title: A human ribonuclease induces apoptosis associated with p21WAF1/CIP1 induction and JNK inactivation.
abstract: BACKGROUND
  Ribonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase.
  
  
  METHODS
  Cytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot.
  
  
  RESULTS
  We show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both ribonucleases in the NCI/ADR-RES cell line by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK. Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAP CONCLUSIONS: We conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK. This mechanism is significantly different from that found for onconase.
authors.affiliation: Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi s/n E-17071 Girona, Spain.
authors.forename: Jessica
authors.initials: J
authors.lastname: Castro
authors.forename: Marc
authors.initials: M
authors.lastname: Ribó
authors.forename: Susanna
authors.initials: S
authors.lastname: Navarro
authors.forename: Maria Victòria
authors.initials: MV
authors.lastname: Nogués
authors.forename: Maria
authors.initials: M
authors.lastname: Vilanova
authors.forename: Antoni
authors.initials: A
authors.lastname: Benito
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D012260:Ribonucleases / Q000235:genetics / Q000494:pharmacology*
chemical_list: C493956:CDKN1A protein, human; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D011994:Recombinant Proteins; D019938:Cyclin D1; D048055:Mitogen-Activated Protein Kinase 8; D012260:Ribonucleases; D020169:Caspases; C058703:ranpirnase