The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, also known as ErbB or HER, plays crucial roles in the development of multicellular organisms. Mutations and over-expression of the ErbB receptors have been implicated in a variety of human cancers. It is widely thought that the ErbB receptors are located in the plasma membrane, and that ligand binding to the monomeric form of the receptors induces its dimeric form for activation. However, it still remains controversial whether prior to ligand binding the receptors exist as monomers or dimers on the cell surface. Using bimolecular fluorescence complementation (BiFC) assays in the present study, we demonstrate that in the absence of bound ligand, all the ErbB family members have preformed, yet inactive, homo- and heterodimers on the cell surface, except for ErbB3 homodimers and heterodimers with cleavable ErbB4, which exist primarily in the nucleus. BiFC assays of the dimerization have also suggested that the ligand-independent dimerization of the ErbB receptors occurs in the endoplasmic reticulum (ER) before newly synthesized receptor molecules reach the cell surface. Based on BiFC and mammalian two-hybrid assays, it is apparent that the intracellular domains of the receptors are responsible for the spontaneous dimer formation. These provide new insights into an understanding of transmembrane signal transduction mediated by the ErbB family members, and are relevant to the development of anti-cancer drugs.

2ϩ -activated potassium (IKCa) and volume-sensitive chloride (ICl.vol) channels, in regulating proliferation of mouse MSCs. We found that inhibition of IKCa with clotrimazole and ICl.vol with 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) reduced cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific short interference (si)RNAs significantly reduced IKCa or ICl.vol density and channel protein and produced a remarkable suppression of cell proliferation (by 24.4 Ϯ 9.6% and 29.5 Ϯ 7.2%, respectively, P Ͻ 0.05 vs. controls). Flow cytometry analysis showed that mouse MSCs retained at G0/G1 phase (control: 51.65 Ϯ 3.43%) by inhibiting IKCa or ICl.vol using clotrimazole (2 M: 64.45 Ϯ 2.20%, P Ͻ 0.05) or NPPB (200 M: 82.89 Ϯ 2.49%, P Ͻ 0.05) or the specific siRNAs, meanwhile distribution of cells in S phase was decreased. Western blot analysis revealed a reduced expression of the cell cycle regulatory proteins cyclin D1 and cyclin E. Collectively, our results have demonstrated that IKCa and ICl.vol channels regulate cell cycle progression and proliferation of mouse MSCs by modulating cyclin D1 and cyclin E expression.

The complete amino acid sequence of bovine S antigen (48-kDa protein) has been determined by cDNA and partial amino acid sequencing. A 1623-base-pair (bp) cDNA contains an open reading frame coding for a protein of 404 amino acids (45,275 Da). Tryptic peptides and cyanogen bromide peptides of native bovine S antigen were purified and partially sequenced. All of these peptides were accounted for in the long open reading frame. Searching of the National Biomedical Research Foundation data bank revealed no extensive sequence homology between S antigen and other proteins. However, there are local regions of sequence similarity with a transducin, including the sites subject to ADP-ribosylation by Bordetella pertussis and cholera toxins and the phosphoryl binding-sites. Secondary structure prediction and circular dichroic spectroscopy show that S antigen is composed predominantly of a-sheet conformation. Acid-catalyzed methanolysis suggests the presence of low levels of carbohydrate in the molecule.

BackgroundProgrammed death-1 (PD-1) immune checkpoint blockade has achieved clinical successes in cancer therapy. However, the response rate of anti-PD-1 agents remains low. Additionally, a subpopulation of patients developed hyperprogressive disease upon PD-1 blockade therapy. Combination therapy with targeted agents may improve immunotherapy. Recent studies show that p53 activation in the myeloid linage suppresses alternative (M2) macrophage polarization, and attenuates tumor development and invasion, leading to the hypothesis that p53 activation may augment antitumor immunity elicited by anti-PD-1 therapy.MethodUsing APG-115 that is a MDM2 antagonist in clinical development as a pharmacological p53 activator, we investigated the role of p53 in immune modulation and combination therapy with PD-1 blockade.ResultsIn vitro treatment of bone marrow-derived macrophages with APG-115 resulted in activation of p53 and p21, and a decrease in immunosuppressive M2 macrophage population through downregulation of c-Myc and c-Maf. Increased proinflammatory M1 macrophage polarization was observed in the spleen from mice treated with APG-115. Additionally, APG-115 has co-stimulatory activity in T cells and increases PD-L1 expression in tumor cells. In vivo, APG-115 plus anti-PD-1 combination therapy resulted in enhanced antitumor activity in Trp53wt, Trp53mut, and Trp53-deficient (Trp53−/−) syngeneic tumor models. Importantly, such enhanced activity was abolished in a syngeneic tumor model established in Trp53 knockout mice. Despite differential changes in tumor-infiltrating leukocytes (TILs), including the increases in infiltrated cytotoxic CD8+ T cells in Trp53wt tumors and M1 macrophages in Trp53mut tumors, a decrease in the proportion of M2 macrophages consistently occurred in both Trp53wt and Trp53mut tumors upon combination treatment.ConclusionOur results demonstrate that p53 activation mediated by APG-115 promotes antitumor immunity in the tumor microenvironment (TME) regardless of the Trp53 status of tumors per se. Instead, such an effect depends on p53 activation in Trp53 wild-type immune cells in the TME. Based on the data, a phase 1b clinical trial has been launched for the evaluation of APG-115 in combination with pembrolizumab in solid tumor patients including those with TP53mut tumors.

Ca(2+) signaling pathways are well studied in cardiac myocytes, but not in cardiac fibroblasts. The aim of the present study is to characterize Ca(2+) signaling pathways in cultured human cardiac fibroblasts using confocal scanning microscope and RT-PCR techniques. It was found that spontaneous intracellular Ca(2+) (Ca(i) (2+)) oscillations were present in about 29% of human cardiac fibroblasts, and the number of cells with Ca(i) (2+) oscillations was increased to 57.3% by application of 3% fetal bovine serum. Ca(i) (2+) oscillations were dependent on Ca(2+) entry. Ca(i) (2+) oscillations were abolished by the store-operated Ca(2+) (SOC) entry channel blocker La(3+), the phospholipase C inhibitor U-73122, and the inositol trisphosphate receptors (IP3Rs) inhibitor 2-aminoethoxydiphenyl borate, but not by ryanodine. The IP3R agonist thimerosal enhanced Ca(i) (2+) oscillations. Inhibition of plasma membrane Ca(2+) pump (PMCA) and Na(+)-Ca(2+) exchanger (NCX) also suppressed Ca(i) (2+) oscillations. In addition, the frequency of Ca(i) (2+) oscillations was reduced by nifedipine, and increased by Bay K8644 in cells with spontaneous Ca(2+) oscillations. RT-PCR revealed that mRNAs for IP3R1-3, SERCA1-3, Ca(V)1.2, NCX3, PMCA1,3,4, TRPC1,3,4,6, STIM1, and Orai1-3, were readily detectable, but not RyRs. Our results demonstrate for the first time that spontaneous Ca(i) (2+) oscillations are present in cultured human cardiac fibroblasts and are regulated by multiple Ca(2+) pathways, which are not identical to those of the well-studied contractile cardiomyocytes. This study provides a base for future investigations into how Ca(2+) signals regulate biological activity in human cardiac fibroblasts and cardiac remodeling under pathological conditions.

• B-cell lymphomas with surface nucleolin-Fas complexes are resistant to Fas-mediated apoptosis through decreased ligand binding.• Expression of nucleolin protects mice from a lethal agonistic Fas challenge, whereas a non-Fas binding nucleolin mutant does not.Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with IntroductionSurvival of individuals with non-Hodgkin's lymphoma (NHL) has improved with recent advancements in chemotherapy regimens, which now include targeted therapies. Despite these advancements, NHL demonstrates frequent relapses and a high mortality rate (30%). 1The principal source of NHL relapse is the survival and expansion of cells resistant to chemotherapy. Stimulation of Fas, a member of the tumor necrosis factor superfamily of apoptosis receptors, by Fas ligand (FasL)-bearing cells or from within damaged cells is an important mechanism of cell elimination, particularly in the lymphoid system. 2,3 Genetic models featuring Fas-disabling mutations develop autoreactive lymphocytes, arising from ineffective negative selection that results in autoimmune disorders and lymphoma. 4,5 Moreover, cells lacking Fas or Fas-defective cells are resistant to customary doses of chemotherapy and radiation. [6][7][8][9] Further investigations determined that Fas is a key component of responses to radiation and chemotherapy regimens, 6 as several forms of chemotherapy, including genotoxic chemotherapy, induce higher expression levels of Fas and/or FasL in order to effectively eliminate tumor cells. 10,11 However, Fas-resistant NHL cells often express normal levels of wild-type Fas and FasL while remaining resistant to Fas activation. The lack of correlation between Fas levels and sensitivity to Fasmediated apoptosis in lymphoid cancer cells indicates additional modulation of the apoptosis pathway. Investigations into the defects of Fas-mediated apoptosis have shown multiple layers of control over Fas signaling. The signaling is initiated by binding of trimeric FasL complexes to a Fas receptor, which recruits the adaptor molecule FADD and subsequently procaspase-8 through the homologous death domain and death effector domain, respectively, to form the death-inducing signaling complex. 3,12 Formation of this complex promotes cleavage and activation of the initiator caspase-8, resulting in activation of an intricate caspase cascade and cell death.13,14 Each of these signaling stages is subjected to different inhibitory mechanisms aimed at pr...

Bone marrow mesenchymal stem cells (MSCs) are used as a cell source for cardiomyoplasty; however, the cellular electrophysiological properties are not fully understood. The present study was to investigate the functional ionic channels in undifferentiated mouse bone marrow MSCs using whole cell patch-voltage clamp technique, RT-PCR, and Western immunoblotting analysis. We found that three types of ionic currents were present in mouse MSCs, including a Ca(2+)-activated K(+) current (I(KCa)), an inwardly rectifying K(+) current (I(Kir)), and a chloride current (I(Cl)). I(Kir) was inhibited by Ba(2+), and I(KCa) was activated by the Ca(2+) ionophore A-23187 and inhibited by the intermediate-conductance I(KCa) channel blocker clotrimazole. I(Cl) was activated by hyposmotic (0.8 T) conditions and inhibited by the chloride channel blockers DIDS and NPPB. The corresponding ion channel genes and proteins, KCa3.1 for I(KCa), Kir2.1 for I(Kir), and Clcn3 for I(Cl), were confirmed by RT-PCR and Western immunoblotting analysis in mouse MSCs. These results demonstrate that three types of functional ion channel currents (i.e., I(Kir), I(KCa), and I(Cl)) are present in mouse bone marrow MSCs.

Hepatocellular carcinomas (HCC) show resistance to chemotherapy and have blunt response to apoptotic stimuli. HCC cell lines express low levels of the Fas death receptor and are resistant to FasL stimulation, whereas immortalized hepatocytes are sensitive. The variable Fas transcript levels and consistently low Fas protein in HCC cells suggest posttranscriptional regulation of Fas expression. The 3 0 -untranslated region (UTR) of Fas mRNA was found to interact with the ribonucleoprotein Human Antigen R (HuR) to block mRNA translation. Silencing of HuR in HCC cells increased the levels of cell surface Fas and sensitized HCC cells to FasL. Two AU-rich domains within the 3 0 -UTR of Fas mRNA were identified as putative HuR-binding sites and were found to mediate the translational regulation in reporter assay. Hydrodynamic transfection of HuR plasmid into mice induced downregulation of Fas expression in livers and established functional resistance to the killing effects of Fas agonist. Human HCC tumor tissues showed significantly higher overall and cytoplasmic HuR staining compared with normal liver tissues, and the high HuR staining score correlated with worse survival of patients with early-stage HCC. Combined, the protumorigenic ribonucleoprotein HuR blocks the translation of Fas mRNA and effectively prevents Fas-mediated apoptosis in HCC, suggesting that targeting HuR would sensitize cells to apoptotic stimuli and reverse tumorigenic properties.Implications: Demonstrating how death receptor signaling pathways are altered during progression of HCC will enable the development of better methods to restore this potent apoptosis mechanism. Mol Cancer Res; 13(5); 809-18. Ó2015 AACR.