Human Pluripotent Stem Cell-Derived Alveolar Organoid with Macrophages.

Alveolar organoids (AOs), derived from human pluripotent stem cells (hPSCs) exhibit lung-specific functions. Therefore, the application of AOs in pulmonary disease modeling is a promising tool for understanding disease pathogenesis. However, the lack of immune cells in organoids limits the use of human AOs as models of inflammatory diseases. In this study, we generated AOs containing a functional macrophage derived from hPSCs based on human fetal lung development using biomimetic strategies. We optimized culture conditions to maintain the iMACs (induced hPSC-derived macrophages) AOs for up to 14 days. In lipopolysaccharide (LPS)-induced inflammatory conditions, IL-1ß, MCP-1 and TNF-α levels were significantly increased in iMAC-AOs, which were not detected in AOs. In addition, chemotactic factor IL-8, which is produced by mononuclear phagocytic cells, was induced by LPS treatment in iMACs-AOs. iMACs-AOs can be used to understand pulmonary infectious diseases and is a useful tool in identifying the mechanism of action of therapeutic drugs in humans. Our study highlights the importance of immune cell presentation in AOs for modeling inflammatory pulmonary diseases.

Protein kinase C-δ interacts with and phosphorylates ARD1.

Protein kinase C-δ (PKCδ) is a diacylglycerol-dependent, calcium-independent novel PKC isoform that is engaged in various cell signaling pathways, such as cell proliferation, apoptosis, inflammation, and oxidative stress. In this study, we searched for proteins that bind PKCδ using a yeast two-hybrid assay and identified murine arrest-defective 1 (mARD1) as a binding partner. The interaction between PKCδ and mARD1 was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. Furthermore, recombinant PKCδ phosphorylated full-length mARD1 protein. The NetPhos online prediction tool suggested PKCδ phosphorylates Ser80 , Ser108 , and Ser114 residues of mARD1 with the highest probability. Based on these results, we synthesized peptides containing these sites and examined their phosphorylations using recombinant PKCδ. Autoradiography confirmed these sites were efficiently phosphorylated. Consequent mass spectrometry and peptide sequencing in combination with MALDI-TOF MS/MS confirmed that Ser80 and Ser108 were major phosphorylation sites. The alanine mutations of Ser80 and Ser108 abolished the phosphorylation of mARD1 by PKCδ in 293T cells supporting these observations. In addition, kinase assays using various PKC isotypes showed that Ser80 of ARD1 was phosphorylated by PKCßI and PKCζ isotypes with the highest selectivity, while Ser108 and/or Ser114 were phosphorylated by PKCγ with activities comparable to that of the PKCδ isoform. Overall, these results suggest the possibility that PKCδ transduces signals by regulating phosphorylation of ARD1.

Ajoene, a Major Organosulfide Found in Crushed Garlic, Induces NAD(P)H:quinone Oxidoreductase Expression Through Nuclear Factor E2-related Factor-2 Activation in Human Breast Epithelial Cells.

BACKGROUND: NAD(P)H:quinone oxidoreductase-1 (NQO1) is a widely-distributed flavin adenine dinucleotide-dependent flavoprotein that promotes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes. This reduces quinone levels and thereby minimizes generation of excess reactive oxygen species (ROS) formed by redox cycling, and concurrent depletion of intracellular thiol pools. Ajoene is derived from crushed garlic. It is formed by a reaction involving two allicin molecules, and is composed of allyl sulfide and vinyl disulfide. Ajoene is present in two isomers, E- and Z-form. METHODS: Expression of antioxidant enzymes and nuclear factor E2-related factor-2 (Nrf2) was measured by Western blot analysis. NQO1 promoter activity was assessed by the luciferase reporter gene assay. ROS accumulation was monitored by using the fluorescence-generating probe 2',7'-dichlorofluorescein diacetate. The intracellular glutathione levels were measured by using a commercially available kit. RESULTS: Z-ajoene significantly up-regulated the expression of representative antioxidant enzyme NQO1 in non-tumorigenic breast epithelial MCF-10A cells at non-toxic concentrations. Z-ajoene enhanced up-regulation and nuclear translocation of Nrf2, which plays a pivotal role in the induction of many genes encoding antioxidant enzymes and other cytoprotective proteins. Z-ajoene treatment also increased the activity of nqo1-promoter harboring antioxidant response element consensus sequences in MCF-10A cells. Silencing of Nrf2 by small interfering RNA abrogated ajoene-induced expression of NQO1. Z-ajoene activated extracellular signal-regulated kinase (ERK). Inhibition of ERK activation by U0126 abrogated ability of Z-ajoene to activate Nrf2 and to induce NQO1 expression. Intracellular ROS accumulation was observed after treatment with Z-ajoene, whereas the E-isoform was not effective. The inhibition of ROS by treatment with N-acetylcysteine, a radical scavenger, abrogated Z-ajoene-induced expression of NQO1 as well as activation of ERK and Nrf2, suggesting that Z-ajoene augments the Nrf2-dependent antioxidant defense via ROS generation and ERK activation. CONCLUSIONS: Z-ajoene induces NQO1 expression in MCF-10A cells through ROS-mediated activation of Nrf2.

Selective Elimination of Culture-Adapted Human Embryonic Stem Cells with BH3 Mimetics.

The selective survival advantage of culture-adapted human embryonic stem cells (hESCs) is a serious safety concern for their clinical application. With a set of hESCs with various passage numbers, we observed that a subpopulation of hESCs at late passage numbers was highly resistant to various cell death stimuli, such as YM155, a survivin inhibitor. Transcriptome analysis from YM155-sensitive (YM155S) and YM155-resistant (YM155R) hESCs demonstrated that BCL2L1 was highly expressed in YM155R hESCs. By matching the gene signature of YM155R hESCs with the Cancer Therapeutics Response Portal dataset, BH3 mimetics were predicted to selectively ablate these cells. Indeed, short-course treatment with a sub-optimal dose of BH3 mimetics induced the spontaneous death of YM155R, but not YM155S hESCs by disrupting the mitochondrial membrane potential. YM155S hESCs remained pluripotent following BH3 mimetics treatment. Therefore, the use of BH3 mimetics is a promising strategy to specifically eliminate hESCs with a selective survival advantage.

A fluorescent chemical probe CDy9 selectively stains and enables the isolation of live naïve mouse embryonic stem cells.

Human and mouse embryonic stem cells (ESCs) differ in terms of their pluripotency status, i.e., naïve vs. primed. This affects various biological properties and leads to several technical hurdles for future clinical applications, such as difficulties in chimera formation, single-cell passaging, and gene editing. In terms of generating functional human tissues and organs via mammalian interspecies chimerism, a fluorescent chemical probe that specifically labels naïve ESCs would help to isolate these cells and monitor their conversion. This study demonstrates that the fluorescent chemical probe compound of designation yellow 9 (CDy9) selectively stains naïve, but not primed, mouse ESCs (mESCs). CDy9 entered cells via Slc13a5, a highly expressed membrane transporter in naïve mESCs. Fluorescence-based cell sorting based on CDy9 staining successfully separated naïve mESCs from primed mESCs. Mice generated using CDy9+ cells isolated during the conversion of mouse epiblast stem cells into naïve mESCs exhibited coat color chimerism. Furthermore, CDy9 specifically stained cells in the inner cell mass of mouse embryos. These findings suggest that CDy9 is a useful tool to isolate functional naïve mESCs.

Quercetin induced ROS production triggers mitochondrial cell death of human embryonic stem cells.

Small molecules to selectively induce cell death of undifferentiated human pluripotent stem cells (hPSCs) have been developed with the aim of lowering the risk of teratoma formation during hPSC-based cell therapy. In this context, we have reported that Quercetin (QC) induces cell death selectively in hESCs via p53 mitochondrial localization. However, the detailed molecular mechanism by which hESCs undergo selective cell death induced by QC remains unclear. Herein, we demonstrate that mitochondrial reactive oxygen species (ROS), strongly induced by QC in human embryonic stem cells (hESCs) but not in human dermal fibroblasts (hDFs), were responsible for QC-mediated hESC's cell death. Increased p53 protein stability and subsequent mitochondrial localization by QC treatment triggered mitochondrial cell death only in hESCs. Of interest, peptidylprolyl isomerase D [PPID, also called cyclophilin D (CypD)], which functions in mitochondrial permeability transition and mitochondrial cell death, was highly expressed in hESCs. Inhibition of CypD by cyclosporine A (CsA) clearly inhibited the QC-mediated loss of mitochondrial membrane potential and mitochondrial cell death. These results suggest that p53 and CypD in the mitochondria are critical for the QC-mediated induction of cell death in hESCs.

Role of MEK partner-1 in cancer stemness through MEK/ERK pathway in cancerous neural stem cells, expressing EGFRviii.

BACKGROUND: Glioma stem cells (GSCs) are a major cause of the frequent relapse observed in glioma, due to their high drug resistance and their differentiation potential. Therefore, understanding the molecular mechanisms governing the 'cancer stemness' of GSCs will be particularly important for improving the prognosis of glioma patients. METHODS: We previously established cancerous neural stem cells (CNSCs) from immortalized human neural stem cells (F3 cells), using the H-Ras oncogene. In this study, we utilized the EGFRviii mutation, which frequently occurs in brain cancers, to establish another CNSC line (F3.EGFRviii), and characterized its stemness under spheroid culture. RESULTS: The F3.EGFRviii cell line was highly tumorigenic in vitro and showed high ERK1/2 activity as well as expression of a variety of genes associated with cancer stemness, such as SOX2 and NANOG, under spheroid culture conditions. Through meta-analysis, PCR super-array, and subsequent biochemical assays, the induction of MEK partner-1 (MP1, encoded by the LAMTOR3 gene) was shown to play an important role in maintaining ERK1/2 activity during the acquisition of cancer stemness under spheroid culture conditions. High expression of this gene was also closely associated with poor prognosis in brain cancer. CONCLUSION: These data suggest that MP1 contributes to cancer stemness in EGFRviii-expressing glioma cells by driving ERK activity.

Technical approaches to induce selective cell death of pluripotent stem cells.

Despite the recent promising results of clinical trials using human pluripotent stem cell (hPSC)-based cell therapies for age-related macular degeneration (AMD), the risk of teratoma formation resulting from residual undifferentiated hPSCs remains a serious and critical hurdle for broader clinical implementation. To mitigate the tumorigenic risk of hPSC-based cell therapy, a variety of approaches have been examined to ablate the undifferentiated hPSCs based on the unique molecular properties of hPSCs. In the present review, we offer a brief overview of recent attempts at selective elimination of undifferentiated hPSCs to decrease the risk of teratoma formation in hPSC-based cell therapy.

Wip1 directly dephosphorylates NLK and increases Wnt activity during germ cell development.

Mice null for wild-type p53-induced phosphatase 1 (WIP1) display defects in testis development and spermatogenesis, resulting in reduced fertility. However, the molecular mechanism underlying these abnormalities in the testis remains uncharacterized. We report that the phosphatase activity of WIP1 increases Wnt activity through Nemo-like kinase (NLK). WIP1 directly interacted with NLK, which is highly homologous to p38 MAPK, a WIP1 substrate, and dephosphorylated its activation site. The WIP1-mediated inhibition of NLK activity markedly decreased the phosphorylation of lymphoid enhancer-binding factor 1 (LEF1), enhancing its interaction with ß-catenin. Additionally, WIP1 depletion impaired germ cell development, as evidenced by the expression of Oct4 and the germ cell-specific markers Ddx4, Nanos3 and Dnd1 during the development of germ cells from Oct4-GFP transgenic (OG2) mouse embryonic stem cells (mESCs). The expression of WIP1, whose level was significantly lower after the differentiation of germ cells from mESCs, occurred in parallel with the expression of germ cell development markers and SRY-box 17 (Sox17), a downstream target of Wnt. These results indicate that WIP1 is essential for germ cell development, which is known to require Wnt activity.

Intact wound repair activity of human mesenchymal stem cells after YM155 mediated selective ablation of undifferentiated human embryonic stem cells.

BACKGROUND: Risk of teratoma formation during human pluripotent stem cell (hPSC)-based cell therapy is one of the technical hurdles that must be resolved before their wider clinical application. To this end, selective ablation of undifferentiated hPSCs has been achieved using small molecules whose application should be safe for differentiated cells derived from the hPSCs. OBJECTIVE: However, the functional safety of such small molecules in the cells differentiated from hPSCs has not yet been extensively validated. METHOD: We used the survivin inhibitor YM155, which induced highly selective cell death of hPSCs for ablating undifferentiated hESCs after differentiation to human mesenchymal stem cells (hMSCs) and examined whether hMSCs remained fully functional after being exposed by YM155. RESULTS: We demonstrated that human mesenchymal stem cells (hMSCs) derived from human embryonic stem cells (hESCs) remained fully functional in vitro and in vivo, while hESCs were selectively ablated. CONCLUSION: These results suggest that a single treatment with YM155 after differentiation of hMSCs would be a valid approach for teratoma-free cell therapy.

Photodynamic Approach for Teratoma-Free Pluripotent Stem Cell Therapy Using CDy1 and Visible Light.

Pluripotent stem cells (PSC) are promising resources for regeneration therapy, but teratoma formation is one of the critical problems for safe clinical application. After differentiation, the precise detection and subsequent elimination of undifferentiated PSC is essential for teratoma-free stem cell therapy, but a practical procedure is yet to be developed. CDy1, a PSC specific fluorescent probe, was investigated for the generation of reactive oxygen species (ROS) and demonstrated to induce selective death of PSC upon visible light irradiation. Importantly, the CDy1 and/or light irradiation did not negatively affect differentiated endothelial cells. The photodynamic treatment of PSC with CDy1 and visible light irradiation confirmed the inhibition of teratoma formation in mice, and suggests a promising new approach to safe PSC-based cell therapy.

Control of stress signaling in stem cells: crossroads of stem cells and cancer.

Tumorigenesis is a relatively rare event in the human body considering the enormous number of cells composing our body and the frequent occurrence of genetic mutations in each cell. Nevertheless, the cells that happen to meet the minimum requirements can be transformed when stressed by a variety of oncogenic stimulations, then progress to form tumors. The vigorous competition between oncogenic signaling and tumor-suppressor defense is a critical determinant of cellular fate, which can be either tumorigenic transformation or cellular senescence/apoptosis depending on "who wins the battle." Recently, a number of cancers have been reported to originate from stem cells, whose self-renewing properties are normally reduced by innate tumor suppressors. Therefore, exploring the innate mechanism by which stem cells modulate tumor suppressors to maintain their "stemness" may provide valuable clues to characterize the distinctive oncogenic susceptibility of stem cells. This review is focused on the recent advances in the field of tumorigenesis of stem cells and on the associated molecular mechanisms.

Repair of Ischemic Injury by Pluripotent Stem Cell Based Cell Therapy without Teratoma through Selective Photosensitivity.

Stem-toxic small molecules have been developed to induce selective cell death of pluripotent stem cells (PSCs) to lower the risk of teratoma formation. However, despite their high efficacies, chemical-based approaches may carry unexpected toxicities on specific differentiated cell types. Herein, we took advantage of KillerRed (KR) as a suicide gene, to selectively induce phototoxicity using visible light via the production of reactive oxygen species. PSCs in an undifferentiated state that exclusively expressed KR (KR-PSCs) were eliminated by a single exposure to visible light. This highly selective cell death in KR-PSCs was exploited to successfully inhibit teratoma formation. In particular, endothelial cells from KR-mPSCs remained fully functional in vitro and sufficient to repair ischemic injury in vivo regardless of light exposure, suggesting that a genetic approach in which KR is expressed in a tightly controlled manner would be a viable strategy to inhibit teratoma formation for future safe PSC-based therapies.

Timely Degradation of Wip1 Phosphatase by APC/C Activator Protein Cdh1 is Necessary for Normal Mitotic Progression.

Wip1 belongs to the protein phosphatase C (PP2C) family, of which expression is up-regulated by a number of external stresses, and serves as a stress modulator in normal physiological conditions. When overexpressed, premature dephosphorylation of stress-mediators by Wip1 results in abrogation of tumor surveillance, thus Wip1 acts as an oncogene. Previously, the functional regulation of Wip1 in cell-cycle progression by counteracting cellular G1 and G2/M checkpoint activity in response to DNA damage was reported. However, other than in stress conditions, the function and regulatory mechanism of Wip1 has not been fully determined. Herein, we demonstrated that protein regulation of Wip1 occurs in a cell cycle-dependent manner, which is directly governed by APC/C(Cdh1) at the end of mitosis. In particular, we also showed evidence that Wip1 phosphatase activity is closely associated with its own protein stability, suggesting that reduced phosphatase activity of Wip1 during mitosis could trigger its degradation. Furthermore, to verify the physiological role of its phosphatase activity during mitosis, we established doxycycline-inducible cell models, including a Wip1 wild type (WT) and phosphatase dead mutant (Wip1 DA). When ectopically expressing Wip1 WT, we observed a delay in the transition from metaphase to anaphase. In conclusion, these studies show that mitotic degradation of Wip1 by APC/C(Cdh1) is important for normal mitotic progression.

Black raspberry-derived anthocyanins demethylate tumor suppressor genes through the inhibition of DNMT1 and DNMT3B in colon cancer cells.

We previously reported that oral administration of black raspberry powder decreased promoter methylation of tumor suppressor genes in tumors from patients with colorectal cancer. The anthocyanins (ACs) in black raspberries are responsible, at least in part, for their cancer-inhibitory effects. In the present study, we asked if ACs are responsible for the demethylation effects observed in colorectal cancers. Three days of treatment of ACs at 0.5, 5, and 25 µg/ml suppressed activity and protein expression of DNMT1 and DNMT3B in HCT116, Caco2 and SW480 cells. Promoters of CDKN2A, and SFRP2, SFRP5, and WIF1, upstream of Wnt pathway, were demethylated by ACs. mRNA expression of some of these genes was increased. mRNA expression of ß-catenin and c-Myc, downstream of Wnt pathway, and cell proliferation were decreased; apoptosis was increased. ACs were taken up into HCT116 cells and were differentially localized with DNMT1 and DNMT3B in the same cells visualized using confocal laser scanning microscopy. Although it was reported that DNMT3B is regulated by c-Myc in mouse lymphoma, DNMT3B did not bind with c-Myc in HCT116 cells. In conclusion, our results suggest that ACs are responsible, at least in part, for the demethylation effects of whole black raspberries in colorectal cancers.

Ibulocydine is a novel prodrug Cdk inhibitor that effectively induces apoptosis in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is frequently associated with abnormalities in cell cycle regulation, leading to increased activity of cyclin-dependent kinases (Cdks) due to the loss, or low expression of, Cdk inhibitors. In this study, we showed that ibulocydine (an isobutyrate prodrug of the specific Cdk inhibitor, BMK-Y101) is a candidate anti-cancer drug for HCC. Ibulocydine has high activity against Cdk7/cyclin H/Mat1 and Cdk9/cyclin T. Ibulocydine inhibited the growth of HCC cells more effectively than other Cdk inhibitors, including olomoucine and roscovitine, whereas ibulocydine as well as the other Cdk inhibitors and BMK-Y101 minimally influenced the growth of normal hepatocyte cells. Ibulocydine induced apoptosis in HCC cells, most likely by inhibiting Cdk7 and Cdk9. In vitro treatment of HCC cells with ibulocydine rapidly blocked phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II, a process mediated by Cdk7/9. Anti-apoptotic gene products such as Mcl-1, survivin, and X-linked IAP (XIAP) are crucial for the survival of many cell types, including HCC. Following the inhibition of RNA polymerase II phosphorylation, ibulocydine caused rapid down-regulation of Mcl-1, survivin, and XIAP, thus inducing apoptosis. Furthermore, ibulocydine effectively induced apoptosis in HCC xenografts with no toxic side effects. These results suggest that ibulocydine is a strong candidate anti-cancer drug for the treatment of HCC.

Potentiation of etoposide-induced apoptosis in HeLa cells by co-treatment with KG-135, a quality-controlled standardized ginsenoside formulation.

Our previous studies demonstrated that KG-135, a quality-controlled red ginseng-specific formulation containing approximately equal amounts of three major ginsenosides (Rk1, Rg3 and Rg5), down-regulated G1 cyclin-dependent kinase in HeLa cells. In the present work, we have found that KG-135 potentates cytotoxicity of etoposide by modulating apoptotic signaling. Co-treatment of etoposide and KG-135 markedly elevated the expression and phosphorylation at the serine 15 residue of p53 as well as the cellular levels of Bax and p21(Waf1/Cip1). The increased accumulation and phosphorylation of p53 (Ser15) were attenuated by treatment of cells with wortmannin, a pan-phosphatidylinositol-3 kinase inhibitor. Moreover, co-treatment of etoposide and KG-135 enhanced mitochondrial localization of Bax. Our results indicate that etoposide-induced apoptosis in HeLa cells can be potentiated in the presence of KG-135 through a mechanism that involves the stabilization of p53 and the stimulation of Bax- and p21-mediated apoptotic signaling pathways. These findings suggest that KG-135 represents a useful candidate adjuvant for the treatment of cancers that could potentially minimize the adverse effects of current clinical chemotherapeutics.

Xylocydine, a novel Cdk inhibitor, is an effective inducer of apoptosis in hepatocellular carcinoma cells in vitro and in vivo.

Hepatocellular carcinoma (HCC) frequently includes abnormalities in cell cycle regulators, including up-regulated cyclin-dependent kinase (Cdks) activities due to loss or low expression of Cdk inhibitors. In this study, we show that xylocydine, a cyclin-dependent kinase (Cdk) specific inhibitor, is a good anti-cancer drug candidate for HCC treatment. Xylocydine (50muM) selectively down-regulates the activity of Cdk1 and Cdk2, accompanied by significant cell growth inhibition in HCC cells. Xylocydine also strongly inhibits the activity of Cdk7 and Cdk9, in vitro as well as in cell cultures, that is temporally associated with apoptotic cell death in xylocydine-induced HCC cells. This is associated with inhibition of phosphorylation of RNA polymerase II at serine residues 5 and 2, which are targets of Cdk7 and Cdk9, respectively. The effects on apoptosis are concomitant with changes in the levels of anti-apoptotic proteins, Bcl-2, XIAP, and survivin, which are markedly down-regulated, and pro-apoptotic molecules, p53 and Bax, which are elevated in HCC cells after treatment with xylocydine. The up-regulated level of p53 was associated with increased stability of the protein, as levels of Ser15 and Ser392 phsophorylated p53 are similarly elevated in the inhibitor treated cells. We demonstrated that xylocydine can effectively suppress the growth of HCC xenografts in Balb/C-nude mice by preferentially inducing apoptosis in the xenografts, whereas the drug did not cause any apparent toxic effect on other tissues. Taken together, these data suggest that the novel Cdk inhibitor xylocydine is a good candidate for an anti-cancer drug for HCC therapy.

ATR (AT mutated Rad3 related) activity stabilizes Cdc6 and delays G2/M-phase entry during hydroxyurea-induced S-phase arrest of HeLa cells.

The Cdc6 protein, a key DNA replication initiation factor, contributes to the long-term maintenance of the S-phase checkpoint by anchoring the Rad3-Rad26 complex to chromatin. Here, we demonstrate that ATR (AT mutated and Rad3 related) activity is essential for maintaining high chromatin levels of the Cdc6 protein, thereby delaying entry into mitosis during hydroxyurea (HU)-induced S-phase arrest of HeLa cells. Downregulation of ATR (AT mutated and Rad3 related) (i.e., using ATR-siRNA) reduced the protein levels of chromatin Cdc6 and significantly increased the cellular levels of phospho-histone H3 (Ser 10), an index of mitosis. Downregulation of Cdc6 was completely restored by pretreatment with MG132, a proteasome inhibitor. Moreover, mitotic entry of MG132-pretreated cells was significantly downregulated. Our results also show that ATR (AT mutated and Rad3 related) kinase phosphorylates Cdc6 at serine residue 6. Thus, this ATR (AT mutated and Rad3 related)-mediated phosphorylation of Cdc6 is likely associated with stabilization of Cdc6 protein, thereby maintaining high levels of chromatin Cdc6 and delaying premature mitotic entry. This novel mechanism likely contributes to the functional regulation of chromatin Cdc6, which delays the cell cycle of hydroxyurea-induced cells to enter mitosis at the S-phase checkpoint.

Phosphorylation of Smac by JNK3 attenuates its interaction with XIAP.

Here we demonstrate that JNK3 can phosphorylate Smac. Smac phosphorylation attenuates its ability to activate apoptosome activity in HeLa S-100 cell lysates. Addition of the X-linked inhibitor of apoptosis protein (XIAP) to the S-100 markedly suppresses apoptosome activity, and this suppressive effect of XIAP is neutralized by adding unphosphorylated Smac, but not phosphorylated Smac. Furtherover, JNK3-mediated phosphorylation of Smac markedly attenuates the interaction between Smac and XIAP, as measured by BIACORE assays and non-denaturing gel shift assays. When JNK3 activity is down-regulated in etoposide-induced HeLa cells by transiently overexpressing a dominant negative version of JNK3 (DN-JNK3), the caspase-3 activity as well as PARP cleavages are markedly enhanced. And the interaction of Smac with XIAP also increases by down-regulating JNK3 activity under the same conditions. These results suggest that JNK3 activity can attenuate the progression of apoptosis through a novel mechanism of action, the down-regulation of interaction between Smac and XIAP.