A recombinant human RNASET2 glycoprotein with antitumorigenic and antiangiogenic characteristics: Expression, purification, and characterization

A recombinant human RNASET2 glycoprotein with antitumorigenic and antiangiogenic characteristics Expression, purification, and characterization Patricia Smirnoff, PhD 1, Levava Roiz, PhD 2, Boaz Angelkovitch, MSc 2, Betty Schwartz, PhD 1, Oded Shoseyov, PhD 2 * 1Institute of Biochemistry, Food Science, and Nutrition, Faculty of Agricultural, Food, and Environmental Quality Sciences, The Hebrew University of Jerusalem, Jerusalem, Rehovot, Israel 2The Robert H. Smith Institute of Plant Science and Genetics in Agriculture, Faculty of Agricultural, Food, and Environmental Quality Sciences, The Hebrew University of Jerusalem, Jerusalem, Rehovot, Israel email: Oded Shoseyov ([email protected]) *Correspondence to Oded Shoseyov, The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agricultural, Food, and Environmental Quality Sciences, The Hebrew University of Jerusalem, PO Box 12, Rehovot 76100, Israel Fax: (011) 972-8-9462283 Keywords antiangiogenic \u95 actin-binding \u95 colon cancer \u95 paclitaxel \u95 RNASET2 Abstract BACKGROUND. Human RNASET2 is a T2-RNase glycoprotein encoded by the RNASET2 gene, which is located on chromosome 6 (6q27). Deletion in 6q27 is associated with several human malignancies. BACKGROUND. A synthetic RNASET2 gene that was optimized for expression in the yeast Pichia pastoris was designed according to the cDNA sequence and was cloned under the control of the methanol-induced promoter fused to the -mating secretion peptide. The recombinant protein was purified from the culture supernatant of transformed P. pastoris through an affinity Sepharose-concanavalin A column. Actin-binding activity was examined by membrane blotting using monoclonal mouse antiactin immunoglobulin M and by cross-linking in solution to G-actin using 1-[3-(dimethylamino)propyl]-3-ethyl-carboimide methiodide. The antiangiogenic activity of RNASET2 (from 0.5 M to 10 M) was assessed by a human umbilical vein endothelial (HUVE) cell assay in the presence of 1 g/mL angiogenin, basic fibroblast growth factor (bFGF), or recombinant human vascular endothelial growth factor (VEGF). Cell colony formation was examined in human colon HT29 cancer cells to assess the antitumorigenic activity of RNASET2 or the enzymatic-inactivated RNASET2 (EI-RNASET2) (1 M each). In an athymic mouse xenograft model, LS174T human cancer cells were injected subcutaneously. When tumors were palpable, the mice were treated for 3 weeks with RNASET2 (1 mg/kg), paclitaxel (10 mg/kg or 15 mg/kg), or a combination of the 2 drugs. RESULTS. The recombinant RNASET2 was identified as a 27-kilodalton glycoprotein that possessed the ability to bind actin in vitro. RNASET2 significantly inhibited clonogenicity in HT29 cells. EI-RNASET2 produced a similar effect, suggesting that its antitumorigenic activity is unrelated to its RNase activity. In HUVE cells, RNASET2 inhibited angiogenin-, bFGF-, and VEGF-induced tube formation in a dose-dependent manner. In athymic mice, RNASET2 inhibited the development of an LS174T-derived xenograft by 40%. A synergistic effect was obtained with combined RNASET2 and paclitaxel treatments. CONCLUSIONS. The current results suggested that RNASET2 represents a new class of antitumorigenic and antiangiogenic drugs, and the findings of this study emphasize the advantage of using agents like RNASET2 in combined therapy. Cancer 2006. \ua9 2006 American Cancer Society

Femmes de cinéma

Ce numéro, consacré aux femmes dans le cinéma latino-américain, propose des panoramas sur toute l’Amérique latine (la place des femmes dans la production, dans la réalisation), des articles sur les pionnières du cinéma, sur des réalisatrices contemporaines, des études plus détaillées sur des aspects cinématographiques de réalisatrices latino-américaines (comme par exemple Lucrecia Martel). Afin de rendre hommage aux réalisatrices latino-américaines, la revue a souhaité leur donner la parole en leur posant 3 questions à propos de leur conception de la création cinématographique. Leurs réponses ouvriront ce numéro. Enfin, une rubrique « reseñas » propose des comptes-rendus de livres récemment publiés à propos du cinéma latino-américain