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Radar-based human motion recognition (HMR) has drawn considerable attention in many social and military applications. Deep learning (DL) is one of the most promising methods in this field, but it requires large-scale labeled training data to ensure the generalization of the network, which is a challenge in radar applications due to the scarcity of labeled data. To overcome this problem, we propose a semisupervised DL algorithm for radar-based HMR with micro-Doppler signatures, which uses a small amount of labeled data and a large amount of unlabeled data to accurately classify typical human motions. We utilize a semisupervised triple generative adversarial nets (Triple-GANs) model and improve it by introducing a connection structure and a loss term related to the Mixup data augmentation. The performance of the proposed method is verified by applying a public radar micro-Doppler spectrogram dataset, including six motions. Experimental results demonstrate that the proposed method can identify six motions with an average accuracy of 90% using only 10% labeled data in the training dataset. Ablation experimental results verify the efficiency of two improvement strategies. Moreover, the proposed method has a higher average accuracy compared with state-of-the-art methods.

Vasoactive intestinal polypeptide (VIP), an anti-inflammatory neuropeptide with pleiotropic cardiovascular effects, induces differentiation of hematopoietic stem cells into regulatory dendritic cells that limit graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We have previously shown that donor plasmacytoid dendritic cells (pDCs) in bone marrow (BM) donor grafts limit the pathogenesis of GVHD. In this current study we show that murine and human pDCs express VIP, and that VIP-expressing pDCs limit T-cell activation and expansion using both in vivo and in vitro model systems. Using T cells or pDCs from transgenic luciferase+ donors in murine bone marrow transplantation (BMT), we show similar homing patterns of donor pDCs and T cells to the major sites for alloactivation of donor T cells: spleen and gut. Cotransplanting VIP-knockout (KO) pDCs with hematopoietic stem cells and T cells in major histocompatibility complex mismatched allogeneic BMT led to lower survival, higher GVHD scores, and more colon crypt cell apoptosis than transplanting wild-type pDCs. BMT recipients of VIP-KO pDCs had more T helper 1 polarized T cells, and higher plasma levels of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-α than recipients of wild-type pDCs. T cells from VIP-KO pDC recipients had increasing levels of bhlhe40 transcripts during the first 2 weeks posttransplant, and higher levels of CyclophilinA/Ppia transcripts at day 15 compared with T cells from recipients of wild-type pDCs. Collectively, these data indicate paracrine VIP synthesis by donor pDCs limits pathogenic T-cell inflammation, supporting a novel mechanism by which donor immune cells regulate T-cell activation and GVHD in allogeneic BMT.

Pabuonqedidae is the most recently erected extinct family of Blattaria, with only a type genus and species reported from the Cretaceous. Pabuonqed magna sp. n., based on a well-preserved specimen from mid-Cretaceous Kachin amber, can be definitely attributed to the genus, but differs from the type species, Pabuonqed eulna Vršanský, 2019, mainly by large body size, filiform bases on antennae, smaller, round eyes, standard pronotum (all plesiomorphies), absence of arolium on mesotarsus (autapomorphies), developed R and narrow cubitus in forewing, and developed Sc and R in hindwing. As the second species of Pabuonqedidae, our new find provides more information on the phylogenic position of Pabuonqedidae and augments the biodiversity of the Cretaceous wood-boring and/or rotten wood-burrowing mole roaches.

Vasoactive intestinal peptide (VIP), an anti-inflammatory neuropeptide with pleiotropic cardio-vascular effects, induces differentiation of hematopoietic stem cells into regulatory dendritic cells that limit graft-versus-host disease (GvHD) in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We have previously shown that donor plasmacytoid dendritic cells (pDCs) in bone marrow (BM) donor grafts limit the pathogenesis of GvHD. In this current study we show that murine and human pDCs express VIP, and that VIP-expressing pDCs limit T cell activation and expansion using both in vivo and in vitro model systems. Using T cells or pDCs from transgenic luciferase+ donors in murine BMT, we show similar homing patterns of donor pDCs and T cells to the major sites for allo-activation of donor T cells: spleen, and gut. Co-transplanting VIP-KO pDCs with hematopoietic stem cells (HSCs) and T cells in MHC mis-matched allogeneic BMT led to lower survival, higher GvHD scores, and more colon crypt cell apoptosis than transplanting wild-type pDCs. BMT recipients of VIP-KO pDCs had more Th1 polarized T cells, and higher plasma levels of GM-CSF and TNF-alpha than recipients of wild-type pDCs. T cells from VIP-KO pDC recipients had increasing levels of bhlhe40transcripts during the first two weeks post-transplant, and higher levels of CyclophilinA/Ppiatranscripts at day 15 compared with T cells from recipients of wild type pDCs. Collectively, these data indicate paracrine VIP synthesis by donor pDCs limits pathogenic T cell inflammation, supporting a novel mechanism by which donor immune cells regulate T cell activation and GvHD in allogeneic BMT.

Invasive bladder cancer (BC) is one of the most lethal malignant urological tumors. Although miR-200a has been reported as an onco-miRNA that targets the PTENgene in endometrioid carcinoma, its biological significance in BC invasion has been poorly explored. In the current study, we found that miR-200a was markedly overexpressed in both human BC tissues and BBN-induced muscle-invasive BC tissues. We further showed that miR-200a overexpression specifically promoted human BC cell invasion, but not migration, viatranscriptional upregulation of matrix metalloproteinase (MMP)-2. Mechanistic studies indicated that the increased phosphorylation of c-Jun mediated the increasing levels of MMP-2mRNA transcription. Further investigation revealed that Dicer was decreased in miR-200a overexpressed BC cells; this resulted in inhibition of miR-16 maturation and consequently led to increased JNK2 protein translation and c-Jun activation. Taken together, the studies here showed that miR-200a overexpression inhibited Dicer expression, in turn, resulted in inhibition of miR-16 maturation, leading to upregulation of JNK2 expression, c-Jun phosphorylation, MMP-2transcription and, ultimately, BC invasion. Collectively, these results demonstrate that miR-200a is an onco-miRNA that is a positive regulator for BC invasion. This finding could be very useful in the ongoing development of new strategies to treat invasive BC patients.

Sex-determining region Y-box 2 (SOX2), a well-known stemness biomarker, is highly expressed in a variety of cancers, including human highly invasive bladder cancer (BC). However, the role of SOX2 may vary in different kinds of malignancy. In the present study, we discovered that ChlA-F, a novel conformation derivative of isolate Cheliensisin A (Chel A), remarkably inhibits the invasive ability of human invasive BC cells through downregulation of SOX2 protein expression. We found that ChlA-F treatment dramatically decreases SOX2 protein expression in human high-grade invasive BC cells. Ectopic expression of SOX2 reversed ChlA-F inhibition of cell invasion ability in human bladder cancer cells, suggesting that SOX2 is a major target of ChlA-F during its inhibition of human BC invasion. Mechanistic studies revealed that ChlA-F downregulates SOX2 at both the protein degradation and protein translation levels. Further studies revealed that ChlA-F treatment induces HuR protein expression and that the increased HuR interacts with USP8mRNA, resulting in elevation of USP8mRNA stability and protein expression. Elevated USP8 subsequently acts as an E3 ligase to promote SOX2 ubiquitination and protein degradation. We also found that ChlA-F treatment substantially increases c-Jun phosphorylation at Ser63 and Ser73, initiating miR-200c transcription. The increased miR-200c directly binds to the 3′-UTR of SOX2mRNA to suppress SOX2 protein translation. These results present novel mechanistic insight into understanding SOX2 inhibition upon ChlA-F treatment and provide important information for further exploration of ChlA-F as a new therapeutic compound for the treatment of highly invasive/metastatic human BC patients.

Background: A virus infection may lead the body to produce more immune cells of particular types or stimulate the production of new ones, both of which may have anti-leukemic effects. There has been no research on whether immune cells stimulated by varicella-zoster virus (VZV) infection have anti-leukemic effects. The objective of this investigation is to assess the impact of VZV infection on patients' long-term survival following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: This retrospective study investigated the association between varicella-zoster virus (VZV) reactivation and outcomes in 219 individuals who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) at the Sun Yat-sen University’s First Affiliated Hospital. According to being diagnosed with VZV infection or not, these patients were grouped into two groups. The comparison of cumulative incidence of relapse, non-recurrent mortality, and overall survival (OS) was conducted between the two groups. Results: Analyzing multivariate data, VZV reactivation was linked to lower relapse incidence in the group containing all individuals (hazard ratio [HR] = 0.27; 95% confidence interval [CI], 0.12–0.64), patients suffering from acute myeloid leukaemia (HR = 0.10; 95% CI, 0.01–0.83), and patients suffering from acute lymphoblastic leukaemia (HR = 0.25; 95% CI, 0.08–0.77). Moreover, VZV reactivation was linked with decreased non-relapse mortality in all individuals (HR = 0.20; 95% CI, 0.05–0.79), but no statistical significance was found for any disease subgroup. Further, VZV reactivation was an independent predictor for improved OS in the group containing all individuals (HR = 0.10; 95% CI, 0.03–0.29), patients suffering from acute myeloid leukaemia (HR = 0.09; 95% CI, 0.01–0.66), and patients suffering from acute lymphoblastic leukaemia (HR = 0.16; 95% CI, 0.04–0.68). Conclusion: This is the first study to show that VZV reactivation following allo-HSCT is an independent predictor for lower relapse rates and improved OS, providing novel therapeutic approaches to improve patients’ long-term survival following allo-HSCT.

ABSTRACTPHLPP1 (PH domain and leucine rich repeat protein phosphatase 1) is a newly identified family of Ser/Thr phosphatases that catalyzes the dephosphorylation of a conserved regulatory motif of the AGC kinases resulting in a tumor suppressive function, while CDKN1B/p27 also acts as a tumor suppressor by regulating cell cycle, senescence, apoptosis, and cell motility. Our most recent studies reveal that CDKN1B is required for PHLPP1 abundance, which contributes to the inhibition of carcinogenic arsenite-induced cell malignant transformation through inhibition of RPS6-mediated Hif1atranslation. However, nothing is known about the mechanisms underlying the crosstalk between these 2 key tumor suppressors in intact cells. Here, for the first time to the best of our knowledge, we show that CDKN1B is able to promote PHLPP1 protein translation by attenuating the abundance of Mir6981, which binds directly to the 5ʹuntranslated region (UTR) of Phlpp1mRNA. Further studies indicate that the attenuation of Mir6981expression is due to macroautophagy/autophagy-mediated degradation of Mir6981in an SQSTM1/p62-dependent fashion. Moreover, we have determined that Sqstm1is upregulated by CDKN1B at the level of transcription viaenhancing SP1 protein stability in an HSP90-depdendent manner. Collectively, our studies prove that: 1) SQSTM1 is a CDKN1B downstream effector responsible for CDKN1B-mediated autophagy; 2) by promoting the autophagy-mediated degradation of Mir6981, CDKN1B exerts a positive regulatory effect on PHLPP1 translation; 3) Mir6981suppresses PHLPP1 translation by binding directly to its mRNA 5ʹ-UTR, rather than classical binding to the 3ʹ-UTR. These findings provide significant insight into understanding the crosstalk between CDKN1B and PHLPP1.Abbreviations:ATG: autophagy related; ACTB: actin beta; BAF: bafilomycin; BECN1: beclin 1; Cdkn1b/p27: cyclin-dependent kinase inhibitor 1B; CHX: cycloheximide; DMEM: dulbecco’s modified eagle medium; FBS: fetal bovine serum; GAPDH: glyceraldehyde −3-phosphate dehydrogenase; Hif1a: hypoxia inducible factor 1, alpha subunit; Hsp90: heat shock protein 90; JUN: Jun proto-oncogene, AP1 transcription factor subunit; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MG132: proteasome inhibitor; Mtor: mechanistic target of rapamycin kinase; Phlpp1: PH domain and leucine rich repeat protein phosphatase 1; Phlpp2: PH domain and leucine rich repeat protein phosphatase 2; Pp2c: protein phosphatase 2 C; RPS6: ribosomal protein S6; Sp1: trans-acting transcription factor 1; Sqstm1/p62: sequestosome 1; TUBA: alpha tubulin; 3ʹ-UTR; 3ʹ-untranslated region; 5ʹ-UTR: 5ʹ-untranslated region.

Over half a million US residents are suffering with bladder cancer (BC), which costs a total $4 billion in treatment annually. Although recent studies report that autophagy-related gene 7 (ATG7) is overexpressed in BCs, the regulatory effects of ATG7 on cancer stem-like phenotypes and invasion have not been explored yet. Current studies demonstrated that the deficiency of ATG7 by its shRNA dramatically reduced sphere formation and invasion in vitro, as well as lung metastasis in vivo in human invasive BC cells. Further studies indicated that the knockdown of ATG7 attenuated the expression of CD44 standard (CD44s), while ectopic introduction of CD44s, was capable of completely restoring sphere formation, invasion, and lung metastasis in T24T(shATG7) cells. Mechanistic studies revealed that ATG7 overexpression stabilized CD44s proteins accompanied with upregulating USP28 proteins. Upregulated USP28 was able to bind to CD44s and remove the ubiquitin group from CD44s’ protein, resulting in the stabilization of CD44s protein. Moreover, ATG7 inhibition stabilized AUF1 protein and thereby reduced tet1mRNA stability and expression, which was able to demethylate usp28promoter, reduced USP28 expression, finally promoting CD44s degradation. In addition, CD44s was defined to inhibit degradation of RhoGDIβ, which in turn promotes BC invasion. Our results demonstrate that CD44s is a key ATG7 downstream regulator of the sphere formation, invasion, and lung metastasis of BCs, providing significant insight into understanding the BC invasions, metastasis, and stem-like properties.

ABSTRACTOur previous studies have demonstrated that XIAP promotes bladder cancer metastasis through upregulating RhoGDIβ/MMP-2 pathway. However, the molecular mechanisms leading to the XIAP upregulation was unclear. In current studies, we found that XIAP was overexpressed in human high grade BCs, high metastatic human BCs, and in mouse invasive BCs. Mechanistic studies indicated that XIAP overexpression in the highly metastatic T24T cells was due to increased mRNA stability of XIAP that was mediated by downregulated miR-200c. Moreover, the downregulated miR-200c was due to CREB inactivation, while miR-200c downregulation reduced its binding to the 3’-UTR region of XIAP mRNA. Collectively, our results demonstrate the molecular basis leading to XIAP overexpression and its crucial role in BC invasion.

Pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) is a tumor suppressor that catalyzes the de-phosphorylation of the AGC kinases, while p27 acts as a tumor suppressor that regulates cell cycle, apoptosis, and cell motility. Our previous studies have identified that PHLPP2 participates in inhibition of transformation of human bronchial epithelial cells following lung carcinogen B[a]P/B[a]PDE exposure. However, nothing was known about the association of p27 with regulation of PHLPP2 expression and the role of PHLPP2 in bladder cancer (BC) invasion. In our current studies, we demonstrated that PHLPP2 inhibited BC invasion through promoting MMP2 degradation via p62-mediated autophagy; and p27 expression was able to stabilize PHLPP2 protein by inhibiting protein degradation of Hsp90, which could directly bind to PHLPP2 and protect it from degradation. More in-depth studies discovered that stabilization of Hsp90 by p27 was mediated by calpain1 proteolysis system, whereas p27 inhibited calpain1 gene transcription by attenuating Jak1/Stat1 cascade in human invasive BC cells. Collectively, we for the first time revealed PHLPP2 downregulation in BCs and its participating in promotion of BC invasion, as well as novel role of p27 and mechanisms underlying its regulation of PHLPP2 protein degradation through Hsp90-dependent manner. Our findings improve our understanding of p27 and PHLPP2 roles and their crosstalk in regulation of BC invasion, which further contributes to improve the current strategy for invasive bladder cancer therapy.

ABSTRACTChronic lung inflammation is accepted as being associated with the development of lung cancer caused by nickel exposure. Therefore, identifying the molecular mechanisms that lead to a nickel-induced sustained inflammatory microenvironment that causes transformation of human bronchial epithelial cells is of high significance. In the current studies, we identified SQSTM1/p62 as a novel nickel-upregulated protein that is important for nickel-induced inflammatory TNF expression, subsequently resulting in transformation of human bronchial epithelial cells. We found that nickel exposure induced SQSTM1 protein upregulation in human lung epithelial cells in vitro and in mouse lung tissues in vivo. The SQSTM1 upregulation was also observed in human lung squamous cell carcinoma. Further studies revealed that the knockdown of SQSTM1expression dramatically inhibited transformation of human lung epithelial cells upon chronic nickel exposure, whereas ectopic expression of SQSTM1 promoted such transformation. Mechanistic studies showed that the SQSTM1 upregulation by nickel was the compromised result of upregulating SQSTM1mRNA transcription and promoting SQSTM1 protein degradation. We demonstrated that nickel-initiated SQSTM1 protein degradation is mediated by macroautophagy/autophagy viaan MTOR-ULK1-BECN1 axis, whereas RELA is important for SQSTM1transcriptional upregulation following nickel exposure. Furthermore, SQSTM1 upregulation exhibited its promotion of nickel-induced cell transformation through exerting an impetus for nickel-induced inflammatory TNFmRNA stability. Consistently, the MTOR-ULK1-BECN1 autophagic cascade acted as an inhibitory effect on nickel-induced TNF expression and cell transformation. Collectively, our results demonstrate a novel SQSTM1 regulatory network that promotes a nickel-induced tumorigenic effect in human bronchial epithelial cells, which is negatively controlled by an autophagic cascade following nickel exposure.

ABSTRACTIsorhapontigenin (ISO) is a new derivative of stilbene isolated from the Chinese herb Gnetum cleistostachyum. Our recent studies have revealed that ISO treatment at doses ranging from 20 to 80 μM triggers apoptosis in multiple human cancer cell lines. In the present study, we evaluated the potential effect of ISO on autophagy induction. We found that ISO treatment at sublethal doses induced autophagy effectively in human bladder cancer cells, which contributed to the inhibition of anchorage-independent growth of cancer cells. In addition, our studies revealed that ISO-mediated autophagy induction occurred in a SESN2 (sestrin 2)-dependent and BECN1 (Beclin 1, autophagy related)-independent manner. Furthermore, we identified that ISO treatment induced SESN2 expression via a MAPK8/JNK1 (mitogen-activated protein kinase 8)/JUN-dependent mechanism, in which ISO triggered MAPK8-dependent JUN activation and facilitated the binding of JUN to a consensus AP-1 binding site in the SESN2promoter region, thereby led to a significant transcriptional induction of SESN2. Importantly, we found that SESN2 expression was dramatically downregulated or even lost in human bladder cancer tissues as compared to their paired adjacent normal tissues. Collectively, our results demonstrate that ISO treatment induces autophagy and inhibits bladder cancer growth through MAPK8-JUN-dependent transcriptional induction of SESN2, which provides a novel mechanistic insight into understanding the inhibitory effect of ISO on bladder cancers and suggests that ISO might act as a promising preventive and/or therapeutic drug against human bladder cancer.

Arsenite exposure is associated with an increased risk of human lung cancer. However, the molecular mechanisms underlying the arsenite-induced human lung carcinogenesis remain elusive. In this study, we demonstrated that arsenite upregulates cyclin D1 expression/activity to promote the growth of human bronchial epithelial Beas-2B cells. In this process, the JNKs (c-Jun N-terminal kinases)/c-Jun cascade is elicited. The inhibition of JNKs or c-Jun by chemical or genetic inhibitors blocks the cyclin D1 induction mediated by arsenite. Furthermore, using a loss of function mutant of p85 (p85, a subunit of PI3K) or dominant-negative Akt (DN-Akt), we showed that PI3K and Akt act as the upstream regulators of JNKs and c-Jun in arsenite-mediated growth promotion. Overall, our data suggest a pathway of PI- 3K/Akt/JNK/c-Jun/cylin D1 signaling in response to arsenite in human bronchial epithelial cells.

The 1% silver clusters containing twistlike helix titania nanoparticles have been synthesized and tested in photocatalytic applications. The special twistlike helix structures were obtained by controlling the hydrolysis of titanium tetrachloride, while variety of routes were investigated to deposit the silver nanoparticles, where impregnation, chemical deposition, and photodeposition methods are adopted to fabricate the Ag/TiO2samples. The influence of the photocatalytic activity of the mesoporous helix materials and the preparation methods on the final structure of the samples was investigated, including the surface area, pore size, silver crystallite size, silver distribution, and silver surface content. The dispersion and particle size of Ag clusters or nanoparticles on Ag/TiO2nanocatalysts were investigated by TEM analysis. The surface Ag nanoparticles contents were determined by XPS results. Diffuse reflectance and photoluminescence analysis were used to investigate the visible light adsorption ability and the active sites amount including photoinduced electrons and holes. The experimental results show that the chemical deposition method is an efficient method to synthesize Ag/TiO2catalysts with highly dispersed nanoparticles of metallic Ag. The Ag/TiO2sample prepared through the chemical deposition method shows the best photocatalytic activity, and its photocatalytic activity is superior to that of P25 and pure TiO2sample. The superior activity of Ag/TiO2photocatalysts can be rationalized in terms of well dispersion and suitable surface content of Ag nanoparticles that will effectively decrease the recombination rate of photoinduced electrons and holes and then increase the photooxidation efficiency of the catalysts.

The chronic lung inflammatory activity and carcinogenicity of nickel compounds have been well documented by previous studies from epidemiology both in vitro and in vivo. However, the molecular mechanism involved in nickelinduced chronic lung inflammation is much less understood. The current study demonstrates that exposure of human bronchial epithelial cells (Beas-2B) to nickel compounds results in the induction of the inflammatory cytokine tumor necrosis factor- (TNF-) and transactivation of nuclear factor of activated T cells (NFAT), nuclear factor-B (NF-B), and activator protein-1 (AP-1). Further studies show that neither overexpression of IKK-KM, a kinase inactive mutant of IKK, nor the ectopic expression of a dominant negative mutant of NFAT could inhibit the TNF- induction by nickel exposure. Overexpression of TAM67, a dominant-negative mutant of c-Jun, dramatically reduced the TNF- induction, suggesting that AP-1 is a mediator of TNF- induction in nickel responses. Our results show that ERKs are AP-1 upstream kinases responsible for TNF- induction by nickel exposure; although JNKs, ERKs, and p38K were all activated in the Beas-2B cells exposed to nickel compounds. Our results demonstrate that inflammatory TNF- could be induced by nickel exposure in Beas-2B cells specifically through an ERKs/AP-1-dependent pathway.

The chronic lung inflammatory activity and carcinogenicity of nickel compounds have been well documented by previous studies from epidemiology both in vitro and in vivo. However, the molecular mechanism involved in nickelinduced chronic lung inflammation is much less understood. The current study demonstrates that exposure of human bronchial epithelial cells (Beas-2B) to nickel compounds results in the induction of the inflammatory cytokine tumor necrosis factor-α (TNF-α) and transactivation of nuclear factor of activated T cells (NFAT), nuclear factor-κB (NF-κB), and activator protein-1 (AP-1). Further studies show that neither overexpression of IKKβ-KM, a kinase inactive mutant of IKKβ, nor the ectopic expression of a dominant negative mutant of NFAT could inhibit the TNF-α induction by nickel exposure. Overexpression of TAM67, a dominant-negative mutant of c-Jun, dramatically reduced the TNF-α induction, suggesting that AP-1 is a mediator of TNF-α induction in nickel responses. Our results show that ERKs are AP-1 upstream kinases responsible for TNF-α induction by nickel exposure; although JNKs, ERKs, and p38K were all activated in the Beas-2B cells exposed to nickel compounds. Our results demonstrate that inflammatory TNF-α could be induced by nickel exposure in Beas-2B cells specifically through an ERKs/AP-1-dependent pathway.

Introduction:Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide known to induce differentiation of regulatory dendritic cells and regulatory T cells. Using allogeneic hematopoietic stem cell transplantation (allo-HSCT) models, we have shown that donor bone marrow (BM) plasmacytoid dendritic cells (pDCs) facilitate HSC engraftment and attenuate pathogenesis of graft vs. host disease (GvHD) through regulation of recipient T cells. However, the mechanism by which pDCs mitigate the GvHD activity of recipient T cells is not clearly understood. Here, we report that donor pDCs limit pathogenic T cell inflammation by VIP production.

Waller: Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Verastem Oncology: Consultancy, Research Funding.

Highly ordered mesoporous MgO catalysts with tunable pore size distribution and fairly good alkaline property were successfully obtained by a double replication procedure. Small-angle XRD, TEM, and nitrogen adsorption and desorption results show that these mesoporous MgO materials possess a highly ordered 2D hexagonal mesostructure with tunable pore diameter that is obtained using different hydrothermal temperature derived SBA-15 and CNK-3 carbon as templates. The alkaline density was determined for all samples from the technique of temperature-programmed desorption of CO 2and FT-IR, and the mesoporous MgO with large pore diameter shows more basic sites than the reference bulk one. Such mesoporous MgO materials have been successfully used as base catalysts in the Baeyer−Villiger oxidation of cyclic ketones to the corresponding lactones. 100% adamantanone conversion and >99% lactone selectivity were obtained over mesoporous MgO-373. The pore diameter has slight influence on the conversion of ketones, since the average pore diameters of mesoporous MgO are large enough to allow the reactants free access to the active sites. In terms of the proposed mechanism, the enhanced medium alkaline properties will be more favorable for the attack by hydrogen peroxide to form a hydroperoxide and percarbonic oxide, and in turn enhancing the catalytic activity. In addition, the higher surface area and large pore volume are attributed to the higher activity of mesoporous MgO-373 than the others, signifying the promising potential applications of these mesoporous MgO materials in alkaline catalysis.

Abstract: It is widely accepted that the consumption of alcohol may lead to hepatic injuries such as hepatic fibrosis and cirrhosis. However, consumption of Maotai, one of the famous liquors in China, is found to have no obvious relevance with hepatic injury as ordinary white wine does in both epidemiological and histopathological studies. Present study used human hepatoma cell line Hep3B to address the mechanisms involved in the resistance of alcohol-induced hepatic injury by Maotai liquor. We found that exposure of Hep3B cells to Maotai residue without ethanol (MRWE) resulted in the increased GST A1 anti-oxidant responsive element (ARE) transcriptional expression, while MRWE treatment did not affect Nrf-2-dependent transcriptional activity. Those findings were further confirmed at all time points and doses tested, suggesting that GST A1 transcription was regulated by MRWE via an Nrf-2-independent pathway. Consistent with GST A1 induction, the phosphorylation of c-Jun, extracellular signal-regulated kinases (ERKs) and p38 kinase (p38 K), were also observed in MRWE-treated Hep3B cells. Furthermore, pretreatment of cells with either PD98059 (an inhibitor specific for MEK1/2-ERKs pathway) or SB202190 (an inhibitor specific for p38 K) led to a significant decrease in the induction of GST A1 transcriptional expression by MRWE treatment. Our results indicate that certain content in MRWE is able to induce GST A1 ARE transcriptional expression, which may provide protective effects for hepatic cells by antagonizing the oxidative stress derived from ethanol via an ERKs- and p38 K-dependent pathway.

Abstract: Epidermal growth factor (EGF) has been reported to act as a tumor promoter in several tissues, such as skin, in association with the induction of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). However, molecular mechanisms involved in these regulations are not well defined. This study addressed a potential role of nuclear factor of activated T cells 3 (NFAT3) in EGF-induced COX-2 and iNOS transcription and cell transformation in mouse epidermal Cl 41 cells. We found that EGF markedly induced anchorage-independent growth (cell transformation) of Cl 41 cells, as well as COX-2 (> 6-fold) and iNOS (> 5-fold) promoter-dependent transcription. The EGF-induced COX-2 transcription was blocked by knockdown of NFAT3 with NFAT3 siRNA, whereas the transcription of iNOS and cell transformation induced by EGF were not affected. Although our recent studies supported that NFAT3 plays an essential role in chemical carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE)-induced cell transformation, the data presented here demonstrated that NFAT3 is required for EGF-induced COX-2 transcription, but neither iNOS transcription nor cell transformation, indicating that the role of NFAT3 in regulating cell transformation is carcinogen-specific.

Dietary freeze-dried black raspberries inhibit tumor induction by N-nitrosomethylbenzylamine in the rat esophagus, but the constituents responsible for this chemopreventive activity have not been identified. We fractionated freeze-dried black raspberries and used mouse epidermal JB6 Cl 41 cells stably transfected with either a nuclear factor kappa B (NFκB)- or an activator protein 1 (AP-1)-luciferase reporter, and treated with racemic anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), to assess the inhibitory effects of the fractions. The ethanol and water extracts of the freeze-dried black raspberries had inhibitory activity and these extracts were fractionated by HPLC to give several bioactive fractions. Further HPLC analysis yielded multiple subfractions, some of which inhibited BPDE-induced NFκB activity. Major constituents of the most active subfractions were identified by their spectral properties and in comparison with standards as cyanidin-3-O-glucoside, cyanidin 3-O-(2G-xylosylrutinoside) and cyanidin 3-O-rutinoside. Analysis of freeze-dried black raspberries indicated that these three components comprised ∼3.4% of the material by dry weight. Consistent with these results, standard cyanidin-3-O-glucoside and cyanidin chloride were also good inhibitors of BPDE-induced NFκB activity. The results of this study demonstrate that cyanidin glycosides of freeze-dried black raspberries are bioactive compounds which could account for at least some of the chemopreventive activity observed in animal models.

Abstract: The results from animal studies have shown that mouse skin is highly susceptible to both ionizing radiation and benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE). Previous studies have also indicated that cyclin D1 plays a crucial role in controlling cell proliferation and tumorigenesis. We, therefore, investigated here the effect of ionizing radiation and B[a]PDE on cyclin D1 transcription and potential involvement of NFAT3 in regulation of cyclin D1 transcription in mouse epidermal Cl 41 cells. We found that B[a]PDE exposure induced a high level of NFAT activation and cyclin D1 transcription in mouse epidermal Cl 41 cells. Ionizing radiation exhibited an enhancement for NFAT activation and cyclin D1 induction by B[a]PDE, even though ionizing radiation by itself had only a marginal effect. By stably knockdown of NFAT3 protein expression using specific NFAT3 small interfering RNA (siRNA), we found that cyclin D1 induction by B[a]PDE or B[a]PDE plus ionizing radiation was dramatically impaired. These results indicate that ionizing radiation is able to enhance cyclin D1 transcription induced by B[a]PDE, and NFAT3 is involved in the regulation of cyclin D1 transcription by B[a]PDE or B[a]PDE plus ionizing radiation.

Skin is a major target of carcinogenic trivalent arsenic (arsenite, As3+). It has been thought that cell proliferation is one of the central events involved in the carcinogenic effect of arsenite. Cyclin D1, a nuclear protein playing a pivotal role in cell proliferation and cell cycle transition from G1 to S phases, has been reported to be induced in human fibroblast by arsenite via uncertain molecular mechanisms. In the present study, the potential roles of PI-3K/Akt/IKKβ/NFκB signal pathway in cyclin D1 induction by arsenite were addressed in mouse epidermal Cl41 cells. We found that exposure of Cl41 cells to arsenite was able to induce cell proliferation, activate PI-3K→Akt/p70S6k signal pathway and increase cyclin D1 expression at both transcription and protein levels. Pre-treatment of Cl41 cells with PI-3K inhibitor, wortmannin, significantly inhibited the phosphorylation of Akt and p70S6k and thereby dramatically impaired the cyclin D1 induction by arsenite, implicating the importance of the PI-3K signal pathway in the cyclin D1 induction by arsenite. Furthermore, inhibition of PI-3K/Akt by overexpression of Δp85 or DN-Akt blocked arsenite-induced IKK phosphorylation, IκBα degradation and cyclin D1 expression, indicating that IKK/NFκB is the downstream transducer of arsenite-triggered PI-3K/Akt cascade. Moreover, inhibition of IKKβ/NFκB signal pathway by overexpression of its dominant negative mutant, IKKβ-KM, also significantly blocked arsenite-induced cyclin D1 expression. Overall, arsenite exposure triggered PI-3K/Akt/IKKβ/NFκB signal cascade which in turn plays essential roles in inducing cyclin D1 expression.

The previous studies have demonstrated that vanadium exposure can cause a variety of biological effects. However, the mechanisms involved in the biological effects caused by vanadium are not well understood. Our previous studies have shown that exposure of mouse epidermal Cl 41 cells to vanadate stimulated the phosphorylation of both ERKs and p38K, and calcium signaling leading NFAT activation. In view of the evidence that ERKs and p38 kinase contribute to VEGF induction, we investigated in the present study the potential roles of ERKs, p38K, and calcium signaling in VEGF induction caused by vanadium exposure. Exposure of Cl 41 cells to vanadium led to VEGF induction in both time- and dose-dependent manners. Pre-treatment of Cl 41 cells with PD98059, an inhibitor of MEK1/2-ERKs pathway, but not SB202190, an inhibitor for p38K pathway, resulted in a dramatic inhibition of VEGF induction by vanadium. More interesting, pre-treatment of Cl 41 cells with intracellular calcium chelator, but not calcium channel blocker, resulted in a dramatic decrease in VEGF induction by vanadium. However, both PI-3K inhibitors and overexpression of Δp85, a dominant negative PI-3K mutant, resulted in only a marginal decrease in VEGF induction by vanadium. Moreover, mTOR, as a downstream molecule of PI-3K, did not attribute to VEGF induction by vanadium because rapamycin pre-treatment did not show any inhibitory effect on VEGF induction. These results indicate that ERKs and intracellular stored calcium release play a critical role in VEGF induction by vanadium. PI-3K is partially involved in VEGF induction by vanadium, while p38K and mTOR are not involved. Those results will help us to understand the molecular mechanisms involved in vanadium-induced biological effects.

Exposure to a highly nickel-polluted environment has the potential to cause a variety of adverse health effects, such as the respiratory tract cancers. Since numerous studies have demonstrated that nickel generally has weak mutagenic activity, research focus had turned to cell signalling activation leading to gene modulation and epigenetic changes as a plausible mechanism of carcinogenesis. Previous studies have revealed that nickel compounds can induce the expression of vascular endothelial growth factor (VEGF), which is a key mediator of angiogenesis both in physiological and pathologic conditions. In the present study, we investigated the potential roles of PI-3K, ERKs, p38 kinase and calcium signalling in VEGF induction by nickel in Cl 41 cells. Exposure of Cl 41 cells to nickel compounds led to VEGF induction in both time- and dose-dependent manners. Pre-treatment of Cl 41 cells with PI-3K inhibitor, wortmannin or Ly294002, resulted in a striking inhibition of VEGF induction by nickel compounds, implicating the role of PI-3K in the induction. However, mTOR, one of downstream molecules of PI-3K, may not contribute to the induction because pre-treatment of Cl 41 cells with its inhibitor, rapamycin, did not show obvious decrease in nickel-induced VEGF expression. Furthermore, pre-treatment of Cl 41 cells with MEK1/2-ERKs pathway inhibitor, PD98059, significantly inhibited VEGF induction by both NiCl2 and Ni3S2, whereas p38 kinase inhibitor, SB202190, did not impair the induction. Pre-treatment of Cl 41 cells with intracellular calcium chelator, but not calcium channel blocker, inhibited VEGF induction by nickel. Collectively these data demonstrate that PI-3K, ERKs and cytosolic calcium, but not p38 kinase, play essential roles in VEGF induction by nickel compounds.

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms by which vanadate mediates adverse effects are not well understood. The present study investigated the vanadate-induced phosphorylation of Akt and p70S6K, two kinases known to be vital for cell survival, growth, transformation, and transition of the cell cycle in mammals. Exposure of mouse epidermal JB6 cells to vanadium led to phosphorylation of Akt and p70S6Kin a time- and dose-dependent manner. Vanadium exposure also caused translocation of atypical isoforms of PKC (λ, ζ) from the cytosol to the membrane, but had no effect on PKCα translocation, suggesting that the atypical PKCs (aPKC) were specifically involved in vanadium-induced cellular response. Importantly, overexpression of a dominant negative mutant PKCλ blocked Akt phosphorylation at Ser473and Thr308, whereas it did not inhibit p70S6kphosphorylation at Thr389and Thr421/Ser424, suggesting that aPKC activation is specifically involved in vanadium-induced activation of Akt, but not in activation of p70S6k. Furthermore, vanadium-induced p70S6kphosphorylation at Thr389and Thr421/Ser424and Akt phosphorylation at Thr308occurred through a PI-3K-dependent pathway because a PI-3K dominant negative mutant inhibited induction as compared with vector control cells. These results indicate that there was a differential role of aPKC in vanadate-induced phosphorylation of Akt and p70S6k, suggesting that signal transduction pathways leading to the activation of Akt and p70S6kwere different.

Abstract The present study investigated the role of reactive oxygen species (ROS) in activation of nuclear factor of activated T cells (NFAT), a pivotal transcription factor responsible for regulation of cytokines, by asbestos in mouse embryo fibroblast PW cells. Exposure of cells to asbestos led to the transactivation of NFAT in a time- and dose-dependent manner. Scavenging of asbestos-induced H2O2 with N-acety-L-cyteine (NAC, a general antioxidant) or catalase (a specific H2O2 inhibitor) resulted in inhibition of NFAT activation. In contrast, an increase in H2O2 generation by the addition of superoxide dismutase (SOD) slightly enhanced asbestos-induced NFAT activation. In addition, pretreatment of cells with sodium formate did not exhibit any inhibition of NFAT activity induced by asbestos. These results demonstrated that H2O2 appeared to play an important role in asbestos-induced NFAT transactivation. Furthermore, it was observed that incubation of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) not only resulted in NFAT activation by itself, but also enhanced asbestos-induced NFAT induction. Pretreatment of cells with cyclosporin A (CSA), a pharmacological inhibitor of the phosphatase calcineurin, blocked both asbestos- and TPA plus asbestos-induced NFAT activation. These data suggest that asbestos is able to induce NFAT activation through H2O2-dependent and CSA-sensitive pathways, which may be involved in asbestos-induced carcinogenesis.

The exposure of mammalian cells to UV irradiation leads to the activation of transcription factors and protein kinases, which are believed to be responsible for the carcinogenic effects of excessive sun exposure. The present study investigated the effect of UV exposure on reactive oxygen species (ROS) generation and protein kinase B (Akt) phosphorylation in epidermal cells and determined if a relationship exists between these UV responses. Exposure of mouse epidermal JB6 Cl 41 cells to UV radiation led to specific phosphorylation of Akt at Ser-473 and Thr-308 in a time-dependent manner. This phosphorylation was confirmed by the observation that overexpression of Akt mutant, Akt-T308/S473A, attenuated phosphorylation of Akt at Ser-473 and Thr-308. UV radiation also generated ROS as measured by electron spin resonance (ESR) in JB6 Cl 41 cells. The generation of ROS by UV radiation was measured further by H2O2and O⨪2fluorescence staining assays. The mechanism of ROS generation involved reduction of molecular oxygen to O⨪2, which generated H2O2through dismutation. H2O2produced ⋅OH via a metal-independent pathway. The scavenging of UV-generated H2O2byN-acety-l-cyteine (NAC, a general antioxidant) or catalase (a specific H2O2inhibitor) inhibited Akt phosphorylation at Ser-473 and Thr-308, whereas the pretreatment of cells with sodium formate (an ⋅OH radical scavenger) or superoxide dismutase (an O⨪2radical scavenger) did not show any inhibitory effects. Furthermore, treatment of cells with H2O2increased UV-induced phosphorylation of Akt at Ser-473 and Thr-308. These results demonstrate that UV radiation generates a whole spectrum of ROS including O⨪2, ⋅OH, and H2O2and induces phosphorylation of Akt at Ser-473. Among the various ROS, H2O2seems most potent in mediating UV-induced phosphorylation of Akt at Ser-473 and Thr-308. It is possible that Akt may play a role in the carcinogenesis effects by UV radiation.

While vanadium compounds are known as potent toxicants as well as carcinogens, the mechanisms of their toxic and carcinogenic actions remain to be investigated. It is believed that an improper cell growth regulation leads to cancer development. The present study examines the effects of vanadate on cell cycle control and involvement of reactive oxygen species (ROS) in these vanadate-mediated responses in a human lung epithelial cell line, A549. Under vanadate stimulation, A549 cells generated hydroxyl radical (·OH), as determined by electron spin resonance (ESR), and hydrogen peroxide (H2O2) and superoxide anion (O•−2), as detected by flow cytometry using specific dyes. The mechanism of ROS generation involved the reduction of molecular oxygen to O•−2by both a flavoenzyme-containing NADPH complex and the mitochondria electron transport chain. The O•−2in turn generated H2O2, which reacted with vanadium(IV) to generate ·OH radical through a Fenton-type reaction (V(IV) + H2O2→ V(V) + ·OH + OH−). The ROS generated by vanadate induced G2/M phase arrest in a time- and dose-dependent manner as determined by measuring DNA content. Vanadate also increased p21 and Chk1 levels and reduced Cdc25C expression, leading to phosphorylation of Cdc2 and a slight increase in cyclin B1expression as analyzed by Western blot. Catalase, a specific antioxidant for H2O2, decreased vanadate-induced expression of p21 and Chk1, reduced phosphorylation of Cdc2Tyr15, and decreased cyclin B1levels. Superoxide dismutase, a scavenger of O•−2, or sodium formate, an inhibitor of ·OH, had no significant effects. The results obtained from the present study demonstrate that among ROS, H2O2is the species responsible for vanadate-induced G2/M phase arrest. Several regulatory pathways are involved: (1) activation of p21, (2) an increase of Chk1 expression and inhibition of Cdc25C, which results in phosphorylation of Cdc2 and possible inactivation of cyclin B1/Cdc2 complex.

The present study investigated the role of reactive oxygen species (ROS) in activation of nuclear factor of activated T cells (NFAT), a pivotal transcription factor responsible for regulation of cytokines, by vanadium in mouse embryo fibroblast PW cells or mouse epidermal Cl 41 cells. Exposure of cells to vanadium led to the transactivation of NFAT in a time- and dose-dependent manner. Scavenging of vanadium-induced H2O2withN-acety-l-cyteine (a general antioxidant) or catalase (a specific H2O2inhibitor) or the chelation of vanadate with deferoxamine, resulted in inhibition of NFAT activation. In contrast, an increase in H2O2generation by the addition of superoxide dismutase or NADPH enhanced vanadium-induced NFAT activation. This vanadate-mediated H2O2generation was verified by both electron spin resonance and fluorescence staining assay. These results demonstrate that H2O2plays an important role in vanadium-induced NFAT transactivation in two different cell types. Furthermore, pretreatment of cells with nifedipine, a calcium channel blocker, inhibited vanadium-induced NFAT activation, whereas A23187and ionomycin, two calcium ionophores, had synergistic effects with vanadium for NFAT induction. Incubation of cells with cyclosporin A (CsA), a pharmacological inhibitor of the phosphatase calcineurin, blocked vanadium-induced NFAT activation. All data show that vanadium induces NFAT activation not only through a calcium-dependent and CsA-sensitive pathway but also involved H2O2generation, suggesting that H2O2may be involved in activation of calcium-calcineurin pathways for NFAT activation caused by vanadium exposure.

Arsenic compounds are a somewhat unique class of metals, which have been considered as both carcinogens and chemotherapeutic agents for cancers. Tumor promotion effects of arsenic are believed to be associated with its transactivational activities on transcription factors, such as AP‐1 and NFκB, while the induction of cell apoptosis and differentiation by arsenic is considered to be a mechanism for the chemotherapeutic effects of arsenic. Here, we found that exposure of cells to arsenite and arsenate leads to transactivation of retinoic acid response elements (RARE) and glucocorticoid response elements (GRE) in mouse epidermal JB6 cells. These inductions occur in a time‐dependent manner. Furthermore, induction of RARE activity by arsenic was synergistically enhanced by co‐treatment of cells with retinoic acid, while GRE activation by arsenic was not affected by combined treatment of cells with fluocinolone acetonide (FA). In consideration of the important role of RARE and GRE in induction of cell differentiation, we speculate that transactivation of RARE and GRE by arsenic may be involved in its induction of cell differentiation and anti-cancer activities in addition to its induction of apoptosis.

Cadmium is a potent and effective carcinogen in rodents and has recently been accepted by IARC (International Agency for Research on Cancer) as a category 1 carcinogen. Cadmium-induced up‐regulation of intracellular signaling pathways leading to increased mitogenesis is thought to be a major mechanism for the carcinogenic activity following chronic cadmium exposure. In the present study, we found that exposure of cells to cadmium induced significant activation of AP‐1 and all three members of the MAP kinase family in mouse epidermal JB6 cells. The induction of AP‐1 activity by cadmium appears to involve activation of Erks, since the induction of AP‐1 activity by cadmium was blocked by pretreatment of cells with PD98058. Interestingly, the induction of AP‐1 by cadmium was greatly enhanced by the chemical tumor promoter, TPA and the growth factor EGF, but not by ultraviolet C radiation. In vivostudies demonstrated that cadmium could also induce transactivation of AP‐1 in AP‐1‐luciferase report transgenic mice. Considering the role of AP‐1 activation in tumor promotion, the results presented in this study provide a possible molecular mechanism for cadmium‐induced carcinogenesis.

Tumor promoting effects of arsenic are believed to be associated with its transactivation activity on transcription factors, such as AP‐1 and NFκB. However, the results from different groups studying the effects of arsenic on NFκB activation are contradictory in different cell models. Since arsenic is a strong skin carcinogen, we have investigated the activation of NFκB by arsenic in a mouse skin epidermal cell line, JB6 cells. Exposure of cells to arsenite or arsenate led to NFκB transactivation in mouse epidermal JB6 NFκB‐luciferase reporter stable transfectants, C141 NFκB mass1. This induction of NFκB activity by arsenic was dose‐ and time‐dependent. The transactivation of NFκB by arsenic appeared to be through activation of Erks and JNKs pathways because increased NFκB activity by arsenic could be dramatically inhibited by either pre‐treatment of cells with PD98059 or overexpression of dominant negative JNK1. That Erks activation is required for arsenic‐induced NFκB transactivation was further supported by the findings that arsenic‐induced NFκB transactivation was impaired in JB6 30.7b cells, which were deficient in Erks.

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms controlling vanadate-induced adverse effects remain to be elucidated. The present study investigated the vanadate-induced p53 activation and involvement of reactive oxygen species (ROS) in p53 activation as well as the role of p53 in apoptosis induction by vanadate. Exposure of mouse epidermal JB6 cells to vanadate led to transactivation of p53 activity in a time- and dose-dependent manner. It also caused mitochondrial damage, apoptosis, and generated ROS. Scavenging of vanadate-induced H2O2byN-acetyl-l-cysteine (a general antioxidant) or catalase (a specific H2O2inhibitor), or the chelation of vanadate by deferoxamine, resulted in inhibition of p53 activation and cell mitochondrial damage. In contract, an increase in H2O2generation in response to superoxide dismutase or NADPH enhanced these effects caused by vanadate. Furthermore, vanadate-induced apoptosis occurred in cells expressing wild-type p53 (p53+/+) but was very weak in p53-deficient (p53−/−) cells. These results demonstrate that vanadate induces p53 activation mainly through H2O2generation, and this activation is required for vanadate-induced apoptosis.

Signal transduction viamitogen-activated protein kinase pathways plays a key role in a variety of cellular responses, including cell proliferation, differentiation, tumor promotion, and cell death. c-Jun N-terminal kinases (JNKs) are identified as members of the mitogen-activated protein kinase family and are known to phosphorylate and activate several transcription factors, including c-Jun, ATF, and Elk-1. However, the role of JNK activation in tumor promotion is not yet defined. Because previous studies have indicated that exposure of JB6 Cl 41 cells to either 12-O-tetradecanoylphorbol-13-acetate (TPA) or tumor necrosis factor-α (TNF-α) results in cell transformation, we investigated the role of JNKs in this biological process by using dominant negative JNK1and the cell transformation model JB6 Cl 41 cells. Incubation of Cl 41 cells with TNF-α led to cell transformation and activation of JNKs. Introduction of the dominant negative mutant of JNK1into JB6 Cl 41 cells specifically inhibited TNF-α-induced activation of JNKs, but not Erks and p38 kinases. Most importantly, expressing dominant negative mutant JNK1inhibited TNF-α-induced cell transformation but not TPA-induced cell transformation. Our results directly demonstrated for the first time that JNK activation is required for TNF-α- but not TPA-induced cell transformation.

Trivalent arsenic (arsenite, As3+) is a human carcinogen, which is associated with cancers of skin, lung, liver, and bladder. However, the mechanism by which arsenite causes cancer is not well understood. In this study, we found that exposure of Cl 41 cells, a well characterized mouse epidermal cell model for tumor promotion, to a low concentration of arsenite (<25 μm) induces cell transformation. Interestingly, arsenite induces Erk phosphorylation and increased Erk activity at doses ranging from 0.8 to 200 μm, while higher doses (more than 50 μm) are required for activation of JNK. Arsenite-induced Erk activation was markedly inhibited by introduction of dominant negative Erk2 into cells, while expression of dominant negative Erk2 did not show inhibition of JNK and MEK1/2. Furthermore, arsenite-induced cell transformation was blocked in cells expressing the dominant negative Erk2. In contrast, overexpression of dominant negative JNK1 was shown to increase cell transformation even though it inhibits arsenite-induced JNK activation. Our results not only show that arsenite induces Erk activation, but also for the first time demonstrates that activation of Erk, but not JNK, by arsenite is required for its effects on cell transformation.

Alcohol abuse is a risk factor for cancers of the gastrointestinal tract, and it also can precipitate psoriasis characterized by hyperproliferation of epidermal cells. Because these effects of alcohol may involve stimulation of cell growth, and ethanol (EtOH) was shown to enhance DNA synthesis in mouse fibroblasts and epidermal cells, we conducted a study to determine whether EtOH can also stimulate mitogenesis in human fibroblasts and keratinocytes. In keratinocytes, EtOH had no effects on mitogenesis after shorter (17‐hr) treatments, but it partially prevented inhibition of DNA synthesis elicited by longer treatments (3‐4 days) with 2 mM calcium (Ca2+), a differentiation‐inducing agent. In contrast, treatment of serum‐starved zinc‐treated (40 μM) human skin fibroblasts with 50‐60 mM EtOH for 17 hr resulted in increased DNA synthesis. EtOH‐induced DNA synthesis was blocked by 1 mM EGTA, a specific Ca2+chelator. Despite the presence of 1.8 mM Ca2+in the cell culture medium, the addition of 1 mM extra Ca2+(final concentration, 2.8 mM) for 17 hr induced DNA synthesis, presumably mediated by Ca2+receptors. In eight independent human skin fibroblast lines examined, treatment with EtOH for 46 hr, but not for 17 hr, invariably enhanced the effects of Ca2+on DNA synthesis, consistent with synergistic stimulation of cell proliferation by EtOH and Ca2+. Neomycin, a Ca2+receptor agonist, and EtOH also exerted synergistic effects on DNA synthesis after longer (46‐hr) treatments. In mouse NIH 3T3 fibroblasts, both EtOH‐ and Ca2+‐enhanced DNA synthesis after 17‐hr treatment, but they stimulated cell proliferation only in combination. The results indicate that in human fibroblasts, EtOH can potentiate the longer‐term effects of high concentrations of Ca2+on DNA synthesis whereas, in keratinocytes, EtOH may inhibit Ca2+‐induced differentiation.

Introduction: Donor plasmacytoid dendritic cells (pDCs) have important roles in the pathogenesis of graft versus host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Vasoactive intestinal peptide (VIP), a potent anti-inflammatory neuropeptide with pleiotropic effects, has shown promise in managing GvHD through induction of regulatory dendritic cells (Chorny, Blood 2006) and regulatory T cells (Delgado, JLB 2005). Our previous data showed that transplantation of hematopoietic stem cells (HSC) lacking VIP expression in enhanced Th1 polarization and antiviral immunity (Li, JI 2011). Whether VIP produced by pDCs plays a key role in preventing GvHD is the focus of the current study.

Waller: Amgen: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Other: Travel expenses, Research Funding; Cerus Corporation: Other: Stock, Patents & Royalties; Chimerix: Other: Stock; Cambium Oncology: Patents & Royalties: Patents, royalties or other intellectual property ; Kalytera: Consultancy.

Waller: Celldex: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cambium Medical Technologies: Consultancy, Equity Ownership; Kalytera: Consultancy; Pharmacyclics: Other: Travel Expenses, EHA, Research Funding.

Introduction:In allogeneic hematopoietic stem cell transplant (AHSCT), donor T cells promote hematopoietic engraftment, reconstitute T-cell immunity and mediate potent beneficial antitumor effects, such as graft versus leukemia (GVL) as well as detrimental graft-versus-host disease (GVHD). We have shown that purified bone marrow pDC to a donor graft composed of purified HSC and T cells significantly improved long-term leukemia-free survival without increasing the risk of GVHD (Lu BLOOD 2012). Vasoactive intestinal peptide (VIP) is a neuropeptide/neurotransmitter, which acts as a major anti-inflammatory factor in animal models of inflammatory and autoimmune diseases. VIP is produced by T cells and dendritic cells under conditions of inflammation (Li Cancer Research 2016). VIP-signaling, thus represents a newly appreciated co-inhibitory pathway involved in T cell activation and expansion and persistence of antigen-specific T cells, but the role of VIP produced by donor dendritic cells in allo-BMT is unclear. Our previous data has shown that production of VIP in pDC improves survival in a murine allo-BMT model. Since the thymus plays a critical role in regenerating naive T cells in which allo-reactive donor T cells undergoes central deletion. We hypothesized that donor pDCs that home to thymus modulate the negative selection of allo-reactive T cells and iTreg production through VIP signaling.

Introduction:Increased numbers of plasmacytoid dendritic cells (pDC) in the bone marrow graft improve clinical outcomes with decreased graft-versus-host disease (GvHD) and improved survival in previous pre-clinical murine experiments (Lu Y, Blood. 2012;119:1075-1085) and in clinic data from BMT CTN 201 (Waller JCO 2014; 32:2365-72). The mechanism by which donor pDC limit severe GvHD and improve survival after allo-BMT is unknown. We have previously described higher levels of donor T cell activation and expansion (measured as bio-luminescent radiance) among recipients of VIP-KO pDC (Li, J.X Blood 2016 128:2154). We hypothesize that blocking VIP signaling augment the magnitude of T cell activation and expansion, and that modulating the VIP pathway may be a potential therapeutic target for regulating GvHD in allo-HSCT.

Introduction