Zohar Mishal

Fenofibrate inhibits angiogenesis in vitro and in vivo

Abstract. Fenofibrate, a peroxisome proliferator-activated receptor (PPAR)-alpha activator, used as a normolipidemic agent, is thought to offer additional beneficial effects in atherosclerosis. Since angiogenesis is involved in plaque progression, hemorrhage, and instability, the main causes of ischemic events, this study was designed to evaluate the action of fenofibrate on angiogenesis. Our results show that fenofibrate (i) inhibits endothelial cell proliferation induced by angiogenic factors, followed at high concentrations by an increase in apoptosis, (ii) inhibits endothelial cell migration in a healing wound model, (iii) inhibits capillary tube formation in vitro, and (iv) inhibits angiogenesis in vivo. Concerning the mechanism of action, the inhibition of endothelial cell migration by fenofibrate can be explained by a disorganization of the actin cytoskeleton. At the molecular level, fenofibrate markedly decreased basic fibroblast growth factor-induced Akt activation and cyclooxygenase 2 gene expression. This inhibition of angiogenesis could participate in the beneficial effect of fenofibrate in atherosclerosis.

Differential intracellular trafficking, secretion and endosomal localization of two IL-15 isoforms

To analyze the intracellular trafficking of two IL‐15 isoforms bearing 48‐ or 21‐amino acid leader peptides (L), we have generated cDNA encoding the two proteins fused at the C terminus with green fluorescent protein (GFP). Confocal microscopy analyses showed that, when transfected in CHO cells, 48L IL‐15/GFP was localized in the Golgi apparatus and in early endosomes, while 21L IL‐15/GFP was detectable only in the cytosol. The presence of 48L IL‐15/GFP in endosomes was confirmed by enzyme‐linked immunosorbent assay on endosome‐enriched subcellular fractions. Exogenous IL‐15 was bound and taken up in endosomes by untransfected CHO cells, indicating that endosomal localization was, at least in part, related to a receptor‐mediated uptake. The 48L IL‐15/GFP fusion protein was efficiently secreted by COS‐7 or CHO cell transfectants, while IL‐15 secretion was less efficient in transfectants expressing 21L IL‐15/GFP or untagged 48L or 21L IL‐15. Treatment with brefeldin A or with inhibitors of N‐linked glycosylation further indicated that the 48L IL‐15/GFP is secreted through the endoplasmic reticulum/Golgi pathway. Our data suggest a different trafficking of the two IL‐15 isoforms and multiple mechanisms controlling IL‐15 secretion.

Endostatin Exhibits a Direct Antitumor Effect in Addition to Its Antiangiogenic Activity in Colon Cancer Cells

Endostatin has been considered a highly specific inhibitor of endothelial cell proliferation and/or migration. To explore the use of endostatin in antiangiogenic gene therapy, we generated a recombinant adenovirus, AdEndo, carrying the gene for mouse endostatin. Injection of 10(9) PFU of AdEndo resulted in a low but significant suppression (25%) of preestablished tumor growth in murine models involving murine Lewis lung carcinoma (LLC) and human breast cancer MDA-MB-231 tumors. Greater anticancer activity was observed when the same dose of AdEndo was injected into two other preestablished murine models involving C51 murine colon cancer and HT29 human colon cancer (55 and 47% tumor growth reduction, respectively). In vitro, endostatin derived from AdEndo-infected MRC-5 fibroblasts inhibited the growth of C51 and HT29 cell lines (72 and 61%, respectively). The extent of this inhibition was comparable to that observed in endothelial cells: 75% for microcapillary endothelial cell line HMEC-1, 52% for human dermal microvascular endothelial cells, 46% for human umbilical vein endothelial cells, and 67% for calf pulmonary arterial endothelial cells. Both endothelial and colon cancer cells showed a clear increase in cell apoptosis (4- to 5-fold for endothelial cells and 5- to 10-fold for colon cancer cells) and an accumulation in the G(1) phase of the cell cycle. This antiproliferative activity was not observed in other tumor cell lines: LLC, MDA-MB-231, murine colon adenocarcinoma MC38, human prostate cancer cell line DU145, and human breast cancer cell line CAL51. Taken together, these results provide evidence that, in addition to its antiangiogenic activity, endostatin exerts a direct anticancer action that appears to be restricted to some tumor cell lines. Thus, endostatin could be used in some colon cancer treatments and its clinical efficacy would depend on the response of tumor cells themselves.

IL-15/IL-15Rα intracellular trafficking in human melanoma cells and signal transduction through the IL-15Rα

There are two IL-15 isoforms and eight isoforms for the IL-15Rα chain whose biological role is poorly understood. Here, we have analysed the intracellular trafficking of IL-15 and IL-15Rα and tried to shed some light on their function(s). In IL-15/GFP CHO transfectants both IL-15 isoforms show nuclear localization. Two melanoma cell lines (MELP and MELREO) spontaneously expressing the IL-15 isoforms, display different intracellular trafficking of the IL-15/IL-15Rα complex. In MELP cells only IL-15Rα is detected inside the nucleus, whereas IL-15 and IL-15Rα assemble at the cell surface and are internalized. Moreover, the transducing molecule TRAF2 co-immunoprecipitates with IL-15Rα and may be deflected to TNFRI using anti-IL-15 blocking mAbs and TNF-α. By contrast, MELREO cells display IL-15Rα and IL-15 nuclear localization but only a partial co-localization of these molecules on the cell surface. In these cells, TRAF2 is strongly associated with IL-15Rα and cannot be deflected by any treatment. Since TRAF2 activates the transcription factor NF-κB, IL-15 through IL-15Rα, could have a role in the control of this pathway. Indeed, anti-IL-15 MaB inhibit the constitutive nuclear localization of NFκB and the phosphorylation of its inhibitor Iκ-Bα. Thus, IL-15Rα controls NF-κB activation, however differences in the intracellular trafficking of the IL-15 and/or IL-15Rα suggest a different biological role for this complex in MELP versus MELREO cells.

Expression of alpha-fetoprotein receptors by human T-lymphocytes during blastic transformation

Alpha-fetoprotein (AFP) and transferrin (Tf) are actively internalized by many growing cells during ontogenic and neoplastic development, including human malignant T- and B-lymphoblastoid cells. Their internalization is, on the contrary, greatly diminished or absent in mature, non-proliferating elements. In the present work, peripheral blood mononuclear cells (PBMCs) and T-lymphocytes, harvested from normal human donors, were induced to blastic transformation with phytohemagglutinin (PHA) and their ability to uptake AFP and Tf was measured and compared with Tf uptake in the same conditions. The capacity of the cells to internalize both proteins was quantified by fluorescence activated cell sorter (FACS) using fluoresceinated derivatives of these proteins. The results obtained show a significant uptake of AFP by T-lymphocytes upon PHA stimulation. The values of AFP incorporation were similar for all the cells studied (PBMCs, T-cells and T4, T8 cell subsets). The time course of AFP uptake paralleled, under the same conditions, the uptake of Tf and the expression of IL2 receptors. AFP uptake increased rapidly from the zero time (resting T-cells) and reached a maximum around 72 hr after PHA activation. Scatchard analysis of kinetic data at 4 degrees C revealed for Hu-AFP one single group of specific binding sites in PHA activated T-lymphocytes with a dissociation constant of 3.03 x 10(-7) M and around 88,000 sites/cell. There results strongly suggest the transitory expression of AFP receptors in T-lymphocytes during blastic transformation.

IL-15 is produced by a subset of human melanomas, and is involved in the regulation of markers of melanoma progression through juxtacrine loops

IL-15 is a novel cytokine active through the IL-2Rβγ. Since several human melanoma cell lines display functional IL-2Rs, we studied the IL-15/melanoma cells interactions. Ten out of 17 melanoma cell lines express the IL-15 transcript and four of them express levels of IL-15 mRNA similar to those detected in control activated monocytes. Nine out of ten cell lines also express two transcripts for the IL-15Rα originated by the alternative splicing of exon′3′. Two melanoma cell lines, MELP and MELREO, derived from patients with rapidly progressive primary melanomas, co-express the two IL-15 transcripts, originated by alternative splicing of exon ′A′. Intracellular Il-15 protein was only detected in these two cells lines and it is mainly retained in the Endoplasmic Reticulum (ER). However, a small amount of IL-15 is also found in the Golgi apparatus and in the early endosomes, suggesting production and intercellular trafficking of endogenous IL-15 protein. Nevertheless, no biologically active IL-15 could be detected in the supernatant of all melanoma cells. The anti IL-15 blocking mAb M111 causes the up regulation of HLA Class I in dense MELP and MELREO cultures. These data suggest that IL-15 is probably active through juxtacrine loops negatively controlling HLA Class I molecules expression. These data offer, for the first time, a likely explanation to the controversal issue of IL-15 secretion and constitute a natural model for understanding IL-15 routing. Moreover, we identify a subset of melanoma cells producing IL-15, possibly involved in tumor escape mechanisms.

IL‐15/IL‐15Rα Intracellular Trafficking in Human Cells and Protection from Apoptosisaa

ABSTRACT: IL‐15 is an immunostimulatory cytokine sharing with IL‐2 the IL‐2Rβγ complex. In vivo, IL‐15 detection in synovial fluids has been associated with the development of rheumatoid arthritis. A debate exists as to whether IL‐15 has the potential to be secreted in meaningful amounts or to act as a pericellular cytokine. Our data show (1) the presence of two IL‐15 isoforms displaying signal peptides of different length and the capacity to be secreted restricted to the isoform bearing the longer one; (2) in cells expressing the two isoforms, the existence of different nuclear localization and intracellular trafficking of IL‐15 and IL‐15Rα and (3) an intercellular microcirculation of IL‐15, not detectable with ELISA kits, but displaying a role as an anti‐apoptotic factor able to induce the deflection of the TNFR associated factor 2 (TRAF) to IL‐15Rα. Our data point to a juxtacrine mechanism of action of IL‐15 and suggest a role for IL‐15/IL‐15Rα in the regulation of apoptosis.

Altered natural killer cell differentiation in CD34+ progenitors from chronic myeloid leukemia patients.

IL-15 and SCF fail to induce NK differentiation and proliferation of CD34+ hematopoietic progenitors from chronic myeloid leukemia patients in contrast to normal stem cells although, both normal and leukemic CD34+ cells display comparable expression of c-kit or IL-15 receptor subunits. Interestingly, confocal microscopy analysis revealed that leukemic and most normal CD34+ cells produce and secrete IL-15, as shown by its trafficking through the Golgi apparatus and early endosomes. However, only leukemic progenitors express the membrane bound IL-15. Colocalization and internalization of IL-15Rβ/γc and IL-15Rα/γc complexes indicated that IL-15 was specifically uptaken by leukemic progenitors. We also demonstrated that in both normal and leukemic progenitors, the signaling kinase Jak3 is constitutively pre-associated with the γc chain. Anti-IL-15 neutralizing mAb treatment resulted in down-regulation of γc chain and disruption of γc/Jak3 interaction in normal but had no effect in leukemic progenitors. Our results suggest the existence in both normal and leukemic CD34+ cells of a constitutive production of a bioactive IL-15 that does not lead to NK differentiation and further indicate that membrane bound IL-15 and constitutive activation of γc are hallmarks of leukemic progenitors.

Scrg1, a novel protein of the CNS is targeted to the large dense-core vesicles in neuronal cells.

Scrapie responsive gene one (Scrg1) is a novel transcript discovered through identification of the genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies. Scrg1 mRNA is distributed principally in the central nervous system and the cDNA sequence predicts a small cysteine‐rich protein 98 amino acids in length, with a N‐terminal signal peptide. In this study, we have generated antibodies against the predicted protein and revealed expression of a predominant immunoreactive protein of 10 kDa in mouse brain by Western blot analysis. We have established CAD neuronal cell lines stably expressing Scrg1 to determine its subcellular localization. Several lines of evidence show that the protein is targeted to dense‐core vesicles in these cells. (i) Scrg1 is detected by immunocytochemistry as very punctate signals especially in the Golgi apparatus and tips of neurites, suggesting a vesicular localization for the protein. Moreover, Scrg1 exhibits a high degree of colocalization with secretogranin II, a dense‐core vesicle marker and a very limited colocalization with markers for small synaptic vesicles. (ii) Scrg1 immunoreactivity is associated with large secretory granules/dense‐core vesicles, as indicated by immuno‐electron microscopy. (iii) Scrg1 is enriched in fractions of sucrose density gradient where synaptotagmin V, a dense‐core vesicle‐associated protein, is also enriched. The characteristic punctate immunostaining of Scrg1 is observed in N2A cells transfected with Scrg1 and for the endogenous protein in cultured primary neurons, attesting to the generality of the observations. Our findings strongly suggest that Scrg1 is associated with the secretory pathway of neuronal cells.

Mineralocorticoid Hormone Signaling Regulates the ‘Epithelial Sodium Channel’ in Fibroblasts from Human Cornea

We investigated the regulation of sodium absorption by steroid hormones in embryologically diverse cells from the human eye. A cell extract from human corneal fibroblasts was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as 82- to 85-kD and 102-kD bands, respectively, by the Western blot technique. In fluorescent, confocal and electron microscopy, the MCR was revealed as a nucleocytoplasmic protein, whereas the ENaC was almost exclusively membrane bound; both appeared aligned along actin filaments of corneal keratocytes, and both were widely colocalized in various cell types of human cornea in situ. Following reverse transcription and amplification of total RNA isolated from corneal fibroblasts, the ENaC and MCR genes in the PCR product were evident as predicted bands of 520 and 843 bp, respectively, whose sequence exhibited 100% identity with those from known human sources. The multiplication of corneal fibroblasts was influenced by both the MCR-specific antagonist RU 26752 and the natural hormone aldosterone, and these steroids also stimulated protein phosphorylation. In quantitative PCR, both the basal and aldosterone-induced levels of ENaC were diminished by the MCR-specific antagonist ZK 91587. Consequently, the ocular sodium channel appears to be regulated by steroid signalling in cells of diverse embryological origins, contrary to the existing notions where (a) this process would be limited exclusively to the epithelial cells and (b) ocular sodium transport would be regulated via the Na+-K+-ATPase in the basolateral membrane.