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Tamoxifen is commonly used to treat patients with ESR/ER-positive breast cancer, but its therapeutic benefit is limited by the development of resistance. Recently, alterations in macroautophagy/autophagy function were demonstrated to be a potential mechanism for tamoxifen resistance. Although MTA1 (metastasis-associated 1) has been implicated in breast tumorigenesis and metastasis, its role in endocrine resistance has not been studied. Here, we report that the level of MTA1 expression was upregulated in the tamoxifen resistant breast cancer cell lines MCF7/TAMR and T47D/TR, and knockdown of MTA1 sensitized the cells to 4-hydroxytamoxifen (4OHT). Moreover, knockdown of MTA1 significantly decreased the enhanced autophagy flux in the tamoxifen resistant cell lines. To confirm the role of MTA1 in the development of tamoxifen resistance, we established a cell line, MCF7/MTA1, which stably expressed MTA1. Compared with parental MCF7, MCF7/MTA1 cells were more resistant to 4OHT-induced growth inhibition in vitro and in vivo, and showed increased autophagy flux and higher numbers of autophagosomes. Knockdown of ATG7 or cotreatment with hydroxychloroquine, an autophagy inhibitor, restored sensitivity to 4OHT in both the MCF7/MTA1 and tamoxifen resistant cells. In addition, AMP-activated protein kinase (AMPK) was activated, probably because of an increased AMP:ATP ratio and decreased expression of mitochondrial electron transport complex components. Finally, publicly available breast cancer patient datasets indicate that MTA1 levels correlate with poor prognosis and development of recurrence in patients with breast cancer treated with tamoxifen. Overall, our findings demonstrated that MTA1 induces AMPK activation and subsequent autophagy that could contribute to tamoxifen resistance in breast cancer.

We aimed to study the role of protein L-isoaspartyl methyltransferase (PIMT) in neuronal differentiation using basic fibroblast growth factor (bFGF)-induced neuronal differentiation, characterized by cell-body shrinkage, long neurite outgrowth, and expression of neuronal differentiation markers light and medium neurofilaments (NF). The bFGF-mediated neuronal differentiation of PC12 cells was induced through activation of mitogen-activated protein kinase (MAPK) signaling molecules [MAPK kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2 (ERK1/2), and p90RSK], and phosphatidylinositide 3-kinase (PI3K)/Akt signaling molecules PI3Kp110β, PI3Kp110γ, Akt, and mTOR. Inhibitors (adenosine dialdehyde and S-adenosylhomocysteine) of protein methylation suppressed bFGF-mediated neuronal differentiation of PC12 cells. PIMT-eficiency caused by PIMT-specific siRNA inhibited neuronal differentiation of PC12 cells by suppressing phosphorylation of MEK1/2 and ERK1/2 in the MAPK signaling pathway and Akt and mTOR in the PI3K/Akt signaling pathway. Therefore, these results suggested that PIMT was critical for bFGF-mediated neuronal differentiation of PC12 cells and regulated the MAPK and Akt signaling pathways. [BMB Reports 2016; 49(8): 437-442]

Protein arginine methyltransferases (PRMTs) mediate the methylation of a number of protein substrates of arginine residues and serve critical functions in many cellular responses, including cancer development, progression, and aggressiveness, T-lymphocyte activation, and hepatic gluconeogenesis. There are nine members of the PRMT family, which are divided into 4 types (types I–IV). Although most PRMTs do not require posttranslational modification (PTM) to be activated, fine-tuning modifications, such as interactions between cofactor proteins, subcellular compartmentalization, and regulation of RNA, via micro-RNAs, seem to be required. Inflammation is an essential defense reaction of the body to eliminate harmful stimuli, including damaged cells, irritants, or pathogens. However, chronic inflammation can eventually cause several types of diseases, including some cancers, atherosclerosis, rheumatoid arthritis, and periodontitis. Therefore, inflammation responses should be well modulated. In this review, we briefly discuss the role of PRMTs in the control of inflammation. More specifically, we review the roles of four PRMTs (CARM1, PRMT1, PRMT5, and PRMT6) in modulating inflammation responses, particularly in terms of modulating the transcriptional factors or cofactors related to inflammation. Based on the regulatory roles known so far, we propose that PRMTs should be considered one of the target molecule groups that modulate inflammatory responses.

The multipotency and anti-inflammatory effects of mesenchymal stem cells (MSCs) make them attractive for cell therapy in regenerative medicine. A large number of MSCs is required for efficient therapy owing to the low homing efficiency of MSCs to target sites. Furthermore, owing to limitations in obtaining sufficient amounts of MSCs, in vitro expansion of MSCs that preserves their differentiation and proliferative potential is essential. The animal factor included in culture media also limits clinical application. In this study, adipose-derived MSCs showed a significantly higher proliferation rate in STK2, a chemically-defined medium, than in DMEM/FBS. The expression of MSC surface markers was increased in the culture using STK2 compared to that using DMEM/FBS. Tri-lineage differentiation analyses showed that MSCs cultured in STK2 were superior to those cultured in DMEM/FBS. In addition, MSCs cultured in STK2 showed a reduced senescence rate, small and homogenous cell size, and were more genetically stable compared to those cultured in DMEM/FBS. Furthermore, secretome analysis showed that the expression of factors related to proliferation/migration, anti-inflammation, and differentiation were increased in STK2 culture medium compared to DMEM/FBS. Taken together, these results suggest that culture using STK2 medium offers many advantages through which it is possible to obtain safer, superior, and larger numbers of MSCs.

Transmethylation is an important reaction that transfers a methyl group in S-adenosylmethionine (SAM) to substrates such as DNA, RNA, and proteins. It is known that transmethylation plays critical roles in various cellular responses. In this study, we examined the effects of transmethylation on tumorigenic responses and its regulatory mechanism using an upregulation strategy of adenosylhomocysteine (SAH) acting as a negative feedback inhibitor. Treatment with adenosine dialdehyde (AdOx), an inhibitor of transmethylation-suppressive adenosylhomocysteine (SAH) hydrolase (SAHH), enhanced the level of SAH and effectively blocked the proliferation, migration, and invasion of cancer cells; the treatment also induced the differentiation of C6 glioma cells and suppressed the neovascular genesis of eggs in a dose-dependent manner. Through immunoblotting analysis, it was found that AdOx was capable of indirectly diminishing the phosphorylation of oncogenic Src and its kinase activity. Interestingly, AdOx disrupted actin cytoskeleton structures, leading to morphological changes, and suppressed the formation of a signaling complex composed of Src and p85/PI3K, which is linked to various tumorigenic responses. In agreement with these data, the exogenous treatment of SAH or inhibition of SAHH by specific siRNA or another type of inhibitor, 3-deazaadenosine (DAZA), similarly resulted in antitumorigenic responses, suppressive activity on Src, the alteration of actin cytoskeleton, and a change of the colocalization pattern between actin and Src. Taken together, these results suggest that SAH/SAHH-mediated transmethylation could be linked to the tumorigenic processes through cross-regulation between the actin cytoskeleton and Src kinase activity.

Anthraquinone compounds are one of the abundant polyphenols found in fruits, vegetables, and herbs. However, thein vivoanti-inflammatory activity and molecular mechanisms of anthraquinones have not been fully elucidated. We investigated the activity of anthraquinones using acute inflammatory and nociceptive experimental conditions. Anthraquinone-2-carboxylic acid (9,10-dihydro-9,10-dioxo-2-anthracenecarboxylic acid, AQCA), one of the major anthraquinones identified from Brazilian taheebo, ameliorated various inflammatory and algesic symptoms in EtOH/HCl- and acetylsalicylic acid- (ASA-) induced gastritis, arachidonic acid-induced edema, and acetic acid-induced abdominal writhing without displaying toxic profiles in body and organ weight, gastric irritation, or serum parameters. In addition, AQCA suppressed the expression of inflammatory genes such as cyclooxygenase- (COX-) 2 in stomach tissues and lipopolysaccharide- (LPS-) treated RAW264.7 cells. According to reporter gene assay and immunoblotting analyses, AQCA inhibited activation of the nuclear factor- (NF-)κB and activator protein- (AP-) 1 pathways by suppression of upstream signaling involving interleukin-1 receptor-associated kinase 4 (IRAK1), p38, Src, and spleen tyrosine kinase (Syk). Our data strongly suggest that anthraquinones such as AQCA act as potent anti-inflammatory and antinociceptive componentsin vivo, thus contributing to the immune regulatory role of fruits and herbs.

Src kinase (Src) is a tyrosine protein kinase that regulates cellular metabolism, survival, and proliferation. Many studies have shown that Src plays multiple roles in macrophage-mediated innate immunity, such as phagocytosis, the production of inflammatory cytokines/mediators, and the induction of cellular migration, which strongly implies that Src plays a pivotal role in the functional activation of macrophages. Macrophages are involved in a variety of immune responses and in inflammatory diseases including rheumatoid arthritis, atherosclerosis, diabetes, obesity, cancer, and osteoporosis. Previous studies have suggested roles for Src in macrophage-mediated inflammatory responses; however, recently, new functions for Src have been reported, implying that Src functions in macrophage-mediated inflammatory responses that have not been described. In this paper, we discuss recent studies regarding a number of these newly defined functions of Src in macrophage-mediated inflammatory responses. Moreover, we discuss the feasibility of Src as a target for the development of new pharmaceutical drugs to treat macrophage-mediated inflammatory diseases. We provide insights into recent reports regarding new functions for Src that are related to macrophage-related inflammatory responses and the development of novel Src inhibitors with strong immunosuppressive and anti-inflammatory properties, which could be applied to various macrophage-mediated inflammatory diseases.

Ginsenoside (G)-F1 is an enzymatic metabolite generated from G-Rg1. Although this metabolite has been reported to suppress platelet aggregation and to reduce gap junction-mediated intercellular communication, the modulatory activity of G-F1 on the functional role of skin-derived cells has not yet been elucidated. In this study, we evaluated the regulatory role of G-F1 on the cellular responses of B16 melanoma cells. G-F1 strongly suppressed the proliferation of B16 cells up to 60% at 200 μg/mL, while only diminishing the viability of HEK293 cells up to 30%. Furthermore, G-F1 remarkably induced morphological change and clustering of B16 melanoma cells. The melanin production of B16 cells was also significantly blocked by G-F1 up to 70%. Interestingly, intracellular signaling events involved in cell proliferation, migration, and morphological change were up-regulated at 1 h incubation but down-regulated at 12 h. Therefore, our results suggest that G-F1 can be applied as a novel anti?skin cancer drug with anti-proliferative and anti-migration features.

The bone and immune systems are closely related through cellular and molecular interactions. Because bone-resorbing osteoclasts (OCs) are derived from the monocyte/macrophage lineage, similar to dendritic cells (DCs), we hypothesized that OCs could serve as antigen-presenting cells (APCs) to activate T cells. In this study, OCs were generated from human monocytes with stimulation by receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Results showed that, similar to DCs, OCs express major histocompatibility complex (MHC) classes I and II, and CD80, CD86, and CD40; and uptake soluble antigens. OCs secrete interleukin-10 (IL-10), transforming growth factor-β (TGF-β), IL-6, and tumor necrosis factor-α (TNF-α), but not IL-12p70. OCs present allogeneic antigens and activate both CD4+ and CD8+ alloreactive T cells in an MHC-restricted fashion. OCs also present soluble protein tetanus toxoid to activate autologous CD4+ T cells. These findings indicate that OCs can function as APCs and activate both CD4+ and CD8+ T cells. Thus, our study provides new insight into the effect of OCs on the immune system and may help develop novel strategies for treating diseases such as rheumatoid arthritis and multiple myeloma, which affect both the bone and immune systems.

Multiple myeloma remains an incurable disease. One of the major problems is that myeloma cells develop drug resistance on interaction with bone marrow stromal cells. In this study, we examined the effects of macrophages (Mφs), a type of stromal cells, on myeloma cell survival and response to chemotherapy. We showed that Mφ, in particular tumor-associated Mφ, is a protector of myeloma cells. The protective effect was dependent on direct contact between Mφs and myeloma cells. Mφs protected both myeloma cell lines and primary myeloma cells from spontaneous and chemotherapy drug-induced apoptosis by attenuating the activation and cleavage of caspase-dependent apoptotic signaling. These findings are clinically relevant because we found that CD68+ Mφs heavily infiltrate the bone marrow of patients with myeloma but not the bone marrow of control patients. Thus, our results indicate that Mφs may contribute to myeloma cell survival and resistance to chemotherapeutic treatment in vivo.

－ Korean mistletoe lectin (KML) is the major component found in Viscum album var. (coloratum) , displaying anti-cancer and immunostimulating activities. Even though it has been shown to boost host immune defense mechanisms, the regulatory roles of KML on the functional activation of macrophages have not been fully elucidated. In this study, regulatory mechanism of KML on macrophage-mediated immune responses was examined in terms of KML-mediated signaling event. KML clearly induced mRNA expression of tumor necrosis factor (TNF)-α, the generation of reactive oxygen species (ROS) and phagocytic uptake in RAW264.7 cells. All of these events were strongly suppressed by U0126, whereas TNF-α mRNA was not diminished by SB203580 and SP600125, indicating ERK as a central enzyme managing KML-induced up-regulation of macrophage functions. Indeed, KML strongly induced the phosphorylation of ERK in a time-dependent manner without altering its total level. Therefore, these data suggest that ERK may be a major signaling enzyme with regulatory property toward various KML-mediated macrophage responses.Keywords: Korean mistletoe lectin

The identification of novel tumor-associated antigens, especially those shared among patients, is urgently needed to improve the efficacy of immunotherapy for multiple myeloma (MM). In this study, we examined whether Dickkopf-1 (DKK1), a protein that is not expressed in most normal tissues but is expressed by tumor cells from almost all patients with myeloma, could be a good candidate. We identified and synthesized DKK1 peptides for human leukocyte antigen (HLA)–A*0201 and confirmed their immunogenicity by in vivo immunization in HLA-A*0201 transgenic mice. We detected, using peptidetetramers, low frequencies of DKK1 peptide-specific CD8-positive (CD8+) T cells in patients with myeloma and generated peptide-specific T-cell lines and clones from HLA-A*0201-positive (HLA-A*0201+) blood donors and patients with myeloma. These T cells efficiently lysed peptide-pulsed but not unpulsed T2 or autologous dendritic cells, DKK1-positive (DKK1+)/HLA-A*0201+ myeloma cell lines U266 and IM-9, and, more importantly, HLA-A*0201+ primary myeloma cells from patients. No killing was observed on DKK1+/HLA-A*0201-negative (HLA-A*0201−) myeloma cell lines and primary myeloma cells or HLA-A*0201+ normal lymphocytes, including B cells. These results indicate that these T cells were potent cytotoxic T cells and recognized DKK1 peptides naturally presented by myeloma cells in the context of HLA-A*0201 molecules. Hence, our study identifies DKK1 as a potentially important antigen for immunotherapy in MM.

The effect of surfactin on the proliferation of LoVo cells, a human colon carcinoma cell line, was examined. Surfactin strongly blocked the proliferation of LoVo cells by inducing pro-apoptotic activity and arresting the cell cycle, according to several lines of evidence on DNA fragmentation, Annexin V staining, and altered levels of poly (ADP-ribose) polymerase, caspase-3, p21WAF1/Cip1, p53, CDK2 and cyclin E. The anti-proliferative activity of surfactin was mediated by inhibiting extracellular-related protein kinase and phosphoinositide 3-kinase/Akt activation, as assessed by phosphorylation levels. Therefore, our data suggest that surfactin may have anti-cancer properties as a result of its ability to downregulate the cell cycle and suppress its survival.

Korean mistletoe lectin (KML) is one of the major active components in Viscum album var. (coloratum), displaying various biological effects such as anti-tumor and anti-metastatic activities. Even though it has been shown to boost host immune defense mechanisms, the immunomodulatory effects of KML on specific immune responses mediated by macrophages have not been fully elucidated. Therefore, in this study, we aimed to demonstrate KML's regulatory roles on macrophage-mediated immune responses. KML clearly blocked lipopolysaccharide (LPS)-induced events [expression of interleukin (IL)-10, nitric oxide (NO) production and phagocytic uptake], and suppressed the normal expression levels of IL-10 (at 2 ng/ml) and tumor necrosis factor (TNF)-alpha (at 10 ng/ml). In contrast, (1) the expression of cytokine (TNF-alpha) and (2) the generation of reactive oxygen species (ROS) induced by LPS were significantly up-regulated with KML co-treatment. In addition, KML itself increased the mRNA levels of IL-3 and IL-23; phagocytic uptake; the surface levels of co-stimulatory molecules (CD80 and CD86), pattern recognition receptors (PRRs) [such as dectin-1 and toll like receptor (TLR)-2] and adhesion molecules [beta1-integrins (CD29) and CD43]; and CD29-mediated cell adhesion events. Finally, according to co-treatment of D-galactose with KML under LPS-induced NO production conditions, KML inhibition seems to be mediated by binding to proteins with D-galactose. Therefore, these data suggest that KML may participate in regulating various macrophage-mediated innate and adaptive responses via binding to surface protein with D-galactose and that some of these may deserve in KML's therapeutic activities such as anti-tumor and anti-microbial effects.

Reelin, a large secreted extracellular matrix glycoprotein, plays a key role in neuronal migration during cortical development and promotes neuronal maturation. The signaling pathway regulating neuronal maturation in the postnatal period are relatively less well understood. In this study, we demonstrated that a heterotrimeric G protein, Go, is a novel target of Reelin-induced signaling to promote neurite outgrowth. In primary hippocampal neurons of Reelin-deficient reeler mice, neurite outgrowth was significantly reduced and rescued upon addition of Reelin. Pertussis toxin (PTX) treatment or transfection with Gαo-siRNA suppressed Reelin-mediated neurite outgrowth in wild-type neurons. Additionally, Reelin treatment led to increased phosphorylation of AKT, GSK3β, and JNK, which were all effectively blocked by the PI3K inhibitor, LY294002. By comparison, PTX specifically blocked JNK activation, but not AKT and GSK3β. Immunoprecipitation assays disclosed that Reelin increases the active forms of both Src and Gαo and promotes their direct association. Notably, Dab1, a cytoplasmic adaptor molecule that mediates Reelin signaling, did not interact with Gαo. Neurite outgrowth by Reelin was induced via activating Src kinase, which directly stimulated Gαo, activity, leading to JNK activation. Based on the collective findings, we suggest that Reelin-dependent signaling mechanisms may be split into Src-AKT-dependent and Src-Go-dependent pathways. Our results additionally provide evidence that Reelin receptors cross-communicate with heterologous G protein-coupled receptors (GPCR) independently of the cognate ligands of GPCR.

Curcumin has been shown to exhibit anti-inflammatory, antimutagenic, and anticarcinogenic activities. However, the modulatory effect of curcumin on the functional activation of primary microglial cells, brain mononuclear phagocytes causing the neuronal damage, largely remains unknown. The current study examined whether curcumin influenced NO production in rat primary microglia and investigated its underlying signaling pathways. Curcumin decreased NO production in LPS-stimulated microglial cells in a dose-dependent manner, with an IC(50) value of 3.7 microM. It also suppressed both mRNA and protein levels of inducible nitric oxide synthase (iNOS), indicating that this drug may affect iNOS gene expression process. Indeed, curcumin altered biochemical patterns induced by LPS such as phosphorylation of all mitogen-activated protein kinases (MAPKs), and DNA binding activities of nuclear factor-kappaB (NF-kappaB) and activator protein (AP)-1, assessed by reporter gene assay. By analysis of inhibitory features of specific MAPK inhibitors, a series of signaling cascades including c-Jun N-terminal kinase (JNK), p38 and NF-kappaB was found to play a critical role in curcumin-mediated NO inhibition in microglial cells. The current results suggest that curcumin is a promising agent for the prevention and treatment of both NO and microglial cell-mediated neurodegenerative disorders.

Mesenchymal stem cells are able to trans-differentiate into nonmesodermal lineage cells. Here, we identified downstream signaling molecules required for acquisition of neuron-like traits by mesenchymal stem cells following the elevation of intracellular cAMP levels. We found that forskolin induced neuron-like morphology and expression of neuron-specific enolase and neurofilament-200 in mesenchymal stem cells. Forskolin sequentially activated protein kinase A and B-regulation of alpha-fetoprotein (Raf), which led to phosphorylation of extracellular signal-regulated kinase. Importantly, blockade of extracellular signal-regulated kinase phosphorylation with a mitogen-activated protein kinase (MAPK) kinase inhibitor abrogated the forskolin-induced morphological changes and induction of neuronal proteins. These results indicate that extracellular signal-regulated kinase/MAPK mediates both cAMP-induced early cytoskeletal rearrangement and the later induction of neuronal markers in mesenchymal stem cells.

We have previously reported on the identification of the endogenous transmethylation inhibitor oligosaccharide-linked acyl carrier protein (O-ACP). In this study, the role of the transmethylation reaction on cell cycle progression was evaluated using various transmethylase inhibitors, including O-ACP. O-ACP significantly inhibited the growth of various cancer cell lines, including NIH3T3, ras-transformed NIH3T3, MDA-MB-231, HT-1376, and AGS. In addition, exposure of ras-transformed NIH3T3 to O-ACP caused cell cycle arrest at the G0/G1 phase, which led to a decrease in cells at the S phase, as determined by flow cytometry. In contrast, transmethylase inhibitors did not affect the expression of p21(WAF1/Cip1), a well known inhibitor of cyclin dependent kinase, indicating that the cell cycle arrest by transmethylase inhibitors might be mediated by a p21(WAF1/Cip1)-independent mechanism. Therefore, O-ACP, a novel transmethylase inhibitor, could be a useful tool for elucidating the novel role of methylation in cell proliferation and cell cycle progression.

We previously reported that apicidin, a novel histone deacetylase inhibitor, inhibited the proliferation of tumor cells via induction of p21(WAF1/Cip1). In this study, we determined the molecular mechanisms by which apicidin induced the p21(WAF1/Cip1) gene expression in HeLa cells. Apicidin induced p21(WAF1/Cip1) mRNA independent of the de novo protein synthesis and activated the p21(WAF1/Cip1) promoter through Sp1-3 site located at -82 and -77 relative to the transcription start site. This transcriptional activation appears to be mediated by protein kinase C (PKC), because calphostin C, a PKC inhibitor, significantly attenuated the activation of p21(WAF1/Cip1) promoter via Sp1 sites, which was accompanied by a marked suppression of p21(WAF1/Cip1) mRNA and protein expression induced by apicidin. Consistent with the transcriptional activation of p21(WAF1/Cip1) promoter by apicidin, apicidin treatment led to the translocation of PKCepsilon from cytosolic to particulate fraction, which was reversed by pretreatment with calphostin C, indicating the involvement of PKC in the transcriptional activation of p21(WAF1/Cip1) via Sp1 sites by apicidin. However, the PKC-mediated transcriptional activation of p21(WAF1/Cip1) by apicidin appears to be independent of the histone hyperacetylation, because apicidin-induced histone hyperacetylation was not affected by calphostin C. Furthermore, a PKC activator, phorbol 12,13-dibutyrate, alone induced the transcriptional activation of p21(WAF1/Cip1) promoter, p21(WAF1/Cip1) mRNA, and protein expression without induction of the histone hyperacetylation, suggesting that the transcriptional activation of p21(WAF1/Cip1) by apicidin might have been mediated by a mechanism other than chromatin remodeling through the histone hyperacetylation. Taken together, these results suggest that the PKC signaling pathway plays a pivotal role in the transcriptional activation of the p21(WAF1/Cip1) gene by apicidin.

The effects of two different classes of calmodulin antagonists on the catalytic activities of purified pyruvate dehydrogenase (PDH) phosphatase and PDH complex (PDC) were studied. In general, PDH phosphatase was more strongly inhibited than PDC by the calmodulin antagonists with the following potency order: fluphenazine > chlorpromazine > thioridazine > triflupromazine. Promazine and two sulfonamides (W-5 and W-7) did not suppress PDH phosphatase activity at 1 mM concentrations, while about 20% of PDC activity was inhibited by these antagonists. Fluphenazine-mediated inhibition of PDH phosphatase was observed with the purified PDC as well as intact mitochondria. Although Ca2+ stimulates PDH phosphatase activity, the addition of exogenous Ca2+ did not overcome the inhibition by calmodulin antagonists. These results suggest that the suppression of PDH phosphatase activity is dependent upon the structure of the individual calmodulin antagonist and appears to be Ca(2+)-independent. Kinetic analysis showed a noncompetitive inhibition of PDH phosphatase by fluphenazine, indicating that it binds to different site(s) from the catalytic site of the enzyme.

Caffeic acid (CA) is a phenolic compound that is frequently present in fruits, grains, and dietary supplements. Although CA has been reported to display various biological activities such as anti-inflammatory, anti-cancer, anti-viral, and anti-oxidative effects, the action mechanism of CA is not yet fully elucidated. In this study, the anti-inflammatory action mechanism of CA was examined in lipopolysaccharide (LPS) treated macrophages (RAW264.7 cells) and HCl/EtOH-induced gastritis. CA was found to diminish nitric oxide (NO) and prostaglandin E2(PGE2) production in LPS-stimulated RAW264.7 cells. Additionally, mRNA levels of tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, and inducible NO synthase (iNOS) were downregulated by CA. CA also strongly suppressed the nuclear translocation of AP-1 family proteins and the related upstream signaling cascade composed of interleukin-1 receptor-associated kinase 1 (IRAK1), IRAK4, TGF-β-activated kinase 1 (TAK1), mitogen-activated protein kinase kinase 4/7 (MKK4/7), and c-Jun N-terminal kinase (JNK). In a direct kinase assay, CA was revealed to directly inhibit IRAK1 and IRAK4. CA also ameliorated HCl/EtOH-induced gastric symptoms via the suppression of JNK, IRAK1, and IRAK4. Therefore, our data strongly suggest that CA acts as an anti-inflammatory drug by directly suppressing IRAK1 and IRAK4.

Tumor cell–derived heat shock proteins are used as vaccines for immunotherapy of cancer patients. However, current approaches require the generation of custom-made products and are clinically ineffective. To improve the applicability of heat shock protein–based immunotherapy in cancers and to enhance clinical efficacy, we explored combinational treatments in a myeloma setting using pooled heterogeneous or allogeneic myeloma cell line–derived glycoprotein 96 (gp96) as universal vaccines, and clearly demonstrated that pooled but not single gp96 from heterogeneous or allogeneic myeloma cell lines was as effective as autologous gp96 in protecting mice from tumor challenge and rechallenge and in treating established myeloma. We showed that interferon γ and CD4+ and CD8+ T cells were required for gp96-induced antimyeloma responses and that pooled gp96 induced broader immune responses that protected mice from developing different myeloma. Furthermore, pooled gp96 plus CpG in combination with anti-B7H1 or anti–interleukin-10 monoclonal antibodies were effective in treating mice with large tumor burdens. Thus, this study strongly suggests that pooled gp96 vaccines from myeloma cell lines can replace gp96 vaccines from autologous tumors for immunotherapy and induce immune responses against broader tumor antigens that may protect against tumor recurrence and development of unrelated tumors in vaccinated myeloma patients.

We have analyzed the importance of substrate methylation by S-adenosylmethionine-dependent methyltransferases for neuronal differentiation of P19 embryonal carcinoma cells. We show that treatment of cells with methyltransferase inhibitor adenosine dialdehyde (AdOx) interferes with neuronal differentiation. Retinoic acid (RA) and AdOx co-treated cells had a decreased number of neurites and a flattened morphology compared with cells differentiated by RA. Also, the amount of neuronal class III tubulin (Tuj1) decreased from 76% to 9.6% with AdOx-treatment. Gene expression levels of wnt-1, brn-2, neuroD, and mash-1 were also down-regulated by AdOx-treatment. But AdOx-treatment did not up-regulate BMP-4 and GFAP genes. Treatment of RA decreased E-cadherin expression during neuronal differentiation. However, in AdOx/RA co-treated cells, E-cadherin expression was restored to the control level. Also, mRNA expression of N-cadherin decreased with AdOx-treatment. Taken together, these data show that methylation reactions might influence the cell-fate decision and neuronal differentiation of P19 cells.

Atiprimod is a novel cationic amphiphilic compound and has been shown to exert antimyeloma effects both in vitro and in mouse experiments. This study was undertaken to evaluate the therapeutic efficacy of atiprimod on mantle cell lymphoma (MCL) and elucidate the mechanism by which it induces cell apoptosis. Atiprimod inhibited the growth and induced apoptosis of MCL cell lines and freshly isolated primary tumor cells in vitro. More importantly, atiprimod significantly inhibited tumor growth in vivo and prolonged the survival of tumor-bearing mice. However, atiprimod also exhibited lower cytotoxicity toward normal lymphocytes. Atiprimod activated c-Jun N-terminal protein kinases (JNK) and up-regulated the level of Bax, Bad, and phosphorylated Bcl-2, resulting in release of apoptosis-inducing factor (AIF) and cytochrome c from mitochondria and activation and cleavage of caspase-9, caspase-3, and PARP. However, AIF, but not activation of caspases or PARP, was responsible for apoptosis in MCL cells because an AIF inhibitor, but not pan-caspase or paspase-9 inhibitors, completely abrogated atiprimod-induced apoptosis. Taken together, our results demonstrate that atiprimod displays a strong anti-MCL activity. Cell apoptosis was induced mainly via activation of the AIF pathway. These results support the use of atiprimod as a potential agent in MCL chemotherapy.

Previous studies demonstrated that circulating dendritic cells (DCs) in myeloma patients were functionally abnormal. However, the phenotype and function of patients' monocyte-derived DCs (MoDCs), which are commonly used for immunotherapy, were poorly defined. This study was undertaken to examine the quality of MoDCs from myeloma patients compared with cells from healthy donors. We found that patient-derived MoDCs are phenotypically and functionally defective. Compared with their normal counterparts, patient-derived, mature MoDCs expressed significantly lower levels of CD1a, CD40, CD80, and HLA-DR and were poor at activating alloreactive T cells, presenting recall antigen, and activating autologous antigen- and myeloma-specific T cells. These abnormalities may be attributed to elevated production of autocrine cytokines such as IL-6, activated p38 and STAT3, and inhibited MEK/ERK signaling pathways in the progenitor cells. Treatment with neutralizing IL-6–specific antibody and, more importantly, p38 inhibitor, or both, could correct these abnormalities. Treating patient-derived cells with these agents not only significantly increased cell yield but also produced MoDCs that were as functional as their normal counterparts. Thus, this study has delineated the mechanistic defects of MoDCs from myeloma patients and identified ways for restoring the function of the cells to improve the efficacy of DC-based immunotherapy in this disease.

Apicidin has been identified as a histone deacetylase (HDAC) inhibitor. Since HDAC inhibitors are emerging as an exciting new class of potential anti-cancer agents, in the present study, we have examined the inhibitory effect of apicidin on cancer invasion and angiogenesis. Apicidin induced di- and tri-acetylated forms of histone H4 and the morphological alteration in v-ras-transformed mouse fibroblast NIH3T3 cells. Apicidin dramatically inhibited the invasion of v-ras-NIH3T3 and human melanoma A2058 cells and it could be associated with its ability to regulate the activities of matrix metalloproteinases. Interestingly, apicidin strongly inhibited the formation of new vessels on chorioallantoic membrane and the tube formation of ECV304 human vascular endothelial cells. This is the first report to show the anti-angiogenic potential of apicidin and it could be developed as a new type of anti-cancer drug.

(3 S,14 S)-Petrocortyne A, a lipid compound (a C(46) polyacetylenic alcohol), from marine sponges ( Petrosia sp.) is potently cytotoxic against several solid tumour cells. In this study, we investigated in vitro anti-inflammatory and pro-aggregative effects of petrocortyne A at non-cytotoxic concentrations on various cellular inflammatory phenomena using the macrophage and monocytic cell lines RAW264.7 and U937. Petrocortyne A blocked tumour necrosis factor-alpha (TNF-alpha) production strongly and concentration-dependently in lipopolysaccharide (LPS)-activated RAW264.7 cells and phorbol 12-myristate 13-acetate (PMA)/LPS-treated U937 cells. It also blocked NO production concentration-dependently in LPS- or interferon (IFN)-gamma-treated RAW264.7 cells. Among the migration factors tested, the compound selectively blocked the expression of hepatocyte growth factor/scatter factor (HGF/SF). On the other hand, as assessed by a cell-cell adhesion assay, petrocortyne A did not block the activation of adhesion molecules induced by aggregative antibodies to adhesion molecules, but suppressed PMA-induced cell-cell adhesion significantly. Intriguingly, petrocortyne A induced U937 homotypic aggregation following long exposure (2 and 3 days), accompanied by weak induction of pro-aggregative signals such as tyrosine phosphorylation of p132 and phosphorylation of extracellular signal-related kinase 1 and 2 (ERK 1/2). Petrocortyne A may thus inhibit cellular inflammatory processes and immune cell migration to inflamed tissue.

We previously reported that apicidin arrested human cancer cell growth through selective induction of p21(WAF1/Cip1). In this study, the apoptotic potential of apicidin and its mechanism in HL60 cells was investigated. Treatment of HL60 cells with apicidin caused a decrease in viable cell number in a dose-dependent manner and an increase in DNA fragmentation, nuclear morphological change, and apoptotic body formation, concomitant with progressive accumulation of hyperacetylated histone H4. In addition, apicidin converted the procaspase-3 form to catalytically active effector protease, resulting in subsequent cleavages of poly(ADP-ribose) polymerase and p21(WAF1/Cip1). Incubation of HL60 cells with z-DEVD-fmk, a caspase-3 inhibitor, almost completely abrogated apicidin-induced activation of caspase-3, DNA fragmentation, and cleavages of poly(ADP-ribose) polymerase and p21(WAF1/Cip1). Moreover, these effects were preceded by an increase in translocation of Bax into the mitochondria, resulting in the release of cytochrome c and cleavage of procaspase-9. The addition of cycloheximide greatly inhibited activation of caspase-3 by apicidin by interfering with cleavage of procaspase-3 and DNA fragmentation, suggesting that apicidin-induced apoptosis was dependent on de novo protein synthesis. Consistent with these results, apicidin transiently increased the expressions of both Fas and Fas ligand. Preincubation with NOK-1 monoclonal antibody, which prevents the Fas-Fas ligand interaction and is inhibitory to Fas signaling, interfered with apicidin-induced translocation of Bax, cytochrome c release, cleavage of procaspase-3, and DNA fragmentation. Taken together, the results suggest that apicidin might induce apoptosis through selective induction of Fas/Fas ligand, resulting in the release of cytochrome c from the mitochondria to the cytosol and subsequent activation of caspase-9 and caspase-3.

We investigated a possible role of reactive oxygen species (ROS) in p70(S6k) activation, which plays an important role in the progression of cells from G(0)/G(1) to S phase of the cell cycle by translational up-regulation of a family of mRNA transcripts that encode for components of the protein synthetic machinery. Treatment of mouse epidermal cell JB6 with H(2)O(2) generated extracellularly by glucose/glucose oxidase led to the activation of p70(S6k) and p90(Rsk) and to phosphorylation of p42(MAPK)/p44(MAPK). The activation of p70(S6k) and p90(Rsk) was dose-dependent and transient, maximal activities being in extracts treated for 15 and 30 min, respectively. Further characterization of ROS-induced activation of p70(S6k) using specific inhibitors for p70(S6k) signaling pathway, rapamycin, and wortmannin revealed that ROS acted upstream of the rapamycin-sensitive component FRAP/RAFT and wortmannin-sensitive component phosphatidylinositol 3-kinase, because both inhibitors caused the inhibition of ROS-induced p70(S6k) activity. In addition, Ca(2+) chelation also inhibited ROS-induced activation of p70(S6k), indicating that Ca(2+) is a mediator of p70(S6k) activation by ROS. However, down-regulation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive protein kinase C (PKC) by chronic pretreatment with TPA or a specific PKC inhibitor Ro-31-8220 did not block the activation of p70(S6k) by ROS, indicating that the activation of TPA-responsive PKC was not required for stimulation of p70(S6k) activity by H(2)O(2) in JB6 cells. Exposure of JB6 cells to platelet-derived growth factor or epidermal growth factor led to a rapid increase in H(2)O(2), phosphorylation, and activation of p70(S6k), which were antagonized by the pretreatment of catalase. Taken together, the results suggest that ROS act as a messenger in growth factor-induced p70(S6k) signaling pathway.

The SNF2/SWI2 ATPase/helicase family comprises proteins from a variety of species, which serve a number of functions, such as transcriptional regulation, maintenance of chromosome stability during mitosis, and various types of DNA repair. Several proteins with unknown functions are also included in this family. The number of genes that belong to this family is rapidly expanding, which makes it easier to analyze the common biological functions of the family members. This study was designed to clone the SNF2/SWI2 helicase-related genes from the fission yeast Schizosaccharomyces pombe in the hope that this would help to elucidate the common functions of the proteins in this family. The hrp1+ (helicase-related gene from S. pombe) gene was initially cloned by PCR amplification using degenerate primers based on conserved SNF2 motifs within the ERCC6 gene, which encodes a protein involved in DNA excision repair. The hrp1+ ORF codes for an 1373-amino acid polypeptide with a molecular mass of 159 kDa. Like other SNF2/SWI2 family proteins, the deduced amino acid sequence of Hrp1 contains DNA-dependent ATPase/7 helicase domains, as well as a chromodomain and a DNA-binding domain. This configuration is similar to that of mCHD1 (mouse chromo-ATPase/helicase-DNA-binding protein 1), suggesting that Hrp1 is a S. pombe homolog of mCHD1, which is thought to function in altering the chromatin structure to facilitate gene expression. Northern blot analysis showed that the hrp1+ gene produces a 4.6-kb transcript, which reaches its maximal level just before the cells enter the exponential growth phase, and then decreases gradually. DNA-damaging agents, such as MMS, MNNG and UV, decrease the rate of transcription of hrp1+. Deletion of the hrp1+ gene resulted in accelerated cell growth. On the other hand, overexpression of Hrp1 caused a reduction in growth rate. These results indicate that hrp1+ may act as a negative regulator of cellular growth.

The presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P was established by western blot analysis. The identity of citrulline formed from L-arginine by the NOS was confirmed by both TLC and HPLC and the other product, NO, by directly measuring the production of nitrogen oxides (NOx) in the reaction mixture. The activity was inhibited by typical NOS inhibitors such as N-nitro-L-arginine methylester and NG,NG-dimethyl-L-arginine with the IC50 of 4.9 x 10(-4) and 2.5 x 10(-4) M, respectively. The NOS activity was maximum at pH 6.5 and at 47.5 degrees C. These results indicate that the NOS of S. aureus ATCC6538P is an isoform distinct from mammalian NOS.

Histone acetylation is linked to the control of chromatin remodeling, which is involved in cell growth, proliferation, and differentiation. It is not fully understood whether cyclic adenosine monophosphate (cAMP), a representative differentiation-inducing molecule, is able to modulate histone acetylation as part of its anticancer activity. In the present study, we aimed to address this issue using cell-permeable cAMP, i.e. dibutyryl cAMP (dbcAMP) and C6 glioma cells. As reported previously, under the conditions of our studies, treatment with dbcAMP clearly arrested C6 cell proliferation and altered their morphology. Its antiproliferative and differentiation-inducing activity in C6 glioma cells involved upregulation of p21WAF/CIP, p27kip1, glial fibrillary acidic protein (GFAP), and Cx43, as well as downregulation of vimentin. Furthermore, dbcAMP modulated the phosphorylation of ERK and Akt in a time-dependent manner and altered the colocalization pattern of phospho-Src and the actin cytoskeleton. Interestingly, dbcAMP upregulated the enzyme activity of histone acetyltransferase (HAT) and, in parallel, enhanced cellular acetyllysine levels. Finally, the hyperacetylation- inducing compound, sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor, displayed similar anticancer activity to dbcAMP. Therefore, our data suggest that antiproliferative and differentiation-inducing activities of dbcAMP may be generated by its enhanced hyperacetylation function

Abstract 2941 Current approaches for vaccination and immunotherapy are often capable of eliciting strong T-cell responses against tumor antigens. However, negative regulatory mechanisms within the tumor microenvironment inhibit antitumor T-cell function, leading to evasion from immune attack. One inhibitory mechanism is upregulation of programmed death-ligand 1 (PD-L1/B7H1) expressed on tumor or stromal cells, which binds to programmed death-1 (PD-1) on activated T cells. PD-1 and B7H1 engagement results in diminished antitumor T-cell responses and correlates with poor outcome in murine and human cancers. To cure mice with large tumor burdens by administering tumor vaccines, we hypothesized that it is necessary not only to enhance the immunogenicity of the vaccines, but also to target the suppressive tumor microenvironment and break immune suppression on effector cells. Therefore, we explored combinational treatments in a myeloma setting by using vaccination and anti-B7H1 blocking mAbs to enhance clinical efficacy of cancer vaccines. To establish mouse tumor models for evaluation of tumor-associated B7H1 in the inhibition of T-cell immunity, first, we examined the expression of B7H1 in four murine myeloma cell lines that originated from Balb/c mice. Results showed that all the myeloma cells expressed a negative T-cell costimulatory molecule B7H1, and blocking surface B7H1 by using a specific mAb (M5H1) enabled tumor-specific CTLs from vaccinated mice to be more efficient at lysing the tumor cells. Thus these results suggest that B7H1 on tumor cells and PD-1 on T cells may form a B7H1/PD-1 molecular shield to prevent lysis by T cells and actively inhibit the cytolytic function of tumor-specific CTLs by expressing B7H1. Next, we evaluated the therapeutic efficacy of DKK1-DNA vaccine in mice with established myeloma. In these experiments, DKK1-DNA vaccine plus CpG was used as a standard vaccine. Anti-B7H1 (M5H1) mAb was used to block negative T-cell signaling in mice receiving DKK1-DNA vaccination. Balb/c mice were first inoculated subcutaneously with the myeloma cells (1 million cells per mouse). Ten days after tumor inoculation, mice were immunized with three injections of the vaccines on days 10, 14, and 17 after tumor inoculation. Mice were also injected intraperitoneally with B7H1 (200 μg per mouse per treatment) on days 1, 4, 7, and 10 following the first vaccination. Mice receiving injections of PBS, isotype IgG (200 μg per mouse per treatment), or CpG- (50 μg per mouse per treatment) alone were used as controls. Results show that mice receiving DKK1-DNA vaccine (plus CpG) together with B7H1 induced more robust tumor growth inhibition (P < 0.01, compared with PBS control mice). The survival of DKK1-CpG vaccine- and vaccine plus B7H1 antibody-treated mice were 60% and 80%, respectively. Thus, our data showed that B7H1 blocking mAbs can further enhance the therapeutic efficacy of the vaccine. Finally, we investigated the mechanism of therapeutic efficacy of vaccine enhanced by anti-B7H1 mAbs. Our results showed that the percentages of IL-10-secreting or Foxp3+ Tregs in the spleens and tumors are significantly decreased in mice vaccinated with DKK1 DNA plus CpG in combination with anti-B7H1 antibodies (P < 0.01, compared with mice receiving DKK1 DNA alone or vaccine plus CpG). These results indicate that myeloma cells are able to induce or recruit regulatory T cells, and the beneficial effects of anti-B7H1 mAbs were derived from their ability to reduce the numbers of Tregs in tumor-bearing mice. Taken together, these results support the concept that B7H1/PD-1 forms a molecular shield to prevent destruction by CTLs and implicate new approaches for immunotherapy of human cancers by B7H1 blocking mAbs. Disclosures: No relevant conflicts of interest to declare.

Abstract 2643 Mantle cell lymphoma (MCL) is a unique subtype of incurable B-cell, non-Hodgkin lymphoma, and its overall survival currently remains only 4–5 years. Management of relapsed or refractory MCL patients is still challenging. Immunotherapy may provide an alternative treatment for patients with MCL. Recent studies demonstrated that PD-1/B7-H1 signaling plays a crucial role in T-cell regulation in various immune responses and is involved in peripheral tolerance, autoimmunity, infection, and antitumor immunity. In the present study, we examined whether B7-H1 plays an important role in immune evasion in MCL. We demonstrated that B7-H1 gene and protein were expressed in most MCL cell lines and primary MCL cells from all patients examined. CD3+ T cells were cultured with irradiated MCL cell lines and primary cells, which were pre-incubated with or without anti-B7-H1 monoclonal antibody or control antibody. The presence of anti-B7-H1 blocking antibody, but not control antibody, increased CD3+ T cell proliferation. We confirmed the effect of B7-H1 in suppression of T cell proliferation by knockdown of B7-H1 gene expression using B7-H1 specific and non-specific control shRNA lentiviral particles. Upon transfection, the B7-H1-specific shRNA reduced both B7-H1 gene and surface protein expression, while the control shRNA did not. The B7-H1 specific shRNA, but not control shRNA, augmented CD3+ T cell proliferation. To address whether B7-H1 contributed to the suppression of host antitumor immunity in MCL, allogeneic CD3+ T cells isolated from normal donors were cocultured with irradiated MCL cell line SP53, control shRNA SP53 (SP53-ctl), or B7-H1 targeted shRNA SP53 (SP53-kd), respectively. After 7 days of coculture, CD3+ T cells were harvested and restimulated with newly irradiated SP53, SP53-ctl or SP53-kd cells. After at least 3 repeated cycles of in vitro restimulation, three cytotoxic T lymphocyte (CTL) lines were generated, and named CTL-SP53, CTL-SP53-ctl and CTL-SP53-kd. CTL-SP53-kd showed increased killing of target cells as compared with CTL-SP53 (P Disclosures: No relevant conflicts of interest to declare.

Abstract 694 Osteoclasts (OCs), the responsible cells for bone resorption, are derived from monocytic precursor cells, with the stimulation of macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor κb ligand (RANKL). The formation and activity of OCs can be either promoted by activated T cell derived RANKL, IL-17, or suppressed by T cell derived IFN-γ, IL-10, and IL-4. On the other hand, OCs express MHC, and costimulatory molecules, secrete IL-10, TGF-β, TNF-α and IL-6 and can act as antigen presenting cells to activate T cells, which indicates that OCs can be considered as immune cells. However, the immune function of OCs is largely unknown, and whether activated T cells can regulate the immune function of OCs is also unclear. In this study, we investigated the effect of OCs on T cell responses, and the cross regulation between activated T cells and immune regulatory OCs. Results showed that autologous OCs could inhibit the proliferation of CD4+ T cells activated by allogeneic antigen, tetanus toxin, staphylococcal enterotoxin B, and anti-CD3/CD28 antibodies. The inhibitory rate range varied from 63% to 88%. To identify the mechanism of OC-mediated T cell suppression, we blocked or inhibited TGF-β, IL-10, (prostaglandin E2) PGE-2, and indoleamine 2,3-dioxygenase (IDO) with neutralizing antibodies or specific inhibitors during the coculture. Results showed that only 1-methyl-DL-tryptophan (1-MT, an IDO inhibitor) could rescue the T cell proliferation, which suggested that OCs mediated the T cell suppression through IDO. To confirm this result, we knocked down IDO expression in OCs with siRNA and found that T cell proliferation was restored completely. As normal OCs didn't express IDO, next we investigated which molecules induced IDO expression in OCs, when cocultured with activated T cells. Results showed that blocking IFN-γ and CD40 ligand (CD40L) could inhibit IDO expression in OCs and rescue the T cell proliferation, and recombinant IFN-γ and soluble CD40L could induce IDO expression in OCs, synergistically. In conclusion, our study identified that OCs can function as immune regulatory cells to suppress T cell proliferation through IDO, which is induced by activated T cell derived IFN-γ and CD40L. This study provides new insight into the reciprocal interaction between OCs and T cells and may be helpful to develop novel therapeutic strategies for diseases involved in both bone and immune systems, such as bone-invasive tumors and autoimmune arthritis. Disclosures: No relevant conflicts of interest to declare.

Abstract 3040 Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by the tumor cells of almost all patients with multiple myeloma (MM) and may be responsible for suppressed osteoblast formation. In our previous studies, we demonstrated that DKK-1 is a potent tumor-associated antigen in MM recognized by cytotoxic T lymphocytes (CTLs), which can effectively lyse autologous myeloma tumor cells in vitro (Qian et al., Blood 2007;110:1587-1594) and eradicate established patient-derived primary myeloma in SCID-hu mice upon adoptive transfer. To examine the potential of DKK1-based immunotherapy in MM, we investigated the efficacy of active vaccination with (murine) DKK1 DNA vaccine in a murine (MOPC-21) myeloma model. A plasmid DNA construct encoding defensin2-sFv was generated, and DKK1 full-length cDNA was cloned from DKK1-expressing mouse stromal cells by reverse transcription–polymerase chain reaction and genetically fused with defensin2 (DNA-vac). We first examined the ability of the vaccines to protect mice from developing myeloma. While 100% of mice vaccinated with vector control or PBS (10 mice for each group) developed tumors, 70% and 40% mice vaccinated with DNA-vac or DNA-vac with CpG (ODN 1826), respectively, developed tumors. These results clearly show that active vaccination with DKK1 DNA vaccines was able to protect mice from tumor challenge, and that combination with CpG was more effective than DNA vaccine alone. On day 90 after tumor challenge, all surviving tumor-free mice were rechallenged with the same myeloma cells and followed for tumor development. By day 180, All surviving mice that were vaccinated with DNA-vac or DNA-vac with CpG have no developed tumors, indicating that active vaccination with DNA vaccines induced strong tumor-specific memory immune responses to protect mice from tumor rechallenge. Next, we examined the therapeutic effects of DNA vaccines in our myeloma mouse model. DNA vaccine alone eradicated established myeloma in 1 out of 5 mice, while DNA vaccine plus CpG eradicated myeloma in 3 out of 5 mice bearing intermediate tumors (≥ 5 mm in diameter). These results indicate that DNA vaccine plus CpG was much more effective at eradicating established myeloma than DNA vaccine alone. Finally, the mechanisms of tumor protection induced by DNA vaccines were investigated. By depleting CD4+ or CD8+ T cells, we showed that CD8+ T cells are required for DNA-induced antimyeloma responses. In DNA-vaccinated mice, splenocytes contained increased numbers of DKK1-specific, IFN-g-secreting and proliferative T cells. The splenic CD8+ T cells exhibited enhanced cytotoxicity against myeloma cells. Furthermore, DKK1-specific CD8+ T cells were shown to be increased in DKK1-DNA vaccinated mice by using DKK1-peptide (P11, P15 and P210) tetramer staining. These results demonstrate the presence of myeloma-specific CTLs in vaccinated mice and show that DKK1 DNA vaccine can induce a potent CTL response capable of killing myeloma cells. Together, our study lays a basis for future clinical trials in MM by using DKK1 as a vaccine for all patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 740 Bone destruction is common in osteolytic tumors such as multiple myeloma. Myeloma cells that reside in the bone cause osteolysis, which is characterized by severe bone pain, multiple fractures, and hypercalcemia in patients and thereby, compromises patient quality of life. While myeloma cells have been proposed to activate osteoclasts and/or inhibit osteoblasts leading to bone resorption, the mechanism underlying myeloma-induced osteolytic bone lesions is still poorly elucidated. As p38 mitogen-activated protein kinase (MAPK) is constitutively activated in myeloma, we hypothesized that p38 MAPK activation in myeloma cells might be responsible for myeloma-induced osteolytic bone lesions. Human myeloma cell lines ARP-1 and MM.1S caused bone lesions in SCID mice after intravenous injection. However, once p38 MAPK was knocked down by small hairpin RNAs (shRNAs) in these cell lines, myeloma was established but failed to cause bone lesions in SCID mice, whereas the wild-type or vector control myeloma cells established myeloma (with similar tumor burdens as myeloma cells with knocked-down p38 MAPK) and caused bone lesions in tibial bones, as measured by radiograph and peripheral quantitative m-computed tomography and further confirmed by histological examination. In vivo examination revealed that the number of bone surface-covering osteoclasts, osteoclast size, cellular nuclear numbers in per osteoclast, and levels of circulating collagen type I (bone resorption marker) and TRAP5b (active osteoclast marker) were all reduced in mice injected with myeloma cells with knocked-down p38 MAPK as compared with controls. Consistently, we found that tumor p38 MAPK had active effects on in vitro osteoclast differentiation and bone resorption, and inhibited osteoblast differentiation and function, suggesting that constitutive activation of p38 MAPK in myeloma cells induces osteoclastogenesis. By functional studies and protein array analysis, we showed that monocyte chemotactic protein-1 (MCP-1) and dickkopf-1 (DKK-1) are upregulated downstream of p38 MAPK activation in myeloma cells and are responsible for enhanced osteoclastogenesis. Our results showed that p38 MAPK activation in myeloma cells upregulate MCP-1 and DKK-1 expression and production, which are released into the microenvironment. MCP-1 enhanced RANK expression on osteoclast precursors and DKK-1 increased RANKL secretion from stromal cells, all of which led to activation of NF-kB and MAPK signaling pathways in osteoclasts. In vivo blockade of MCP-1 and DKK-1 by specific antibodies significantly abrogated tumor p38 MAPK-induced osteoclast activation and bone lesions in established myeloma-SCID mice. Thus, our results have elucidated a novel mechanism that p38 MAPK activity in myeloma cells contributes to myeloma-induced osteolytic bone lesions. This study indicates that disruption of tumor p38 MAPK may be a new therapeutic approach to treat osteolytic bone lesions in myeloma. Disclosures: No relevant conflicts of interest to declare.

Abstract 4804 Multiple myeloma is a B-cell malignancy characterized by the proliferation of plasma cells in the bone marrow. It is the second most common hematological malignancy and is still largely incurable. One of the major problems is that myeloma cells develop drug resistance upon interaction with bone marrow stromal cells. To better understand the importance of different stromal cell components in the bone marrow microenvironment, we examined the effects of macrophages on myeloma cell survival and myeloma cell response to chemotherapy. We report here that macrophages, in particular tumor-associated macrophages obtained by culturing macrophages with myeloma cell culture supernatants, are a protector of myeloma cells. Macrophages protected both myeloma cell lines and primary myeloma cells isolated from patients from spontaneous and chemotherapy drug-induced apoptosis via attenuating the activation of caspase-dependent apoptotic signaling. The protective effect was dependent on direct contact between macrophages and myeloma cells. Although tumor-associated macrophages secreted large amounts of IL-6, which is the most important survival factor for myeloma cells, our results showed that IL-6 neutralizing antibodies fail to significantly affect the protective effects of tumor-associated macrophages. The reduced numbers of apoptotic tumor cells in the cocultures were not the result of macrophage-uptake of apoptotic cells, because macrophages with or without the ability to phagocytose apoptotic cells provide similar protection to myeloma cells against chemotherapy-induced apoptosis. These findings are clinically relevant, because we examined bone marrow biopsies of patients by immunochemical analysis and found that CD68+ macrophages are heavily infiltrated in the bone marrow (tumor bed) of patients with myeloma but not control patients. Thus, our results indicate that macrophages are an important component of the bone marrow stromal cells and may contribute to myeloma cell survival and resistance to chemotherapeutic treatment in vivo. Disclosures: No relevant conflicts of interest to declare.

Abstract 465 Immunotherapy may complement the current treatments for lymphomas. The lack of suitable shared lymphoma-associated antigens limits its applicability. Therefore, identification and utilization of novel and more potent tumor-associated antigens, particularly those shared among patients, are urgently needed to improve the efficacy of immunotherapy in the diseases. Recent studies have shown that Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by myeloma and other tumor cells, and is absent from normal tissues and organs except placenta and prostate. In the present study we demonstrated that DKK1 is also overexpressed in mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Using DKK1 peptide-pulsed dendritic cells (DCs), we successfully generated HLA-A*0201+ DKK1-specific CTL lines and clones in vitro. These CTLs effectively lysed DKK1+/HLA-A*0201+ lymphoma cell lines Jeko-1 and Granta 519 cells, but not DKK1-/HLA-A*0201+ BJAB, RL and Mino cells nor DKK1+/HLA-A*020- CA46 and Daudi cells. Furthermore, the T-cell clones efficiently killed DKK1+/HLA-A*0201+ primary B-cell lymphoma cells from patients but not lymphoma cells from DKK1–/HLA-A*0201+ patients. HLA-ABC or HLA-A*0201 blocking mAbs significantly inhibited T cell-mediated cytotoxicity against peptide-pulsed T2 cells (P < .01, compared with medium control). No inhibitory effect was observed with mAb against HLA-DR and isotype control IgG. The results indicate that the cytotoxicity was attributed to MHC class I and more specifically, HLA-A*0201-restricted CD8+ CTLs. The CTLs did not kill DKK1–/HLA-A*0201+ DCs, B cells, or PBMCs, These results suggest that the CTLs recognized DKK1 peptides that are naturally processed and presented in the context of HLA-A*0201 molecules on lymphoma cells. To determine the in vivo antitumor activity, NOD-SCID and SCID-hu mice were used for lymphoma cell lines and primary lymphoma cells, respectively. Mice were treated with DKK1-specific CTLs after tumor established in NOD-SCID and SCID-hu mice. Control mice were treated with naïve CD8+ T cells or PBS alone. Tumor burden was measured according to levels of circulating human B2M, and survival rates were determined. Low levels (< 50 ng/ml) of circulating human B2M were detected in group treated DKK1-specific CTLs, while high levels (≥ 150 ng/ml) of circulating human B2M were detected in control mice. In SCID-hu model, X-ray examination showed that established tumors were eradicated in 60% mice treated with DKK1-specific CTLs, while large tumor burdens were found in all control mice. In NOD-SCID model, 40% of mice survived with the treatment of DKK1-specific CTLs. TUNEL assay further confirmed that tumor cells were lysed by DKK1-specific CTLs not naïve CD8+ T cells. These results indicate that DKK1-specific CTLs are able to eradicate established, patient-derived primary B- cell lymphoma in the hosts and adoptive transfer of DKK1-specific CTLs may be used for B-cell lymphoma therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 918 Bone is a dynamic tissue that is constantly being remodeled by bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OB). Bone destruction, observed in patients with autoimmune disease rheumatoid arthritis and malignancies such as multiple myeloma, is believed to be caused by hyperactivation of OCs. OCs differentiate from hematopoietic monocytic precursors under stimulation by the cytokines RANKL (receptor activator of nuclear factor κB ligand) and M-CSF (macrophage colony-stimulating factor), which are produced primarily by bone marrow stromal cells, OBs, and activated T cells. As OCs are derived from monocytes/macrophages lineage similar to dendritic cells (DCs), we hypothesized that OCs could serve as antigen-presenting cells (APCs) to activate T cells. In this study, OCs were generated from human monocytes with the stimulation of RANKL and M-CSF in vitro. DCs derived from monocytes with the stimulation of GM-CSF and IL-4 were used as positive controls. First, we examined the phenotype of OCs by flow cytometry, and OCs were detached by non-enzymatic cell dissociation solution. Results showed that OCs expressed MHC class I and II molecules and costimulatory molecules important for APCs such as CD80, CD86 and CD40. The expression of these molecules could be upregulated by LPS and IFN-γ. Second, we showed by PCR that OCs expressed IL-10, TGF-β, IL-6 and TNF-α, but not IL-12p35 and p40. Third, we examined the ability of OCs to present alloantigens and activate alloreactive T cells in a mixed lymphocyte reaction assay. OCs could present allogeneic antigens and activate both CD4+ and CD8+ alloreactive T cells to proliferate as detected by [3H]thymidine incorporation and CFSE dilution assay. The activation was restricted by MHC class I and II molecules. Finally, we recruited tetanus toxoid (TT)-immunized healthy donors to test whether OCs could uptake exogenous soluble antigens and present them to CD4+ T cells. OCs pulsed with TT could activate autologous specific CD4+ T cells, which was MHC II molecule restricted. These findings indicate that OCs could function as APCs and activate both CD4+ and CD8+ T cells. Thus, our study provides new insight to the effect of OCs on the immune system especially T cells. This is not only important for a better understanding of the crosstalk between the bone and immune system but also may help develop novel strategies for treating diseases such as rheumatoid arthritis and multiple myeloma, which affect both the bone and immune systems. Disclosures: No relevant conflicts of interest to declare.

Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by the tumor cells of most multiple myeloma (MM) patients and contributes to osteolytic bone disease by inhibiting the differentiation of osteoblasts. DKK1 has been targeted as a myeloma-associated antigen by immunotherapeutical approaches based on DKK1-directed monoclonal antibodies and DKK1-specific cytolytic T lymphocytes (CTLs). The present study was conducted to develop a treatment strategy through adoptive transfer of DKK1-specific CTLs in a SCID-hu system. Using DKK1 peptide-pulsed dendritic cells (DCs), we successfully generated DKK1-specific CTL lines and clones from HLA-A2+ MM patients in vitro. These CTLs not only had cytolytic activity against DKK1 peptide-pulsed T2 cells, but also significantly lysed autologous myeloma tumor cells, HLA-A2+ and DKK1+ MM cell lines U266 and IM-9, and primary tumor cells in vitro. No killing was observed against HLA-A2+ normal lymphocytes, including B cells and HLA-A2+ DKK1- XG1 or HLA-A2- myeloma cell lines or primary myeloma cells from patients. To determine the in vivo antitumor activity, SCID-hu mice were engrafted with primary MM cells expressing high levels of DKK1 from patients and treated with DKK1-specific CTLs after one month of tumor injection. Control mice were treated with naïve CD8+ T cells or PBS alone. All mice were examined by X-ray, tumor burden was measured according to levels of circulating human IgG, and survival rates were determined. All mice established human myeloma within one month after tumor inoculation, at which time the level of circulating human IgG was greater than 600 ng/ml in mouse serum. After another 3 weeks, the level of circulating human IgG was greater than 2,500 ng/ml in the serum of all control mice, while only 40% mice treated with DKK1-specific CTLs had high levels of circulating human IgG. X-ray examination showed that osteolytic bone disease and a large tumor burden were found in all control mice, while the established tumor was eradicated in 60% mice treated with DKK1-specific CTLs. Results of immunofluorescence studies showed that only tumor-reactive DKK1-specific CD8+ T cells were able to infiltrate into the tumor and mediate apoptosis in tumor cells. All control mice succumbed to myeloma within one month of establishment of the myeloma, while 9 out of 15 mice treated with DKK1-specific CTLs were macroscopically disease-free at the end of the experiment. These results suggest that DKK1-specific CTLs are able to eradicate established patient-derived primary myeloma in the host and that adoptive transfer of DKK1-specific CTLs may be used for myeloma therapy.

Despite advances in therapy for multiple myeloma (MM), it remains an incurable malignancy. Therefore, immunotherapy may be an option for new treatments to stabilize or even eradicate minimal residual tumors achieved after high-dose chemotherapy supported by autologous stem cell transplantations. Idiotype protein (Id), secreted by myeloma cells, is a tumor-specific antigen, and Id-based immunotherapy has been explored in patients with MM and other B-cell tumors for inducing and/or enhancing Id-specific immune responses that could control the tumor cells. Although previous studies have demonstrated that Id-specific CD8+ cytotoxic T lymphocytes (CTLs) are able to lyse myeloma cells, it is unclear whether other types of Id-specific T cells, such as CD4+ type-1 (Th1) and type-2 helper (Th2) cells, are also able to suppress or kill myeloma tumor cells. As Id-based immunotherapy may induce the responses by all of these T-cell subsets, it is important to understand the functions of these T cells in the context of tumor cells. In this study, we used the 5TGM1 myeloma murine model to generate Id-specific T-cell subsets. Mice were immunized with mature dendritic cells pulsed with Id conjugated with KLH. Using limiting dilution assay, we obtained and identified T-cell clones of different subsets based on surface expression of CD4+ or CD8+ molecules and secretion of IFN-γ or IL-4 in response to Id protein. CTL, Th1, and Th2 T-cell clones were selected, expanded, and subjected to functional tests. The standard 51Cr release and Annexin-V-binding assays were used to examine the cytotoxic activity of these T cells. CTL and Th1 but not Th2 cells specifically lysed 5TGM1 myeloma cells and Id-pulsed but not unpulsed DCs. We examined the suppressive activity of the T-cell clones against myeloma cells by measuring 3H-thymidine incorporation and Id protein secretion. 5TGM1 myeloma cells cultured with irradiated CTL or Th1 cells displayed significantly lower growth rate and secretion of Id protein, while 5TGM1 myeloma cells cultured with irradiated Th2 showed increased proliferation and production of Id protein compared with the tumor cells cultured with irradiated, irrelevant T cells. The in vivo function of these T cells on the tumor cells was also examined in the mouse model. After adoptive transfer of the T cells into 5TGM1 tumor-bearing C57BL/KaLwRij mice, 80% and 60% of mice that received CTL or Th1 cells respectively survived without further increase in serum Id proteins. In contrast, all mice that received PBS or normal splenocytes as controls, or Id-specific Th2 cells developed myeloma with a drastic increase in serum Id proteins. Compared with controls, mice that received Th2 cells had larger tumor burdens and shorter survival time. Thus, these results demonstrate that Id-specific CTL and Th1 but not Th2 responses are beneficial to patients receiving Id-based immunotherapy. Further studies are needed to develop novel strategies to induce only Th1 and CTL responses following Id vaccination.

Introduction: Mantle cell lymphoma (MCL) has a poor outcome and is a therapeutic challenge. Preclinical evaluation of investigational agents for MCL has been limited by lack of suitable animal models that mimic the natural history of human MCL and provide the microenvironment in which MCL cells thrive. Since MCL usually involves the bone marrow, we developed an in vivo mouse model for primary human MCL cells in severe combined immunodeficient mice (SCID-hu), which have been implanted with human fetal bone. Materials and Methods: Human primary MCL cells were obtained and isolated from spleen, lymph nodes, bone marrow aspirates, or peripheral blood of six different MCL patients. Purified patient primary MCL cells were directly inoculated into human fetal bone chip or injected into mouse tail vein. Immunohistochemical staining with anti-human CD20 or cyclin D1 antibodies and detection of circulating human beta 2-microglobulin (B2M) in mouse serum were used to monitor the engraftment, growth, and immigration of human primary MCL cells in SCID-hu mice. Results: A total of 30 SCID-hu mice and 5 SCID mice were used. Twenty of SCID-hu mice received inoculation of 0.5 – 5 × 106 of patient primary MCL cells (2–5 SCID-hu mice/patient sample) into human fetal bone chips implanted in mice subcutaneously. Five of SCID-hu mice and 5 of SCID mice (without human fetal hone chips) were injected intravenously with 5 × 106 of patient MCL cells. The same number of cells were injected into human bone chips in 5 SCID-hu mice with equal volume of PBS as controls. Successful primary MCL cell engraftment was observed in 15 out of 20 SCID-hu mice after injection of these cells into human fetal bones. But only one out of 5 SCID-hu mice had successful engraftment after the intravenous injection of primary MCL cells into mouse tail vein. Importantly, none of SCID mice had successful engraftment after intravenous injection of 5 × 106 of primary MCL cells. These data indicated that human fetal bone provides a critical microenvironment for the survival and growth of primary MCL cells. Increasing levels of circulating human B2M in mouse serum were found after successful engraftment and growth of human primary MCL cells in SCID-hu mice. Immunohistochemical staining with anti-human CD20 and cyclin D1 antibodies confirmed that, similar to the human disease, primary MCL cells homed to mouse lymph nodes, spleen, bone marrow, and gastrointestinal tract but not to mouse liver. Treatment of MCL-bearing SCID-hu mice with atiprimod suppressed B2M secreted by functioning human MCL cells and induced tumor regression. Conclusion: Human primary MCL cells from patient were able to successfully engraft in SCID-hu mice, and mimiced the natural organ involvement of human disease homing to mouse lymph node, spleen, bone marrow, and gastrointestinal tract but not to liver. This in vivo model mimiced the natural biological features of human MCL and is therefore useful for preclinical evaluation of new therapeutic agents.

Mantle cell lymphoma (MCL) has poor outcome and is a therapeutic challenge. Atiprimod, a cationic amphiphilic compound, inhibits the proliferation of most human tumor cell lines. Our study was undertaken to evaluate the therapeutic efficacy of Atiprimod on MCL cells and elucidate the mechanism by which it induces cell apoptosis. Four human MCL cell lines, SP53, MINO, Grant 519, and Jeko-1; freshly isolated primary MCL cells from three MCL patients; and normal peripheral blood mononuclear cells (PBMCs) from five healthy donors were treated with Atiprimod. A 3H-thymidine incorporation assay showed that Atiprimod not only inhibited the growth of the MCL cell lines SP53, MINO, Grant 519, and Jeko-1, but also freshly isolated patient MCL cells in a dose-dependent manner. Interestingly, Atiprimod also inhibited the proliferation of PHA-, PMA/ionomycin-, or anti-CD3 mAb/anti-CD28 mAb-activated, but not resting PBMCs from healthy donors. Flow cytometry analysis with fluorescence-labeled Annexin V and propidium iodide showed that Atiprimod induced apoptosis in both the cell lines and primary MCL cells in time-dependent and dose-dependent manners. Notably, Atiprimod did not induce apoptosis of normal T cells and B cells. Atiprimod was also effective and therapeutic in a MCL mouse model established in severe combined immunodeficient (SCID) mice. SP53 cells (5 × 106) were inoculated subcutaneously into the right leg of SCID mice. Three weeks later, after palpable tumors developed, mice were treated intraperitoneally with either vehicle alone (PBS) or Atiprimod (25 mg/kg per day) for 6 consecutive days. Tumor growth was significantly inhibited after Atiprimod treatment compared with vehicle control, and the survival time of tumor-bearing mice was significantly prolonged in the treatment group. Western blot analysis showed that apoptosis of MCL cells was induced through the activation of caspase-9, caspase-3, and PARP pathway. Furthermore, the expression of phosphorylated Bcl-2, Bax, Bad, Bax-xL, and cytochrome C was increased in Atiprimod-treated MCL cells. Pretreatment of cells with caspase-9 inhibitor (z-LEHD) or pan-caspase inhibitor (z-VAD) but not caspase-8 inhibitor (z-IETD) completely blocked Atiprimod-induced apoptosis. In conclusion, Atiprimod inhibits growth and induces apoptosis of MCL cells in vitro and in vivo. Cell apoptosis was induced via activation of the mitochondrial signaling pathway. The therapeutic efficacy of Atiprimod on MCL cells in the mouse model supports the use of Atiprimod as a potential agent in MCL chemotherapy.

Previous studies have demonstrated that circulating dendritic cells (DCs) in myeloma patients are functionally abnormal. However, the phenotypic and functional properties of monocyte-derived DCs (MoDCs) from myeloma patients, which are commonly used for immunotherapy in patients, are still poorly defined. This study was undertaken to examine the quality of MoDCs from myeloma patients compared with cells from healthy donors. We analyzed and compared cells from 12 newly diagnosed, stage II or III myeloma patients and 10 healthy donors. MoDCs were generated by culturing adherent cells or purified CD14+ monocytes in medium containing GM-CSF and IL-4. A cocktail of IL-1β, IL-6, TNF-α, and PGE2 was used to induce DC maturation. We found that patient-derived MoDCs are phenotypically and functionally defective. Compared with their normal counterpart, patient-derived, immature and mature MoDCs expressed significantly lower levels of CD1a, CD40, CD80, and HLA-DR. The mature cells were also poor at activating alloreactive T cells, presenting recall antigen and activating autologous antigen-specific T cells, and inducing myeloma-specific cytotoxic T-lymphocyte response. These abnormalities may be attributed to elevated production of autocrine cytokines such as IL-6, constitutively activated p38 and STAT3, and inhibited MEK/ERK signaling pathways in the progenitor cells. Treatment with neutralizing IL-6-specific antibody and more importantly, p38 inhibitor, or both, could correct these abnormalities. Treating patient-derived cells with these agents not only significantly increased cell yield, but also produced MoDCs that were as functional as their normal counterpart. We further showed that the p38 inhibitor inhibited the activity of p38 and drastically increased MEK1/2 and ERK1/2 phosphorylation and activation, and a combination of IL-6 neutralizing antibody with the p38 inhibitor further upregulated phosphorylation of MEK and ERK. By examining the role of STAT3 in MoDC differentiation, we found that STAT3 activation did not impair differentiation of patient-derived cells. These results indicate that the p38 inhibitor improved the generation and function of the MoDCs via inhibiting p38 and activating Raf/MEK/ERK signaling pathways, independent of the STAT3 pathway. Thus, this study has delineated the mechanistic defects of MoDCs from myeloma patients, and identified ways for restoring the function of the cells to improve the efficacy of DC-based immunotherapy against multiple myeloma.