Regulation of cell death in mitotic neural progenitor cells by asymmetric distribution of prostate apoptosis response 4 (PAR-4) and simultaneous elevation of endogenous ceramide

Cell death and survival of neural progenitor (NP) cells are determined by signals that are largely unknown. We have analyzed pro-apoptotic signaling in individual NP cells that have been derived from mouse embryonic stem cells. NP formation was concomitant with elevated apoptosis and increased expression of ceramide and prostate apoptosis response 4 (PAR-4). Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis. Apoptotic cells also stained for proliferating cell nuclear antigen (a nuclear mitosis marker protein), but not for nestin (a marker for NP cells). In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(-)/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis. The other cell was nestin(+), but PAR-4(-), and was not apoptotic. Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain.     

A novel isoform of prostate apoptosis response 4 (PAR-4) that co-distributes with F-actin and prevents apoptosis in neural stem cells

The elevated expression of prostate apoptosis response-4 (PAR-4) induces apoptosis in differentiating mouse embryonic stem (ES) cells. In embryoid body (EB) cells and the E15.5 stage of embryonic mouse brain, PAR-4 is expressed as two isoforms (38 and 33 kDa). Using mouse EB-derived RNA as a template we have cloned and characterized a novel isoform of PAR-4 (PAR-4/p33) that lacks exon 3 and shows a bona fide splice junction of exons 2 and 4. The molecular mass for PAR-4/p33 is estimated to be 33 kDa, corresponding to the short form found in the EB cells and E15.5 mouse brain. The fluorescent fusion protein of PAR-4/p33 is mainly found in the cytosol and is co-distributed with F-actin filaments, while that of the 38 kDa full length PAR-4/p38 is predominantly translocated to the nucleus. In contrast to the full length PAR-4 (PAR-4/p38), ectopic expression of PAR-4/p33 does not result in the activation of caspase 3 and the induction of apoptosis. PAR-4/p33 forms a complex with PAR-4/p38, which inhibits its nuclear translocation and the induction of apoptosis. PAR-4/p33 is suggested to be a dominant negative isoform of PAR-4/p38 and may regulate PAR-4-dependent apoptosis.     

Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell-derived neural transplants

The formation of stem cell-derived tumors (teratomas) is observed when engrafting undifferentiated embryonic stem (ES) cells, embryoid body-derived cells (EBCs), or mammalian embryos and is a significant obstacle to stem cell therapy. We show that in tumors formed after engraftment of EBCs into mouse brain, expression of the pluripotency marker Oct-4 colocalized with that of prostate apoptosis response-4 (PAR-4), a protein mediating ceramide-induced apoptosis during neural differentiation of ES cells. We tested the ability of the novel ceramide analogue N-oleoyl serinol (S18) to eliminate mouse and human Oct-4(+)/PAR-4(+) cells and to increase the proportion of nestin(+) neuroprogenitors in EBC-derived cell cultures and grafts. S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of beta-tubulin III. However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm. Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.     

Regulation of apoptosis during neuronal differentiation by ceramide and b-series complex gangliosides

Lipid analysis of gestational day E14.5 mouse brain revealed elevation of ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide was coincident with the first appearance of b-series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F-11) cells with sialyltransferase-II (ST2) resulted in a 70% reduction of ceramide-induced apoptosis. This was most likely due to an 80% reduced expression of prostate apoptosis response-4 (PAR-4). PAR-4 expression and apoptosis were restored by preincubation of ST2-transfected cells with N-butyl deoxinojirimycin (NB-DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 mitogen-activated protein (MAPK) kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining for complex gangliosides, but only low staining for apoptosis (TUNEL) and PAR-4. Apoptosis and PAR-4 expression, however, were elevated in the ventricular zone which only weakly stained for complex gangliosides. Whole cell patch clamping revealed a 2-fold increased calcium influx in ST2-transfected cells, the blocking of which with nifedipine restored apoptosis to the level of untransfected cells. In serum-free culture, supplementation of the medium with IGF-1 was required to maintain MAPK phosphorylation and the anti-apoptotic effect of BCG expression. BCG-enhanced calcium influx and the presence of insulin-like growth factor-1 may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide-induced apoptosis by up-regulation of MAPK and reduction of PAR-4 expression.     

Reactive oxygen species-mediated activation of JNK and down-regulation of DAXX are critically involved in penta-O-galloyl-beta-d-glucose-induced apoptosis in chronic myeloid leukemia K562 cells

Although 1,2,3,4,6-penta-O-galloyl-beta-d-glucose (PGG) was well known to have antitumor activities in breast, prostate, kidney, liver cancers and HL-60 leukemia via regulation of caspase 3, p53, S-phase kinase-associated protein 2 (Skp2) and insulin receptor signaling, the underlying mechanism of PGG-induced apoptosis linked with reactive oxygen species (ROS) mediated c-Jun N-terminal kinase (JNK) and DAXX was never elucidated in chronic myeloid leukemia (CML) K562 cells until now. Herein PGG significantly decreased the viability of CML cell lines such as K562 and KBM-5 without hurting normal peripheral blood lymphocytes (PBLs). PGG increased the number of TUNEL-positive cells and the sub-G1 cell population as well as activated caspase cascades including caspase-8, -9 and -3 in K562 cells. Interestingly, a significant activation of JNK by PGG was observed by MULTIPLEX assay and Western blotting. Conversely, JNK inhibitor D-JNKi suppressed the cleavages of caspase 3 and PARP induced by PGG in K562 cells. Also, PGG dramatically enhanced generation of ROS and reduced the expression of death-domain-associated protein (DAXX). Of note, ROS inhibitor acetyl-l-cysteine (NAC) reversed JNK-dependent apoptosis and DAXX inhibition induced by PGG. Overall, these findings suggest that ROS-dependent JNK activation and DAXX downregulation are critically involved in PGG-induced apoptosis in K562 cells.     

Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells

We have reported that ceramide mediates binding of atypical protein kinase C (PKC) zeta to its inhibitor protein, PAR-4 (prostate apoptosis response-4), thereby inducing apoptosis in differentiating embryonic stem cells. Using a novel method of lipid vesicle-mediated affinity chromatography, we showed here that endogenous ceramide binds directly to the PKCzeta.PAR-4 complex. Ceramide and its analogs activated PKCzeta prior to binding to PAR-4, as determined by increased levels of phosphorylated PKCzeta and glycogen synthase kinase-3beta and emergence of a PAR-4-to-phosphorylated PKCzeta fluorescence resonance energy transfer signal that co-localizes with ceramide. Elevated expression and activation of PKCzeta increased cell survival, whereas expression of PAR-4 promoted apoptosis. This suggests that PKCzeta counteracts apoptosis, unless its ceramide-induced activation is compromised by binding to PAR-4. A luciferase reporter assay showed that ceramide analogs activate nuclear factor (NF)-kappaB unless PAR-4-dependent inhibition of PKCzeta suppresses NF-kappaB activation. Taken together, our results show that direct physical association with ceramide and PAR-4 regulates the activity of PKCzeta. They also indicate that this interaction regulates the activity of glycogen synthase kinase-3beta and NF-kappaB.     

沉默DAXX基因表达促进人卵巢癌细胞凋亡和衰老

DAXX是Fas死亡结构域相关蛋白(Fas death-associated protein, DAXX),可与Fas死亡受体的死亡域结合.通过激活c-Jun NH2末端激酶通路,DAXX增强Fas介导的凋亡,同时也增强转录生长因子β依赖的凋亡.本研究通过免疫组化检测人正常卵巢组织和卵巢癌组织中DAXX蛋白表达,然后通过Tet-on诱导体系构建DAXX基因沉默的慢病毒,感染卵巢癌细胞后,加入嘌呤霉素筛选稳定表达的OV2008细胞,四环素诱导DAXX基因沉默,通过免疫印迹和定量PCR检测DAXX基因干扰效果,MTT检测细胞增殖,克隆形成实验检测克隆形成能力,免疫印迹和免疫荧光方法检测DNA损伤蛋白的变化,SA-β-gal检测细胞衰老.结果表明,在人正常卵巢组织中DAXX低表达,而在人卵巢癌组织中DAXX高表达,随着四环素浓度的增加DAXX的表达量降低,DAXX基因沉默抑制卵巢癌OV2008细胞的增殖和克隆形成. 免疫印迹结果显示,DAXX基因沉默可诱导DNA损伤相关蛋白p-H2AX和p-CHK2高表达.SA-β-gal检测结果表明,DAXX基因沉默可诱导OV2008细胞发生衰老,免疫印迹检测发现,p21和p27在 DAXX沉默的卵巢癌细胞中高表达.综上结果,DAXX沉默可以抑制卵巢癌细胞的增殖和克隆形成,同时也促进卵巢癌细胞发生DNA损伤和衰老.该研究为卵巢癌的基因治疗提供新的思路.     

p62 forms a ternary complex with PKCzeta and PAR-4 and antagonizes PAR-4-induced PKCzeta inhibition.

It has been reported that prostate apoptosis response-4 (PAR-4) binds to and inhibits protein kinase Czeta (PKCzeta) which phosphorylates IkappaB kinase beta (IKKbeta) for nuclear factor kappaB (NFkappaB) activation, while p62 binds to and recruits PKCzeta to the NFkappaB signaling complex. Thus, a mechanism to coordinate the two binding proteins for the regulation of PKCzeta is expected to exist. The present data show that p62 and PAR-4 do not compete for PKCzeta binding but directly interact each other and form a ternary complex with PKCzeta. Furthermore, p62 not only enhances the catalytic activity of PKCzeta but also reactivates catalytically inactive PAR-4-bound PKCzeta. As the result, over-expression of p62 protects cells from PAR-4-mediated inactivation of NFkappaB and apoptotic death. Thus, the regulatory role of p62 for free and PAR-4-bound PKCzeta is important in activation of NFkappaB.     

Poster Session 3

Lipid analysis of gestational day E14.5 mouse brain revealed elevation of ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide was coincident with the first appearance of β-series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F-11) cells with sialyltransferase-II (ST2) resulted in a 70% reduction of apoptosis that was induced with the novel ceramide analog (NCA) N-oleoyl serinol (S18). This was most likely due to an 80% reduced expression of prostate apoptotis response-4 (PAR-4). PAR-4 expression and apoptosis were restored by preincubation of ST2-transfected cells with N-butyl deoxinojirimycin (NB-DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 MAPK-kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining for complex gangliosides, but only low staining for apoptosis (TUNEL) and PAR-4. Apoptosis and PAR-4 expression, however, were elevated in the ventricular zone, which only weakly stained for complex gangliosides. Complex gangliosides may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide-induced apoptosis by up-regulation of MAPK and reduction of PAR-4 expression. NCAs may be useful to analyse the molecular mechanisms that underlie or counteract ceramide-induced apoptosis during neuronal development.
Acknowledgements:   Supported by grants MH61934-04 (to E.B.) and NS11853 (to R.K.Y.).     

p53siRNA therapy reduces cell proliferation, migration and induces apoptosis in triple negative breast cancer cells

p53 protein is probably the best known tumor suppressor. Earlier reports proved that human breast cancer cells expressing mutant p53 displayed resistance to apoptosis. This study is intended to investigate, the potential applications of RNA interference (RNAi) to block p53 expression, as well as its subsequent effect on cell growth, apoptosis and migration on a triple negative human breast cancer cell line (Hs578T). p53siRNA significantly reduced cell index (CI) compared to the control and we observed an inhibition of cellular migration in the interval of time between 0 and 30 h, as shown in the data obtained by dynamic evaluation using the xCELLigence System. Also, by using PCR-array technology, a panel of 84 key genes involved in apoptosis was investigated. Our studies indicate that the knockdown of p53 expression by siRNA modulates several genes involved in cell death pathways and apoptosis, showing statistically significant gene expression differences for 22 genes, from which 18 were upregulated and 4 were downregulated. The present research also emphasizes the important role of BCL-2 pro-apoptotic family of genes (Bim, Bak, and Bax) in activating apoptosis and reducing cell proliferation by p53siRNA treatment. Death receptors cooperate with BCL-2 pro-apoptotic genes in reducing cell proliferation. The limited success may be due to the activation of the antiapoptotic gene Mcl-1, and it may be associated with the resistance of triple negative breast cancer cells to cancer treatment. Thus, targeting p53siRNA pathways using siRNA may serve as a promising therapeutic strategy for the treatment of breast cancers.     

ZIPK: a unique case of murine-specific divergence of a conserved vertebrate gene

Zipper interacting protein kinase (ZIPK, also known as death-associated protein kinase 3 [DAPK3]) is a Ser/Thr kinase that functions in programmed cell death. Since its identification eight years ago, contradictory findings regarding its intracellular localization and molecular mode of action have been reported, which may be attributed to unpredicted differences among the human and rodent orthologs. By aligning the sequences of all available ZIPK orthologs, from fish to human, we discovered that rat and mouse sequences are more diverged from the human ortholog relative to other, more distant, vertebrates. To test experimentally the outcome of this sequence divergence, we compared rat ZIPK to human ZIPK in the same cellular settings. We found that while ectopically expressed human ZIPK localized to the cytoplasm and induced membrane blebbing, rat ZIPK localized exclusively within nuclei, mainly to promyelocytic leukemia oncogenic bodies, and induced significantly lower levels of membrane blebbing. Among the unique murine (rat and mouse) sequence features, we found that a highly conserved phosphorylation site, previously shown to have an effect on the cellular localization of human ZIPK, is absent in murines but not in earlier diverging organisms. Recreating this phosphorylation site in rat ZIPK led to a significant reduction in its promyelocytic leukemia oncogenic body localization, yet did not confer full cytoplasmic localization. Additionally, we found that while rat ZIPK interacts with PAR-4 (also known as PAWR) very efficiently, human ZIPK fails to do so. This interaction has clear functional implications, as coexpression of PAR-4 with rat ZIPK caused nuclear to cytoplasm translocation and induced strong membrane blebbing, thus providing the murine protein a possible adaptive mechanism to compensate for its sequence divergence. We have also cloned zebrafish ZIPK and found that, like the human and unlike the murine orthologs, it localizes to the cytoplasm, and fails to bind the highly conserved PAR-4 protein. This further supports the hypothesis that murine ZIPK underwent specific divergence from a conserved consensus. In conclusion, we present a case of species-specific divergence occurring in a specific branch of the evolutionary tree, accompanied by the acquisition of a unique protein–protein interaction that enables conservation of cellular function.     

The pro-apoptotic protein Par-4 facilitates vascular contractility by cytoskeletal targeting of ZIPK

Par-4 (prostate apoptosis response 4) is a pro-apoptotic protein and tumour suppressor that was originally identified as a gene product up-regulated during apoptosis in prostate cancer cells. Here, we show, for the first time, that Par-4 is expressed and co-localizes with the actin filament bundles in vascular smooth muscle. Furthermore, we demonstrate that targeting of ZIPK to the actin filaments, as observed upon PGF-2α stimulation, is inhibited by the presence of a cell permeant Par-4 decoy peptide. The same decoy peptide also significantly inhibits PGF-2α induced contractions of smooth muscle tissue. Moreover, knockdown of Par-4 using antisense morpholino nucleotides results in significantly reduced contractility, and myosin light chain and myosin phosphatase target subunit phosphorylation. These results indicate that Par-4 facilitates contraction by targeting ZIPK to the vicinity of its substrates, myosin light chain and MYPT, which are located on the actin filaments. These results identify Par-4 as a novel regulator of myosin light chain phosphorylation in differentiated, contractile vascular smooth muscle.     

Interdependent regulation of p53 and miR-34a in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) has an incidence 4/100 000 people in the western world and is one of the first cancers reported to be associated with deregulated miRNA expression. microRNAs are small non coding RNAs that are important regulators of protein expression through binding to their untranslated 3'-UTR region. The miR-34 family was demonstrated to be induced by the tumor suppressor p53 and to elicit p53-like responses like senescence, cell cycle arrest and apoptosis depending on the cell type. We have shown in a recent paper that miR-34a is severely increased in the TCL1-mouse model of CLL. This finding was reflected in human CLL. Moreover, it is demonstrated that its expression is dependent on the presence of the SNP309 in the intronic promoter of the MDM2 gene. In addition, low miR-34a expression was associated with shorter time to treatment (log-rank P = 0.003) in CLL. When reintroduced into CLL cells, miR-34a was able to induce apoptosis. Interestingly, this was dependent on an intact p53 pathway. Here, we present data showing that knockdown of p53 in HCT-116 cells severely reduces miR-34a induced apoptosis. In conclusion, miR-34a is proposed as a marker for the activity of the p53 pathway in CLL.