arrested cells proceeded in anaphase. After 4 h, the multiple α- and γ-tubulin aggregates coalesced into two well-defined spindles in a bipolar mitotic spindle organization. Overall, our findings suggest that the maintenance of microtubular integrity plays a relevant role in stabilising the pericentriolar matrix, whose dismantling can be associated with apoptosis after exposure to microtubule depolymerising agents.     

Giardia lamblia EB1 is a functional homolog of yeast Bim1p that binds to microtubules

Giardia lamblia, with two nuclei and a distinct polarized morphology, is an interesting organism for investigating how distribution of its microtubule (MT) is controlled during its cell cycle. In this study, we identified the end-binding protein 1 (EB1) of G. lamblia, a well-known microtubule-associated protein that organizes MTs in eukaryotes. Immunofluorescence assays using recombinant EB1 (rEB1)-specific antibodies demonstrated EB1 localization in nuclear membrane as well as in some cytoskeletal structures such as axomenes and median bodies of trophozoites of G. lamblia. Complementation experiments using the BIM1 knock-out mutant of yeast, the yeast homolog of mammalian EB1, showed that giardial EB1 was able to carry out a homologous function in controlling MT dynamics. In addition, rEB1 of G. lamblia co-precipitated with MTs by an in vitro binding assay, thereby demonstrating that G. lamblia EB1 is a MT-associated protein. These results, taken together, suggest that G. lamblia EB1 is a functional homolog of eukaryotic EB1 and is likely to be a determinant for MT distribution.     

Oncogenic BRAF(V600E) inhibits BIM expression to promote melanoma cell survival

Somatic activating mutations of BRAF are the earliest and most common genetic abnormality detected in the genesis of human melanoma. However, the mechanism(s) by which activated BRAF promotes melanoma cell cycle progression and/or survival remain unclear. Here we demonstrate that expression of BIM, a pro-apoptotic member of the BCL-2 family, is inhibited by BRAF-->MEK-->ERK signaling in mouse and human melanocytes and in human melanoma cells. Trophic factor deprivation of melanocytes leads to elevated BIM expression. However, re-addition of trophic factors or activation of a conditional form of BRAF(V600E) leads to rapid inhibition of BIM expression. In both cases, inhibition of BIM expression was dependent on the activity of MEK1/2 and the proteasome. Consistent with these observations, pharmacological inhibition of BRAF(V600E) or MEK1/2 in human melanoma cells (using PLX4720 and CI-1040 respectively) led to a striking elevation of BIM expression. Re-activation of BRAF-->MEK-->ERK signaling led to phosphorylation of BIM-EL on serine 69 and its subsequent degradation. Interestingly, endogenous expression of BIM in melanoma cells was insufficient to induce apoptosis unless combined with serum deprivation. Under these circumstances, inhibition of BIM expression by RNA interference provided partial protection from apoptosis. These data suggest that regulation of BIM expression by BRAF-->MEK-->ERK signaling is one mechanism by which oncogenic BRAF(V600E) can influence the aberrant physiology of melanoma cells.