title: Cell-penetrating TAT-FOXO3 fusion proteins induce apoptotic cell death in leukemic cells.
abstract: FOXO proteins are Akt-regulated transcription factors involved in the control of cell cycle, DNA repair, stress defense, apoptosis, and tumor suppression. We reported that plasmid-based overexpression of constitutively active FOXO3 in cells from chronic lymphocytic leukemia (CLL) reduced their survival, suggesting that increasing FOXO3 activity in hematologic malignancies may represent a promising therapeutic strategy. The transactivating transcription factor (TAT) protein transduction domain (PTD) derived from the HIV TAT protein was shown to efficiently deliver macromolecular cargo in various cell types. In this study, wild-type FOXO3 and FOXO3 mutated on Akt sites [FOXO3 T32A/S253A/S315A or TM (triple mutant)] were fused to the TAT-PTD. Using biochemical techniques, flow cytometry, and microscopy analysis, we found a rapid and dose-dependent cell penetration into leukemic cells of unlabeled and fluorescein isothiocyanate-labeled TAT-FOXO3 fusion proteins followed by their accumulation within nuclear and cytoplasmic compartments. Treatment with TAT-FOXO3 TM-but not wild-type TAT-FOXO3-proteins induced Jurkat and K562 leukemic cell death and affected cell viability of other hematologic malignancies including primary cells from CLL. Cell transduction with TAT-FOXO3 TM induced apoptotic cell death as shown by morphologic changes, Annexin V/7-AAD (7-amino-actinomycin D) staining, activation of effector caspases, and PARP cleavage, caspase blockade through the use of the inhibitor Z-VAD, and expression of Bim and p27(KIP1). By contrast, TAT-FOXO3 TM blocked cell proliferation of primary T cells, without affecting their viability. Together, our data show that cell penetrating TAT-FOXO3 TM fusion proteins constitute novel potential therapeutic agents in the treatment of lymphoproliferative disorders and hematologic malignancies.
authors.affiliation: INSERM UMR 576, Hôpital de l'Archet, 151 Route de Saint-Antoine de Ginestiére, B.P. 3079, 06202, Nice Cedex 3, France.
authors.forename: Makram
authors.initials: M
authors.lastname: Essafi
authors.forename: Alice D
authors.initials: AD
authors.lastname: Baudot
authors.forename: Xavier
authors.initials: X
authors.lastname: Mouska
authors.forename: Jill-Patrice
authors.initials: JP
authors.lastname: Cassuto
authors.forename: Michel
authors.initials: M
authors.lastname: Ticchioni
authors.forename: Marcel
authors.initials: M
authors.lastname: Deckert
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D048708:Cell Growth Processes / Q000187:drug effects; D045744:Cell Line, Tumor; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000096:biosynthesis / Q000276:immunology; D004305:Dose-Response Relationship, Drug; D016650:Fluorescein-5-isothiocyanate / Q000493:pharmacokinetics; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D015696:Gene Products, tat / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D020672:I-kappa B Proteins / Q000096:biosynthesis / Q000276:immunology; D019169:Jurkat Cells; D020014:K562 Cells; D007938:Leukemia / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008213:Lymphocyte Activation / Q000187:drug effects; D051058:Oncogene Protein v-akt / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D014161:Transduction, Genetic; D020298:U937 Cells
chemical_list: C436291:CDKN1B protein, human; C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D015696:Gene Products, tat; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D015514:Oncogene Proteins, Fusion; D050760:Cyclin-Dependent Kinase Inhibitor p27; D051058:Oncogene Protein v-akt; D016650:Fluorescein-5-isothiocyanate