Marco R. Soria

Ribosome inactivating proteins from plants: present status and future prospects

Plant ribosome–inactivating proteins (RIPs) N–glycosidases which cleave the N–glycosidic bond of adenine in a specfic ribosomal RNA sequence. Most commonly RIPs are single–chain proteins (type 1 RIPs), but some (type 2 RIPs) possess a galactose–specific lectin domain that binds to cell surfaces. The latter RIPs are potent toxins, the best known of which is ricin. RIPs have antiviral and abortifacient activities, and, in a widespread application, can also be linked to antibodies or ligands to form immunotixins or conjugates specifically toxic to a given type of cell.

A new Chinese hamster ovary cell line expressing α2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins

Chinese hamster ovary (CHO) cells are widely employed to produce glycosylated recombinant proteins. Our group as well as others have demonstrated that the sialylation defect of CHO cells can be corrected by transfecting the alpha2,6-sialyltransferase (alpha2,6-ST) cDNA. Glycoproteins produced by such CHO cells display both alpha2,6- and alpha2,3-linked terminal sialic acid residues, similar to human glycoproteins. Here, we have established a CHO cell line stably expressing alpha2,6-ST, providing a universal host for further transfections of human genes. Several relevant parameters of the universal host cell line were studied, demonstrating that the alpha2,6-ST transgene was stably integrated into the CHO cell genome, that transgene expression was stable in the absence of selective pressure, that the recombinant sialyltransferase was correctly localized in the Golgi and, finally, that the bioreactor growth parameters of the universal host were comparable to those of the parental cell line. A second step consisted in the stable transfection into the universal host of cDNAs for human glycoproteins of therapeutic interest, i.e. interferon-gamma and the tissue inhibitor of metalloproteinases-1. Interferon-gamma purified from the universal host carried 40.4% alpha2,6- and 59.6% alpha2,3-sialic acid residues and showed improved pharmacokinetics in clearance studies when compared to interferon-gamma produced by normal CHO cells.

Ber-H2 (anti-CD30)-saporin immunotoxin : a new tool for the treatment of Hodgkin's disease and CD30+ lymphoma : in vitro evaluation

An immunotoxin containing an anti‐CD30 monoclonal antibody (Ber‐H2) and saporin, a ribosome‐inactivating protein type 1, is described. It specifically inhibits protein synthesis by Hodgkin derived target cell lines with a very high efficiency (IC50 ranging from 5 × 10–12 M to 5.10∼14 M, assaporin), while irrelevant immunotoxins do not. Present results suggest that this immunotoxin could be used for in vivo therapy as well as for ex vivo bone marrow purging in Hodgkins disease and CD304+ lymphomas.

EDF-1, a Novel Gene Product Down-regulated in Human Endothelial Cell Differentiation

Endothelial cell differentiation is a crucial step in angiogenesis. Here we report the identification of EDF-1, a novel gene product that is down-regulated when endothelial cells are induced to differentiate in vitro. The cDNA encodingEDF-1 was isolated by RNA fingerprinting from human endothelial cells exposed to human immunodeficiency virus type 1 Tat, a viral protein known to be angiogenic. The deduced amino acid sequence of EDF-1 encodes a basic intracellular protein of 148 amino acids that is homologous to MBF1 (multiprotein-bridgingfactor 1) of the silkworm Bombyx mori and to H7, which is implicated in the early developmental events of Dictyostelium discoideum. Interestingly, human immunodeficiency virus type 1 Tat, which affects endothelial functions, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and culture on fibrin gels, which promote endothelial differentiationin vitro, all down-regulate EDF-1 expression both at the RNA and protein levels. In addition, the inhibition of EDF-1 translation by an antisense anti-EDF-1 construct results in the inhibition of endothelial cell growth and in the transition from a nonpolar cobblestone phenotype to a polar fibroblast-like phenotype. These data suggest that EDF-1 may play a role in the regulation of human endothelial cell differentiation.

THE AMINO-TERMINAL FRAGMENT OF HUMAN UROKINASE DIRECTS A RECOMBINANT CHIMERIC TOXIN TO TARGET CELLS: INTERNALIZATION IS TOXIN MEDIATED

In contrast to two‐chain urokinase (uPA), a chemical conjugate between uPA and native saporin (a cytotoxic plant seed ribosome‐inactivating protein) did not require plasminogen activator inhibitors to be internalized. To dissect this pathway, we constructed a chimera consisting of the amino‐terminal fragment (ATF) of human urokinase fused to a saporin isoform (SAP‐3). The chimeric ATF‐SAP toxin was expressed in Escherichia coli, purified, and characterized for its ribosome‐inactivating activity. Besides being a potent inhibitor of protein synthesis in cell‐free assays, ATF‐SAP was specifically cytotoxic toward cells expressing human uPAR. Competition experiments indicated that both the human uPAR and the LDL‐related receptor protein are involved in mediating the cell killing ability of ATF‐SAP. We conclude that neither plasminogen activator inhibitors nor the catalytic moiety of urokinase are necessary to initiate these internalization pathways. Thus, saporin may play a role similar to plasminogen activator inhibitors in its ability to trigger internalization of uPAR‐bound ligands through endocytic receptors.—Fabbrini, M. S., Carpani, D., Bello‐Rivero, I., Soria, M. R. The amino‐terminal fragment of human urokinase directs a recombinant chimeric toxin to target cells: internalization is toxin‐mediated. FASEB J. 11, 1169–1176 (1997)

OVER-EXPRESSION OF HEPATOCYTE GROWTH FACTOR IN HUMAN KAPOSI'S SARCOMA

Kaposis sarcoma is a highly vascularized multifocal tumor which frequently appears as a complication of HIV infection. It has been suggested that a disorder in the cytokine network may contribute to the development of the disease. We examined the expression of several cytokines in human sporadic Kaposis sarcoma specimens, as well as in spindle cells cultured from human lesions, and consistently found high levels of expression of hepatocyte growth factor (HGF). In addition, human lesion‐derived spindle cells synthesize and secrete biologically active hepatocyte growth factor and express the hepatocyte‐growth‐factor receptor (c‐MET). Moreover, elevated levels of transforming growth factor β1 (TGFβ1) mRNA were found in lesions of human sporadic Kaposis sarcoma and in lesion‐derived spindle cells which also over‐express urokinase. Since HGF, TGFβ1 and urokinase are all involved in capillary‐vessel organization, dysregulation of these interacting agents may play a role in the initiation and/or progression of Kaposis sarcoma, stimulating the growth of spindle cells and recruiting endothelial cells into the lesion.

Genetic engineering of α2,6-sialyltransferase in recombinant CHO cells and its effects on the sialylation of recombinant interferon-γ.

The CHO cell line has achieved considerable commercial importance as a vehicle for the production of human therapeutic proteins, but is known to lack a functional copy of the gene coding for α2,6-sialyltransferase (EC 2.4.99.1). The cDNA for rat α2,6-ST was expressed in a recombinant CHO cell line making interferon-γ, using a novel in vitro amplification vector. The enzyme was expressed efficiently, and resulted in up to 60% of the total sialic acids on interferon-γ being linked in the α2,6-conformation. This sialic acid linkage distribution was more akin to that seen in natural human glycoproteins. In the most successful cell clones, expression of α2,6-sialyltransferase improved the overall level of sialylation by up to 56%, and had no adverse effects on cell growth, IFN-γ productivity or other aspects of IFN-γ glycosylation. These experiments demonstrate how the glycosylation machinery of rodent cells can be genetically manipulated to replicate human tissues.

Na-butyrate increases the production and α2,6-sialylation of recombinant interferon-γ expressed by α2,6- sialyltransferase engineered CHO cells

A non-human like glycosylation pattern in human recombinant glycoproteins expressed by animal cells may compromise their use as therapeutic drugs. In order to correct the CHO glycosylation machinery, a CHO cell line producing recombinant human interferon- γ (IFN) was transformed to replace the endogenous pseudogene with a functional copy of the enzyme α2,6-sialyltransferase (α2,6-ST). Both the parental and the modified CHO cell line were propagated in serum-free batch culture with or without 1 mM sodium butyrate. Although Na-butyrate inhibited cell growth, IFN concentration was increased twofold. The IFN sialylation status was determined using linkage specific sialidases and HPLC. Under non- induced conditions, IFN expressed by α2,6-engineered cells contained 68% of the total sialic acids in the α2,6- conformation and the overall molar ratio of sialic acids to IFN was 2.3. Sodium butyrate addition increased twofold the molar ratio of total sialic acids to IFN and 82% of total sialic acids on IFN were in the α2,6-conformation. In contrast, no effect of the sodium butyrate was noticed on the sialylation of the IFN secreted by the α2,6-ST deficient parental cell line. This study deals for the first time with the effect of Na-butyrate on CHO cells engineered to produce human like sialylation.

Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism

A fluorescent derivative of a chimeric toxin between human pro‐urokinase and the plant ribo‐some‐inactivating protein saporin (p‐uPA‐SapTRITC), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase‐inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein‐related receptor protein (LRP) family, and involves a macro‐quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two‐step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.—Ippoliti, R., Lendaro, E., Benedetti, P. A., Torrisi, M. R., Belleudi, F., Carpani, D., Soria, M. R., Fabbrini, M. S. Endocytosis of a chimera between human pro‐urokinase and the plant toxin saporin: an unusual internalization mechanism. FASEB J. 14, 1335–1344 (2000)

FGF-2 stimulates migration of Kaposi's sarcoma-like vascular cells by HGF-dependent relocalization of the urokinase receptor

The spindle‐shaped cell line TTB was recently isolated from highly vascularized skin lesions of BKV/HIV‐1 tat transgenic mice and shown to possess an autocrine loop for hepatocyte growth factor (HGF). We show that fibroblast growth factor‐2 (FGF‐ 2) stimulates TTB cell migration and promotes polarization of uPAR at the leading edge of migrating cells. FGF‐stimulated TTB cells presented the typical migratory phenotype, with a triangular cell shape and concomitant breakdown of actin stress fibers and smooth muscle‐specific actin isoform. FGF‐2‐stimulated migration was blocked by antibodies against urokinase‐type plasminogen activator (uPA) or uPA receptor (uPAR) and by neutralizing anti‐HGF antibodies. The latter also inhibited uPAR relocalization at the cell surface of FGF‐2‐treated TTB cells. This points to a crosstalk between FGF‐2 and HGF that might mediate TTB cell migration by modulating the localization of cell surface uPAR.—Cavallaro, U., Wu, Z., Di Palo, A., Montesano, R., Pepper, M. S., Maier, J. A. M., Soria, M. R. FGF‐2 stimulates migration of Kaposis sarcoma‐like vascular cells by HGF‐dependent relocalization of the urokinase receptor. FASEB J. 12, 1027–1034 (1998)