Dependent Signal Transduction Research Articles

Identification and characterization of the cDNAs encoding the two subunits of Chinese mitten crab (Eriocheir sinensis) calcineurin: Their implications in stress and immune response

Calcineurin (CN), the only Ca2+/calmodulin-activated serine/threonine protein phosphatase, is a key effector participating in Ca2+-dependent signal transduction pathways in a number of cellular processes under normal, stress and pathological conditions. However, the expression and the relevance of CN in stress and immune response have not been characterized in crustaceans. Here, we identified the cDNAs that encode the two subunits of CN (termed EsCN-A and EsCN-B, respectively) in Chinese mitten crab Eriocheir sinensis and analysed their expression patterns in response to stress and immune challenges. The catalytic subunit EsCN-A is comprised of 511 amino acids with a theoretical molecular mass of 57.5 kDa and shows 80% sequence identity with human beings CN-A alpha isoform, while the regulatory subunit EsCN-B protein is composed of 170 amino acids with an estimated molecular mass of 19.3 kDa and shares 88% sequence identity with human beings CN-B type 1. Tissue distribution analysis reveals that both EsCN-A and EsCN-B mRNA transcripts are expressed in all tested tissues with the greatest expression in hepatopancreas and the lowest expression in haemocytes. In addition, both EsCN-A and EsCN-B genes could be significantly up-regulated but with different expression patterns by ambient salinity (15‰ and 30‰ salinities) and pH (pH 6 and 8.5) stresses in gill, hepatopancreas, haemocytes, intestine and muscle. Furthermore, EsCN-A and EsCN-B were up-regulated by LPS and Poly(I:C) immune stimulations in E. sinensis haemocytes in vitro. Moreover, EsCN-A and EsCN-B mRNA were significantly up-regulated in haemocytes, gill, hepatopancreas, intestine and muscle in response to Edwardsiella tarda challenge in vivo. Finally, we revealed the importance of EsCN in LPS-induced nitric oxide production in E. sinensis haemocytes. Together our observations suggest that EsCN, the important downstream effector of CaM-mediated signalling pathway(s), may possess vital roles in stress and immune response in the Chinese mitten crab.

Ca2+-dependent Signal Transduction

Ca2+-dependent Signal Transduction

RP5-833A20.1/miR-382-5p/NFIA-dependent signal transduction pathway contributes to the regulation of cholesterol homeostasis and inflammatory reaction.

Cardiovascular disease caused by atherosclerosis is the number one cause of death in Western countries and threatens to become the major cause of morbidity and mortality worldwide. Long noncoding RNAs are emerging as new players in gene regulation, but how long noncoding RNAs operate in the development of atherosclerosis remains unclear. Using microarray analysis, we found that long noncoding RNA RP5-833A20.1 expression was upregulated, whereas nuclear factor IA (NFIA) expression was downregulated in human acute monocytic leukemia macrophage-derived foam cells. Moreover, we showed that long noncoding RNA RP5-833A20.1 may decreases NFIA expression by inducing hsa-miR-382-5p expression in vitro. We found that the RP5-833A20.1/hsa-miR-382-5p/NFIA pathway is essential to the regulation of cholesterol homeostasis and inflammatory responses in human acute monocytic leukemia macrophages. Lentivirus-mediated NFIA overexpression increased high-density lipoprotein cholesterol circulation, reduced low-density lipoprotein cholesterol, and very-low-density lipoprotein cholesterol circulation, decreased circulation of inflammatory cytokines, including interleukin-1β, interleukin-6, tumor necrosis factor-α, and C-reactive protein, enhanced reverse cholesterol transport, and promoted regression of atherosclerosis in apolipoprotein E-deficient mice. Our findings indicated that the RP5-833A20.1/miR-382-5p/NFIA pathway was essential to the regulation of cholesterol homeostasis and inflammatory reactions and suggested that NFIA may represent a therapeutic target to ameliorate cardiovascular disease.

Relationship between nitric oxide- and calcium-dependent signal transduction pathways in growth hormone release from dispersed goldfish pituitary cells

Nitric oxide (NO) and Ca2+ are two of the many intracellular signal transduction pathways mediating the control of growth hormone (GH) secretion from somatotropes by neuroendocrine factors. We have previously shown that the NO donor sodium nitroprusside (SNP) elicits Ca2+ signals in identified goldfish somatotropes. In this study, we examined the relationships between NO- and Ca2+-dependent signal transduction mechanisms in GH secretion from primary cultures of dispersed goldfish pituitary cells. Morphologically identified goldfish somatotropes stained positively for an NO-sensitive dye indicating they may be a source of NO production. In 2h static incubation experiments, GH release responses to the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) were attenuated by CoCl2, nifedipine, verapamil, TMB-8, BHQ, and KN62. In column perifusion experiments, the ability of SNP to induce GH release was impaired in the presence of TMB-8, BHQ, caffeine, and thapsigargin, but not ryanodine. Caffeine-elicited GH secretion was not affected by the NO scavenger PTIO. These results suggest that NO-stimulated GH release is dependent on extracellular Ca2+ availability and voltage-sensitive Ca2+ channels, as well as intracellular Ca2+ store(s) that possess BHQ- and/or thapsigargin-inhibited sarcoplasmic/endoplasmic reticulum Ca2+-ATPases, as well as TMB-8- and/or caffeine-sensitive, but not ryanodine-sensitive, Ca2+-release channels. Calmodulin kinase-II also likely participates in NO-elicited GH secretion but caffeine-induced GH release is not upstream of NO production. These findings provide insights into how NO actions many integrate with Ca2+-dependent signalling mechanisms in goldfish somatotropes and how such interactions may participate in the GH-releasing actions of regulators that utilize both NO- and Ca2+-dependent transduction pathways.

Suppression effect of lithium on astroglial differentiation through JAK/STAT3 dependent signal transduction pathway

Objective To investigate the effect of Lithium on astrogliogenesis by neural stem/progenitor cells (NSCs) and its canonical regulating JAK/STAT3 singal transduction pathway.Methods Under NSCs differentiating condition,we firstly detected the P-Tyr-705 STAT3 among total STAT3 expression to test the lithium effect on JAK/STAT3 activation,then we used AICAR,which was an specific STAT3 agonist,to up-regulate STAT3 activity.After validating the effect of AICAR on inducing STAT3 activation and astrogliogenesis,we tested whether Lithium could block the effect of AICAR by detecting P-Tyr-705 STAT3 and GFAP expression.Results Lithium dramatically reduced the JAK/STAT3 activation of differentiating NSCs,P-Tyr-705 STAT3 expression increased by 17 folds (P ＜0.01).Pre-treatment of 3 mmol/L Lithium abolished the induced JAK/STAT3 activation after 24h and GFAP expressing on 3 days (P ＜ 0.05),as detected by Western blotting and immunocytochemistry.Conclusions Lithium could suppress astroglial differentiation through JAK/STAT3 dependent signal transduction pathway. Key words: Lithium; Astrocytes ; Neural stem cells ; STAT3 transcription factor

Sesquiterpene dimmer (DSF-27) inhibits the release of neuroinflammatory mediators from microglia by targeting spleen tyrosine kinase (Syk) and Janus kinase 2 (Jak2): Two major non-receptor tyrosine signaling proteins involved in inflammatory events

Non-receptor protein tyrosine kinases (NRPTKs)-dependent inflammatory signal transduction cascades play key roles in immunoregulation. However, drug intervention through NRPTKs-involved immunoregulation mechanism in microglia (the major immune cells of the central nervous system) has not been widely investigated. A main aim of the present study is to elucidate the contribution of two major NRPTKs (Syk and Jak2) in neuroinflammation suppression by a bioactive sesquiterpene dimmer (DSF-27). We found that LPS-stimulated BV-2 cells activated Syk and further initiated Akt/NF-κB inflammatory pathway. This Syk-dependent Akt/NF-κB inflammatory pathway can be effectively ameliorated by DSF-27. Moreover, Jak2 was activated by LPS, which was followed by transcriptional factor Stat3 activation. The Jak2/Stat3 signal was suppressed by DSF-27 through inhibition of Jak2 and Stat3 phosphorylation, promotion of Jak/Stat3 inhibitory factors PIAS3 expression, and down-regulation of ERK and p38 MAPK phosphorylation. Furthermore, DSF-27 protected cortical and mesencephalic dopaminergic neurons against neuroinflammatory injury. Taken together, our findings indicate NRPTK signaling pathways including Syk/NF-κB and Jak2/Stat3 cascades are potential anti-neuroinflammatory targets in microglia, and may also set the basis for the use of sesquiterpene dimmer as a therapeutic approach for neuroinflammation via interruption of these pathways.

Induction of Phagocytosis and Intracellular Signaling by an Inhibitory Channel Catfish Leukocyte Immune-Type Receptor: Evidence for Immunoregulatory Receptor Functional Plasticity in Teleosts

Immunoregulatory receptors are categorized as stimulatory or inhibitory based on their engagement of unique intracellular signaling networks. These proteins also display functional plasticity, which adds versatility to the control of innate immunity. Here we demonstrate that an inhibitory catfish leukocyte immune-type receptor (IpLITR) also displays stimulatory capabilities in a representative myeloid cell model. Previously, the receptor IpLITR 1.1b was shown to inhibit natural killer cell-mediated cytotoxicity. Here we expressed IpLITR 1.1b in rat basophilic leukemia-2H3 cells and monitored intracellular signaling and functional responses. Although IpLITR 1.1b did not stimulate cytokine secretion, activation of this receptor unexpectedly induced phagocytosis as well as extracellular signal-related kinase 1/2- and protein kinase B (Akt)-dependent signal transduction. This novel IpLITR 1.1b-mediated response was independent of an association with the FcRγ chain and was likely due to phosphotyrosine-dependent adaptors associating with prototypical signaling motifs within the distal region of its cytoplasmic tail. Furthermore, compared to a stimulatory IpLITR, IpLITR 1.1b displayed temporal differences in the induction of intracellular signaling, and IpLITR 1.1b-mediated phagocytosis had reduced sensitivity to EDTA and cytochalasin D. Overall, this is the first demonstration of functional plasticity for teleost LITRs, a process likely important for the fine-tuning of conserved innate defenses.

Cellular Metabolism of Estradiol in Models for Select Molecular Subtypes of Clinical Breast Cancer

The mitogenic ovarian steroid hormone 17β-estradiol (E2) is associated with progression of Estrogen Receptor Positive (ER+) breast cancer, and ER represents a major therapeutic target for endocrine therapy. In addition to the ER dependent signal transduction, cellular oxidative metabolism of the ER ligand E2 generates several metabolites with pleotropic growth modulatory effects on breast cancer cells, providing valuable leads for novel therapeutic approach. The molecular classification of clinical breast cancer has defined cancer subtypes based on differential expression of the genes for hormones and growth factor receptors, thereby facilitating subtype targeted therapeutic interventions. However, de novo or acquired resistance to conventional endocrine and targeted small molecule based treatment limits the therapeutic efficacy and promotes therapy resistant disease progression. These aspects emphasize the need for identification of new efficacious non-toxic lead compounds. The present review summarizes critical experiments conducted to i) develop and optimize human tissue derived cell culture models for select molecular subtypes of clinical breast cancer, ii) determine the status of homeostatic growth control, cellular metabolism of 17β-estradiol (E2) and cancer risk in the developed models, and iii) evaluate the therapeutic efficacy and identify possible mechanisms of action of select herbal extracts/phyto-chemical. Additionally, this review discusses the evidence for the role of E2 metabolism in breast carcinogenesis and therapy. The data generated from the cell culture experiments demonstrate that the models for select molecular sub-types exhibit aberrant hyperproliferation, altered cellular metabolism of E2 and enhanced cancer risk. Select mechanistically distinct herbal extracts and natural phyto-chemical at their respective maximum cytostatic concentrations modulate cellular metabolism of E2 favoring generation of anti-proliferative metabolites and inhibit anchorage independent growth, thus reducing cancer risk. Collectively, these data validate the present mechanism based cell culture approach to identify and prioritize novel efficacious lead compounds for subtype targeted therapy of clinical breast cancer.

Key Nodes In FLT3 Dependent Signaling Determine Growth and Survival Of Childhood Acute Lymphoblastic Leukemia

The balanced regulation of complex signaling networks plays an important role in cell proliferation, survival and apoptosis. Receptor tyrosine kinase (RTK) dependent signal transduction has been implicated in the pathogenesis of many malignancies including acute lymphoblastic leukemia (ALL) of childhood. Moreover, deregulated RTK activity was observed following targeted inhibition of constitutively active kinases or kinase-dependent pathways in a variety of malignancies, which conferred pharmacological resistance. In ALL, fms-like-tyrosine kinase 3 (FLT3) and platelet-derived growth factor receptor beta (PDGFRβ) are targeted for mutation (Roberts et al., 2012). However, in primary ALL the RTK dependent signaling state is poorly defined and the occurrence of relapse in the context of genotype-directed monotherapy regimens targeting RTKs, such as FLT3, underlines the need for an activity-based approach to RTK signaling in leukemia comprising the identification of critical downstream target proteins.To select for driver RTKs in ALL, we combined the analyses of RTK expression in primary ALL (n=102), ALL cell lines and normal hematopoietic cells with the characterization of ligand dependent cell proliferation as well as shRNA mediated RTK repression in vitro and in vivo. We observed aberrant RTK expression patterns in ALL cells compared to normal lymphoid progenitor and stem cell populations as well as mature T- and B-lymphocytes. RNA interference mediated repression of growth promoting RTKs FLT3 and PDGFRβ in primary ALL led to a loss or reduction of the affected cell population in vivo. To identify critical signaling nodes we performed a phosphoproteomic characterization by iTRAQ (isobaric tags for relative and absolute quantification)-based mass spectrometry of the signal transduction of selected driver RTKs in the corresponding primary ALL samples. Primary ALL cells were propagated in NSG mice after xenotransplantation, and regulated phosphoproteins were identified after ligand stimulation. Our network-directed approach to RTK signaling in ALL thus allowed for the identification of downstream signaling nodes implicated in aberrant RTK activity.We identified a total of 2241 phosphoproteins and observed a striking diversity of RTK driven signaling processes in primary ALL exhibiting only a marginal overlap between phosphoregulated proteins which illustrates the inter-individual heterogeneity and the challenge for non-combinatorial therapies. Despite a predominant receptor and cell type specific composition of potentiated signaling networks our phosphoproteomic analyses identified p21-activated protein kinase PAK2 as a novel key nodal point in FLT3 dependent signaling in ALL. The importance of PAK protein family members in the regulation of cell proliferation and survival and the emerging role of PAK proteins in the pathogenesis of a broad range of tumors suggests a hitherto unanticipated function in the malignant transformation of ALL and the signal transduction of FLT3 (Ye and Field, 2012).As a kinase PAK2 represents a druggable target and may be suited for combinatorial intervention strategies targeting FLT3 signaling in order to induce synthetic lethality. Inhibition of group I PAK kinases (PAK1, 2, 3) using the allosteric inhibitor IPA-3 and RNA interference mediated repression of PAK2 led to the loss of ALL cells due to an impaired cell proliferation and an increased apoptosis. Notably, PAK2 depleted ALL cells showed an elevated sensitivity towards pharmacological FLT3 inhibition which underlines the potential role of PAK2 as a novel target in ALL and the need for novel small molecule PAK inhibitors with higher specificity and improved applicability in vivo. Disclosures:No relevant conflicts of interest to declare.

Cyclic GMP regulates M3AChR activity at plasma membranes from Airway smooth muscle

Muscarinic acetylcholine receptors MAChRs from Bovine Tracheal Smooth Muscle (BTSM) plasma membranes are responsible for the cGMP rise and signal-amplitude peaks associated with smooth muscle contraction present in bronchial asthma. These MAChRs bind [(3)H]QNB and exhibit the classic G Protein Coupled-Receptor (GPCR) behavior towards muscarinic agonist and antagonists that is sensitive to sensitive to GTP analogs. Interestingly, the [(3)H]QNB binding activity was stimulated by cGMP and ATP, and was enhanced by IBMX and Zaprinast, inhibitors of cGMP-PDE. Cyclic GMP plus ATP affected the agonist-antagonist muscarinic binding activities. Thus, the high affinity agonist (Carbamylcholine) binding sites disappeared, whereas, 4-DAMP, a M3 selective antagonist displayed an additional high affinity-binding site. In contrast, non-selective (atropine) and M2-selective (methoctramine and gallamine) antagonists revealed one low binding site. Moreover, the 4-DAMP-mustard alkylation of the MAChRs blocked the cGMP effect indicating that the M3AChR is the main receptor target of cGMP. Interestingly, these cGMP effects were potentiated by an activator (Sp-8-pCPT-cGMPS), and diminished by an inhibitor (Rp-8-pCPT-CGMPS), of cGMP-dependent protein kinase (PKG-II), which was detected by Western blotting using specific PKG II antibodies. Finally, plasma membrane M3AChRs were phosphorylated in a cGMP-dependent manner and this novel post-translational reversible modification at M3AChRs may act as a feedback mechanism to terminate the cGMP dependent muscarinic signal transduction cascades at the sarcolema of BTSM.

Abstract A233: Pharmacodynamic Transcriptional markers of carboxyamidotriazole orotate (CTO) exposure in anagen hair.

Abstract Background: Carboxyamidotriazole Orotate (CTO) is the orotic acid salt of carboxyamodotriazole (CAI)which is an inhibitor of calcium -dependent intracellular and extracellular signal transduction pathways, also possessing antiproliferative, antiangiogenic and anti-invasive properties. The activity of CTO alone and combination with temozolomide or 5-fluorouracil was demonstrated in human glioblastoma, melanoma and colon tumor xenografts. In Phase I (NCT01107522), nine patients pretreated with 2 to 8 targeted and non-targeted drugs and having refractory tumors responded to CTO at doses ranging 75mg/m2/day through 427mg/m2/day, and achieved stable disease for different periods (3 to 14 months). Four pretreated tumors were found to have different genomic mutations (PI3KCA, EGFR multiple, BRAFV600 and NRAS) consistent with CTO's suggested mechanism of action to inhibit multiple tyrosine kinases in addition to modulating calcium signaling pathways and calcium-dependent signal transduction pathways, as yet uncharacterized. Method: Using Epistem's ex vivo plucked anagen hair assay platform global gene expression evaluation by microarray analysis was performed to assess transcriptional response to varying doses of CTO (2, 5 and10 μM). Plucked anagen scalp hairs from five healthy donors were exposed to varying doses of CTO over a 24 hr period. Total RNA was isolated form the hair bulbs post culture and used to assess global transcriptional alterations. Results:Significant biologically relevant alteration of the anagen hair bulb transcriptome, ranging from -100 fold to +25 fold differential expression of some mRNAs was observed at CTO levels shown to be clinically relevant. Importantly, transcriptional signatures associated with inhibition of EGFR, MEK, HDAC and HSP90 were strongly suppressed at all doses of CTO. In addition, genes associated with non-voltage dependent calcium signaling were strongly suppressed, together with those associated with RAS and Growth Factor Signature. Modest suppression of transcription signatures of WNT signaling was evident at longer CTO exposure. In contrast, activation of tumor suppressor signatures associated with P53 was observed. Conclusion: These data provide multiple pharmacodynamics markers associated with the suggested mechanism of action of CTO e.g., inhibition of genes of non-voltage dependent calcium signaling and multiple onco-pathways, and also activation of signatures associated with tumor suppressors. These results in the anagen hair assay may provide the molecular MOA of CTO's observed clinical benefit in a broad spectrum of malignant tumors with different genomic types and a tool to design customized combination therapies of CTO with other agents. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A233. Citation Format: Rashida A. Karmali, Gino Miele. Pharmacodynamic Transcriptional markers of carboxyamidotriazole orotate (CTO) exposure in anagen hair. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A233.

KHARON1 Mediates Flagellar Targeting of a Glucose Transporter in Leishmania mexicana and Is Critical for Viability of Infectious Intracellular Amastigotes

The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle.

FRI0044 IL-1 dependent SYNDECAN-4 signal transduction is mediated by its heparan sulfate side chains in RA synovial fibroblasts

BackgroundIn rheumatoid arthritis (RA) pathogenesis, activated synovial fibroblasts (SFs) play an important role, because of their tumor-like and agressive behavior. This phenotype is promoted by a steady stimulation with growth-factors...

Different classes of CB1 ligands bias CB1‐dependent signal transduction

Biased agonism describes the ligand‐dependent selectivity of different signal transduction pathways by molecules that act as agonists at the same receptor. This functional selectivity may be exploited to alter the balance between signaling pathways downstream of a receptor. Agonists of the type 1 cannabinoid receptor (CB1) are structurally diverse and include the aminoalkylindole WIN55,212–2, the endocannabinoid N‐arachidonoyl ethanolamine (anadamide), and the Δ9‐tetrahydrocannabinol (THC)‐like, CP55,940. Given the differences in potency and efficacy of these agonists, we sought to determine whether activation of CB1 by these ligands resulted in biased signal transduction in a cell culture model of striatal neurons. We found that the THC‐like compound CP55,940 enhanced the interaction between CB1 and β‐arrestin2 compared to vehicle, anandamide, and WIN55,212–2, which resulted in CB1 internalization and persistent pERK signalling. In contrast, the endocannaibnoid anandamide and the aminoalkylindole WIN55,212–2 enhanced the interaction between CB1 and Gαi, and promoted a rapid and transient increase in pERK signaling compared to vehicle or CP55,940. Based on these data, the therapeutic efficacy of cannabinoid agonists may be maximized by selecting agents with specific potency, affinity, and signaling bias.

Epigenetic regulation of cholinergic receptor M1 (CHRM1) by histone H3K9me3 impairs Ca2+ signaling in Huntington’s disease

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by an expanded trinucleotide CAG repeat in the gene coding for huntingtin. Deregulation of chromatin remodeling is linked to the pathogenesis of HD but the mechanism remains elusive. To identify what genes are deregulated by trimethylated histone H3K9 (H3K9me3)-dependent heterochromatin, we performed H3K9me3-ChIP genome-wide sequencing combined with RNA sequencing followed by platform integration analysis in stable striatal HD cell lines (STHdhQ7/7 and STHdhQ111/111) cells. We found that genes involving neuronal synaptic transmission including cholinergic receptor M1 (CHRM1), cell motility, and neuronal differentiation pathways are downregulated while their promoter regions are highly occupied with H3K9me3 in HD. Moreover, we found that repression of CHRM1 gene expression by H3K9me3 impairs Ca(2+)-dependent neuronal signal transduction in stable cell lines expressing mutant HD protein. Thus, our data indicate that the epigenetic modifications, such as aberrant H3K9me3-dependent heterochromatin plasticity, directly contribute to the pathogenesis of HD.

Overexpression of CDCA2 in Human Squamous Cell Carcinoma: Correlation with Prevention of G1 Phase Arrest and Apoptosis

Cell division cycle associated 2 (CDCA2) recruits protein phosphatase 1 to chromatin to antagonize activation of ataxia telangiectasia mutated (ATM)-dependent signal transduction. ATM kinase plays a critical role in the DNA damage response and its phosphorylation cascade to inhibit the p53-MDM2 interaction, which releases p53 to induce p21 and G1 cell-cycle arrest. However, the relevance of CDCA2 to human malignancy including oral squamous cell carcinoma (OSCC) is unknown. In the current study, we found that CDCA2 expression was up-regulated in OSCC cell lines. Functional studies with shRNA system showed that knockdown of CDCA2 significantly (P<0.05) inhibited cellular proliferation compared with the control cells by arresting cell-cycle progression at the G1 phase and up-regulating the cyclin-dependent kinase inhibitors (p21Cip1, p27Kip1, p15INK4B, and p16INK4A). CDCA2 knockdown also promoted apoptosis after treatment with the DNA damage reagent, cisplatin. In clinical samples, the CDCA2 protein expression level in primary OSCCs was significantly (P<0.05) greater than in matched normal oral tissues (67/85, 79%). Furthermore, CDCA2-positive cases were correlated significantly (P<0.05) with high cancer progression. Our results showed for the first time that CDCA2 frequently is overexpressed in OSCCs and might be associated closely with OSCC progression by preventing cell-cycle arrest and apoptosis.

Synthesis and Nrf2-inducing activity of the isothiocyanates iberverin, iberin and cheirolin

Numerous studies have reported a potent induction of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent gene expression by the isothiocyanate sulforaphane. However, little is known regarding the Nrf2-inducing activities of the lower structure-related sulforaphane homologues, such as iberverin, iberin and cheirolin, which exhibit different sulfur oxidation states. Therefore, in this study we synthesized the isothiocyanates iberverin, iberin and cheirolin with a high yield and purity and determined their Nrf2-inducing activity in NIH3T3 fibroblasts. Iberverin, iberin and cheirolin significantly induced Nrf2 nuclear translocation. The increase in nuclear Nrf2 levels was accompanied by a significant increase in heme oxygenase 1 (HO-1) and γ-glutamylcysteine synthetase (γGCS) mRNA and protein levels. Overall, iberverin, iberin and cheirolin exhibited a similar potency to sulforaphane in inducing Nrf2-dependent gene-expression. Furthermore, our data suggest that the induction of Nrf2 by iberverin, iberin and cheirolin may have occurred via an extracellular signal-related kinase (ERK)-dependent signal-transduction pathway.

Abstract B84: Activated c-Src enhances hypoxia in human glioblastoma multiforme and promotes tumor angiogenesis

Abstract Exhibiting substantial vascularization and propensity to infiltrate surrounding brain tissue, glioblastoma multiforme (GBM) is the most aggressive primary brain tumor. Even with aggressive intervention afflicted individuals uniformly receive poor prognosis. Growth factor receptor tyrosine kinases (RTKs) are frequently amplified, overexpressed, and constitutively activated in malignant gliomas, and because targeting these molecules has produced promising results in the treatment of several human neoplasms, RTKs are emerging as increasingly viable targets in effective treatment of GBM. Functional ambiguity between normal and constitutively activated RTK signalling compounds the challenges of identifying suitable targets, however. Likewise, the functions of many reported downstream signalling molecules, namely Src family non-receptor tyrosine kinases (SFKs), remain cryptic. Our lab has previously developed a clinically relevant in vivo model that reliably maintains the genomic profiles of corresponding patient tumors, and employing this model, we are able to accurately investigate mechanisms of RTK signalling in human GBM. Ongoing studies suggest that tumor overexpression and activation of the epidermal growth factor receptor (EGFR) promotes the invasive phenotype in human GBM potentially via c-Src dependent signal transduction. In fact, we have observed SFK activation downstream of EGFR. To further investigate this, we implanted GBM patient tumor cells from two individuals with distinct EGFR protein expression, tumor phenotype, and c-Src activity into the brains of nude rats. Minimally expressing EGFR tumor cells from a patient displaying an angiogenic in vivo phenotype were transduced to stably overexpress activated c-Src. Likewise, EGFR-overexpressing tumor cells from a patient displaying an invasive, non angiogenic in vivo phenotype were transduced to stably overexpress kinase-inactive-dominant-negative c-Src. To our surprise, compared with corresponding xenograft control tumors, xenograft tumors overexpressing constitutively activated c-Src showed substantially increased contrast enhancement on MRI, enhanced histological features of angiogenesis, and revealed marked increases in molecular markers of tumor hypoxia and angiogenesis on western blot. Meanwhile, compared with corresponding xenograft control tumors, xenograft tumors overexpressing kinase-inactive-dominant-negative c-Src did not show increased contrast enhancement on MRI, showed no histological evidence of angiogenesis, and maintained a non-angiogenic molecular profile on western blot. We hereby report that activation of the SFK c-Src enhances tumor hypoxia, promotes the aggressive angiogenic tumor phenotype, and is subsequently not likely a primary downstream SFK substrate of overexpressed EGFR in human GBM. Overexpression of wild type EGFR may, instead, signal through a different SFK to promote the invasive phenotype. These results suggest that, more so than invasion, c-Src activation may be a valuable diagnostic and prognostic indicator of malignant progression. Furthermore, these results indicate that c-Src activation may be a strategic target in the therapeutic intervention of human malignant glioma. Citation Format: Eskil Eskilsson, Krishna M. Talasila, Gro V. Røsland, Lina Leiss, Narve Brekkå, Per Øystein Sakariassen, Per Øyvind Enger, Rolf Bjerkvig, Hrvoje Miletic. Activated c-Src enhances hypoxia in human glioblastoma multiforme and promotes tumor angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B84.

Autonomous changes in the swimming direction of sperm in the gastropodStrombus luhuanus

The sperm of the gastropod Strombus luhuanus show dimorphism. The eusperm have a nucleus and fertilize the egg, whereas the other type of sperm, parasperm, are anucleate and are thought to assist fertilization. Here we report the autonomous changes in the swimming pattern of S. luhuanus eusperm. In artificial seawater, the eusperm collected from S. luhuanus sperm ducts formed sperm bundles and initially swam backward with asymmetric flagellar waveforms to detach from the bundles. One hour later, the sperm began to swim forward and in a circle. After an additional 1 h incubation, the sperm swam straight, with a change in the flagellar waveforms from asymmetric to symmetric. Spontaneous backward swimming with symmetric waveforms was also observed. The eusperm stored in the female seminal receptacle were motile and showed forward symmetric swimming with spontaneous backward swimming, which appeared necessary for detachment from the wall of receptacle. All of these motility changes were observed in the absence of parasperm, suggesting that these changes autonomously occur in eusperm. Our waveform analysis of these swimming patterns revealed that only the swimming with symmetric waveform showed reverse propagation of the flagellar waveforms. Both types of backward swimming were diminished in Ca(2+)-free seawater and in seawater containing Ni(2+), indicating the regulation of swimming direction by Ca(2+)-dependent signal transduction.

New insights into the molecular mechanisms underlying sensitivity/resistance to the atypical retinoid ST1926 in acute myeloid leukaemia cells: The role of histone H2A.Z, cAMP-dependent protein kinase A and the proteasome

ST1926 is an atypical retinoid and a promising anti-tumour agent with selective apoptotic activity on the leukaemic blast. The anti-tumour activity of the compound has been associated with its capacity to induce DNA double stranded breaks. Target profiling by affinity chromatography coupled to mass spectrometry led to the identification of histone H2A.Z as a protein capable of binding ST1926 specifically. The result was confirmed by studies involving Surface Plasmon Resonance (SPR). This indicates that H2A.Z is a primary target of ST1926 and links the perturbations of the histone pathway observed by microarray analysis to the DNA damage and apoptotic responses caused by the atypical retinoid. Comparison of the whole-genome gene-expression profiles of the ST1926-sensitive NB4 and the ST1926-resistant NB4.437r cell lines demonstrated differential expression of numerous genes. Network analysis of the data indicated enrichment of the cellular pathways controlling cAMP (cyclic adenosinemonophosphate)-dependent signal transduction, proteasome-dependent protein degradation and nuclear histones in NB4.437r cells. Pharmacological inhibition of cAMP-dependent protein kinase A with H89 partially reverted resistance of NB4.437r cells to ST1926. Conversely, inhibition of the proteasome with MG132 or bortezomib blocked the apoptotic response afforded by ST1926 in the NB4 cell line. This last effect was associated with a dramatic reduction in the DNA damage caused by the atypical retinoid. The results corroborate the idea that DNA damage is an important determinant of ST1926 apoptotic activity. More importantly, they demonstrate a proactive role of the proteasome in the DNA damaging and ensuing apoptotic response observed upon the challenge of acute myeloid leukaemia cells with ST1926.