Bim plays a crucial role in synergistic induction of apoptosis by the histone deacetylase inhibitor SBHA and TRAIL in melanoma cells

The wide variation in sensitivity of cancer cells to TRAIL- or histone deacetylase (HDAC) inhibitor – induced apoptosis precludes successful treatment of cancer with these agents. We report here that TRAIL and SBHA synergistically induce apoptosis of melanoma cells as revealed by quantitative analysis using the normalized isobologram method. This is supported by enhanced activation of caspase-3 and cleavage of its substrates, PARP and ICAD. Co-treatment with SBHA and TRAIL did not enhance formation of the death-inducing signaling complex (DISC) and processing of caspase-8 and Bid, but potentiated activation of Bax and release of Cytochrome C and Smac/DIABLO from mitochondria into the cytosol. SBHA down-regulated Bcl-X_L, Mcl-1 and XIAP, but up-regulated Bax, Bak, and the BH3-only protein Bim_EL. Up-regulation of the latter by SBHA was attenuated by the presence of TRAIL, which was inhibitable by the pan-caspase inhibitor z-VAD-fmk. Inhibition of Bim by siRNA attenuated conformational changes of Bax, mitochondrial apoptotic events, and activation of caspase-3, leading to marked inhibition of the synergy between SBHA and TRAIL. Thus, Bim plays an essential role in synergistic induction of apoptosis by SBHA and TRAIL in melanoma. This work was supported by the NSW State Cancer Council, the Melanoma and Skin Cancer Research Institute Sydney, the Hunter Melanoma Foundation, NSW, and the National Health and Medical Research Council, Australia. X.D. Zhang is a Cancer Institute NSW Fellow.     

Elevated Mcl-1 inhibits thymocyte apoptosis and alters thymic selection

T cells developing in the thymus undergo rigorous positive and negative selection to ensure that those exported to peripheral lymphoid organs bear T-cell receptors (TCRs) capable of reacting with foreign antigens but tolerant of self. At each checkpoint, whether a thymocyte survives or dies is determined by antiapoptotic and proapoptotic Bcl-2 family members. We used Mcl-1 transgenic (tg) mice to investigate the impact of elevated expression of antiapoptotic Mcl-1 on thymocyte apoptosis and selection, making a side-by-side comparison with thymocytes from BCL-2tg mice. Mcl-1 was as effective as Bcl-2 at protecting thymocytes against spontaneous cell death, diverse cytotoxic insults and TCR–CD3 stimulation-driven apoptosis. In three different TCR tg models, Mcl-1 markedly enhanced positive selection of thymocytes, as did Bcl-2. In H-Y TCR tg mice, elevated Mcl-1 and Bcl-2 were equally effective at inhibiting deletion of autoreactive thymocytes. However, in the OT-1tg model where deletion is mediated by a peripheral antigen whose expression is regulated by Aire, Mcl-1 was less effective than Bcl-2. Thus, the capacity of Mcl-1 overexpression to inhibit apoptosis triggered by TCR stimulation apparently depends on the thymocyte subset subject to deletion, presumably due to differences in the profiles of proapoptotic Bcl-2 family members mediating the deletion.     

S1219 residue of 53BP1 is phosphorylated by ATM kinase upon DNA damage and required for proper execution of DNA damage response

B lymphocyte receptor-mediated apoptosis is associated with increased expression of the BimL isoform of Bim. The mechanisms involved in the regulation of BimL protein expression are still unknown. We report that BimL expression following BCR activation is not associated with a specific increase of BimL mRNA but rather to the intron retention structure of the BimEL mRNA. Indeed, expression of a BimEL cDNA leads in Hela cells leads to the production of both BimEL and BimL proteins. Mutation of the intron-splicing GT sequence present in the exon 3 results in the production of only BimEL protein. Ectopic expression of BimEL cDNA resulted in a large increase of BimL expression upon BCR-stimulation, whereas cells transfected with the GT/AA mutated form of BimEL only produced BimEL proteins upon BCR-activation. These data showed that BimL expression induced by BCR activation may result from the splicing of BimEL mRNA independently of Bim promoter regulation.     

Bim-mediated apoptosis and PD-1/PD-L1 pathway impair reactivity of PD1(+)/CD127(-) HCV-specific CD8(+) cells targeting the virus in chronic hepatitis C virus infection

PD-1 molecule promotes anergy and IL-7 receptor (CD127) induces an anti-apoptotic effect on T cells. Correlation between PD-1/CD127 phenotype and hepatitis C virus (HCV)-specific CD8⁺ cell reactivity in resolved infection (RI) after treatment and persistent HCV-infection (PI) was analysed. Directly ex vivo, PD-1 and CD127 expression on HCV-specific CD8⁺ cells displayed a positive and negative correlation, respectively with viraemia. Proliferation after stimulation on PD-1⁻/CD127⁺ cells from RI cases was preserved, while it was impaired on PD-1⁺/CD127⁻ cells from PI patients. PD1⁺/CD127⁺ population was observed in PI, and these maintained expansion ability but they did not target the virus. Frequency of PI cases with HCV-specific CD8⁺ cell proliferation increased after anti-PD-L1 and anti-apoptotic treatment. Bim expression on HCV-specific CD8⁺ cells from PI patients was enhanced. In conclusion, during chronic HCV infection non-reactive HCV-specific CD8⁺ cells targeting the virus are PD-1⁺/CD127⁻/Bim⁺ and, blocking apoptosis and PD-1/PD-L1 pathway on them enhances in vitro reactivity.     

Bim protein degradation contributes to cisplatin resistance

Cisplatin is the first-line chemotherapy for the treatment of several cancers. However, the development of cisplatin resistance represents a major clinical problem, and the mechanisms of acquired resistance are not fully understood. Here we show that degradation of the Bcl-2 homology 3-only proapoptotic protein Bim plays an important role in cisplatin resistance in ovarian cancer. Specifically, we show that treatment of ovarian cancer cells with cisplatin caused Bim phosphorylation and subsequent degradation and that its degradation is associated with cisplatin resistance. We also show that cisplatin treatment caused the activation of ERK, which correlated with Bim phosphorylation and degradation. By inhibiting ERK phosphorylation with the MEK inhibitor and knocking down ERK expression with siRNA, we show that Bim phosphorylation and degradation were blocked, which suggests that Bim is phosphorylated by ERK and that such phosphorylation is responsible for cisplatin-induced Bim degradation. We show that ERK was activated in cisplatin-resistant OV433 cells as compared with their counterpart parental OV433 cells. We also show that Bim was phosphorylated and degraded in cisplatin-resistant OV433 cells but not in the parental OV433 cells. Importantly, we show that inhibition of Bim degradation by the proteasome inhibitor MG132 sensitized resistant OV433 cells to cisplatin-induced death. Taken together, our data indicate that degradation of Bim via ERK-mediated phosphorylation can lead to cisplatin resistance. Therefore, these findings suggest that cisplatin resistance can be overcome by the combination of cisplatin and the proteasome inhibitors in ovarian cancer cells.     

Anti-apoptotic gene Bcl2 is required for stapes development and hearing

In this paper we describe novel and specific roles for the apoptotic regulators Bcl2 and Bim in hearing and stapes development. Bcl2 is anti-apoptotic while Bim is pro-apoptotic. Characterization of the auditory systems of mice deficient for these molecules revealed that Bcl2^−/− mice suffered severe hearing loss. This was conductive in nature and did not affect sensory cells of the inner ear, with cochlear hair cells and neurons present and functional. Bcl2^−/− mice were found to have a malformed, often monocrural, porous stapes (the small stirrup-shaped bone of the middle ear), but a normally shaped malleus and incus. The deformed stapes was discontinuous with the incus and sometimes fused to the temporal bones. The defect was completely rescued in Bcl2^−/−Bim^−/− mice and partially rescued in Bcl2^−/−Bim^+/− mice, which displayed high-frequency hearing loss and thickening of the stapes anterior crus. The Bcl2^−/− defect arose in utero before or during the cartilage stage of stapes development. These results implicate Bcl2 and Bim in regulating survival of second pharyngeal arch or neural crest cells that give rise to the stapes during embryonic development.     

The unknown face of IRE1α – Beyond ER stress

IRE1α (Inositol Requiring kinase Enzyme 1 alpha), a transmembrane protein localized to the endoplasmic reticulum (ER) is a master regulator of the unfolded protein response (UPR) pathway. The fate determining steps during ER stress-induced apoptosis are greatly attributed to IRE1α’s endoribonuclease and kinase activities. Apart from its role as a chief executioner in ER stress, recent studies have shown that upon activation in the presence or absence of ER stress, IRE1α executes multiple cellular processes such as differentiation, immune response, progression and repression of the cell cycle. Besides its crucial role in protein misfolding, the versatile contributions of IRE1α in other cellular functions are greatly unknown. In this review, we have discussed the structural conservation of IRE1 among eukaryotes, the mechanisms underlying its activation and the recent understandings of the non-apoptotic functions of IRE1 other than ER stress-induced cell death.     

miR-106b-93-25簇对子宫内膜癌细胞的调控作用研究

目的:检测mi R-106b-93-25基因簇对子宫内膜癌细胞增殖及凋亡的影响,并探讨其机制。方法:q RT-PCR检测临床子宫内膜癌标本及癌旁正常组织中mi R-106b、mi R-93和mi R-25及其宿主基因MCM7的表达情况。将micro RNA及其拮抗剂转染ECC-1细胞后,MTT实验检测ECC-1细胞增殖情况,流式细胞术检测ECC-1细胞周期及细胞凋亡情况。荧光素酶报告系统验证mi R-106b和mi R-25分别直接调控p21和Bim。结果:临床标本子宫内膜癌组织与癌旁正常组织相比mi R-106b-93-25簇及其宿主基因MCM7的表达明显增高。mi R-106b-93-25簇能够促进ECC-1细胞增殖,减少凋亡。转染mi R-106b和mi R-93的细胞出现明显的S期阻滞,过表达mi R-25的细胞凋亡明显减少。mi R-106b-93-25簇通过抑制靶基因p21和Bim的表达,引起促增殖、抗凋亡作用。结论:mi R-106b-93-25簇能够促进子宫内膜癌细胞增殖,抑制凋亡,并使细胞发生S期阻滞。mi R-106b-93-25簇在子宫内膜癌的发生与发展中具有重要的作用。