Endothelin-1 decreases gap junctional intercellular communication by inducing phosphorylation of connexin 43 in human ovarian carcinoma cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50–75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC

Catalytic mechanism and role of hydroxyl residues in the active site of theta class Glutathione-S-Transferases: Investigation of Ser-9 and Tyr-113 in a Glutathione S-Transferase from the australian sheep blowfly Lucilia cuprina

Abstract Spectroscopic and kinetic studies have been performed on the Australian sheep blowfly Lucilia cuprina glutathione S-transferase (Lucilia GST; EC 2.5.1.18) to clarify its catalytic mechanism. Steady state kinetics of Lucilia GST are non-Michaelian, but the quite hyperbolic isothermic binding of GSH suggests that a steady state random sequential Bi Bi mechanism is consistent with the anomalous kinetics observed. The rate-limiting step of the reaction is a viscosity-dependent physical event, and stopped-flow experiments indicate that product release is rate-limiting. Spectroscopic and kinetic data demonstrate thatLucilia GST is able to lower the pK a of the bound GSH from 9.0 to about 6.5. Based on crystallographic suggestions, the role of two hydroxyl residues, Ser-9 and Tyr-113, has been investigated. Removal of the hydroxyl group of Ser-9 by site-directed mutagenesis raises the pK a of bound GSH to about 7.6, and a very low turnover number (about 0.5% of that of wild type) is observed. This inactivation may be explained by a strong contribution of the Ser-9 hydroxyl group to the productive binding of GSH and by an involvement in the stabilization of the ionized GSH. This serine residue is highly conserved in the Theta class GSTs, so the present findings may be applicable to all of the family members. Tyr-113 appears not to be essential for the GSH activation. Stopped-flow data indicate that removal of the hydroxyl group of Tyr-113 does not change the rate-limiting step of reaction but causes an increase of the rate constants of both the formation and release of the GSH conjugate. Tyr-113 resides on α-helix 4, and its hydroxyl group hydrogen bonds directly to the hydroxyl of Tyr-105. This would reduce the flexibility of a protein region that contributes to the electrophilic substrate binding site; segmental motion of α-helix 4 possibly modulates different aspects of the catalytic mechanism of theLucilia GST

Temperature adaptation of glutathione S-transferase P1-1. A case for homotropic regulation of substrate binding.

Human glutathione S-transferase P1-1 (GST P1-1) is a homodimeric enzyme expressed in several organs as well as in the upper layers of epidermis, playing a role against carcinogenic and toxic compounds. A sophisticated mechanism of temperature adaptation has been developed by this enzyme. In fact, above 35 degrees C, glutathione (GSH) binding to GST P1-1 displays positive cooperativity, whereas negative cooperativity occurs below 25 degrees C. This binding mechanism minimizes changes of GSH affinity for GST P1-1 because of temperature fluctuation. This is a likely advantage for epithelial skin cells, which are naturally exposed to temperature variation and, incidentally, to carcinogenic compounds, always needing efficient detoxifying systems. As a whole, GST P1-1 represents the first enzyme which displays a temperature-dependent homotropic regulation of substrate (e.g. GSH) binding

Recombinant Anti-Human Melanoma Antibodies Are Versatile Molecules

The low cost, high versatility, and reliable production of bacterially produced recombinant antibody fragments speeds up the development of tumor-targeting agents. High-quality recombinant anti-melanoma antibodies are much sought after in the scientific community. We cloned the murine antibody 225.28S, currently used in radioimmunoimaging of human melanoma lesions, in single-chain Fv configuration (scFv) for soluble expression in bacteria. The recombinant antibody fragment conserved the binding specificity of the parental antibody. In order to arm the scFv(225.28S) with biologically useful effector functions, we developed vectors for soluble expression of scFv(225.28S) in bacteria that allow both covalent and noncovalent chemical antibody modification at positions that do not interfere with antigen binding. An expression vector was developed that appends a cysteine residue at the C-terminal extremity of the recombinant antibody, thus allowing reaction with thiol-specific reagents, including 99mTc labeling, at a position that does not interfere with antigen binding. The scFv(225.28S) was also successfully expressed with a casein kinase II substrate tag that enables efficient and stable 32P labeling. For noncovalent antibody modification, we developed an expression vector that appends the human calmodulin gene at the C-terminal extremity of scFv(225.28S). The calmodulin domain is poorly immunogenic and can be targeted with chemically modified high-affinity calmodulin ligands. The recombinant anti-human melanoma antibodies described in this article should prove useful “building blocks” for the development of anti-melanoma diagnostic and therapeutic strategies

Endothelin-1 Inhibits Prolyl Hydroxylase Domain 2 to Activate Hypoxia-Inducible Factor-1α in Melanoma Cells

The endothelin B receptor (ET(B)R) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1alpha is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation.Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ET(B)R, enhance the expression and activity of HIF-1alpha and HIF-2alpha that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-alpha stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1alpha oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ET(B)R markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKT-mammalian target of rapamycin (mTOR) pathway is required for ET(B)R-mediated PHD2 inhibition, HIF-1alpha, HIF-2alpha, and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1alpha, ET(B)R expression is associated with low PHD2 levels. In melanoma xenografts, ET(B)R blockade by ET(B)R antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1alpha, and HIF-2alpha expression, and an increase in PHD2 levels.In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1alpha stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ET(B)R may represent a potential therapeutic treatment of melanoma by impairing HIF-1alpha stability

Cell-to-Cell Signaling Influences the Fate of Prostate Cancer Stem Cells and Their Potential to Generate More Aggressive Tumors

An increasing number of malignancies has been shown to be initiated and propelled by small subpopulations of cancer stem cells (CSC). However, whether tumor aggressiveness is driven by CSC and by what extent this property may be relevant within the tumor mass is still unsettled. To address this issue, we isolated a rare tumor cell population on the basis of its CD44+CD24− phenotype from the human androgen-independent prostate carcinoma cell line DU145 and established its CSC properties. The behavior of selected CSC was investigated with respect to the bulk DU145 cells. The injection of CSC in nude mice generated highly vascularized tumors infiltrating the adjacent tissues, showing high density of neuroendocrine cells and expressing low levels of E-cadherin and β-catenin as well as high levels of vimentin. On the contrary, when a comparable number of unsorted DU145 cells were injected the resulting tumors were less aggressive. To investigate the different features of tumors in vivo, the influence of differentiated tumor cells on CSC was examined in vitro by growing CSC in the absence or presence of conditioned medium from DU145 cells. CSC grown in permissive conditions differentiated into cell populations with features similar to those of cells held in aggressive tumors generated from CSC injection. Differently, conditioned medium induced CSC to differentiate into a cell phenotype comparable to cells of scarcely aggressive tumors originated from bulk DU145 cell injection. These findings show for the first time that CSC are able to generate differentiated cells expressing either highly or scarcely aggressive phenotype, thus influencing prostate cancer progression. The fate of CSC was determined by signals released from tumor environment. Moreover, using microarray analysis we selected some molecules which could be involved in this cell-to-cell signaling, hypothesizing their potential value for prognostic or therapeutic applications

Anisotropy and chemical composition of ultra-high energy cosmic rays using arrival directions measured by the Pierre Auger Observatory

The Pierre Auger Collaboration has reported evidence for anisotropy in the distribution of arrival directions of the cosmic rays with energies $E>E_{th}=5.5\times 10^{19}$ eV. These show a correlation with the distribution of nearby extragalactic objects, including an apparent excess around the direction of Centaurus A. If the particles responsible for these excesses at $E>E_{th}$ are heavy nuclei with charge $Z$, the proton component of the sources should lead to excesses in the same regions at energies $E/Z$. We here report the lack of anisotropies in these directions at energies above $E_{th}/Z$ (for illustrative values of $Z=6,\ 13,\ 26$). If the anisotropies above $E_{th}$ are due to nuclei with charge $Z$, and under reasonable assumptions about the acceleration process, these observations imply stringent constraints on the allowed proton fraction at the lower energies

Polyamines and ripening of photoreceptor outer-segment in chicken embryo.

Polyamines and their related monoacetyl derivatives were studied in rod outer segment (ROS) and cone outer segment (COS) of photoreceptor cells from chick embryo retina during eye development (7th-18th days). Putrescine was found to be necessary, in the second phase of retinogenesis, to sustain both ROS and COS differentiation and, after acetylation, gamma-aminobutyric acid synthesis. On the Other hand, spermidine and even more spermine intervene in the third phase of development when photoreceptors mature. Moreover, the presence of Nl-acetylspermidine already at the 7th day indicates that in the outer segment of photoreceptor cells too, as in the whole retina, putrescine synthesis comes about by two pathways. One pathway involves ornithine decarboxylase; the other, spermidine/spermine N1-acetyltransferase and FAD-dependent polyamine oxidase activities that convert spermidine to putrescine via N1-acetylspermidine. These different biosynthetic pathways are probably also decisive in permitting gamma-aminobutyric acid synthesis, which is very important in the ripening process of neural retina