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Int J Hematol. 2012 Mar;95(3):299-310. doi: 10.1007/s12185-012-1013-1. Epub 2012 Feb 5.

Impaired cell adhesion, apoptosis, and signaling in WASP gene-disrupted Nalm-6 pre-B cells and recovery of cell adhesion using a transducible form of WASp.

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1
Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University Graduate School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.

Abstract

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disease affecting cell morphology and signal transduction in hematopoietic cells. The function of Wiskott-Aldrich syndrome protein (WASp) and its partners in protein interaction have been studied intensively in mice; however, detailed biochemical characterization of its signal transduction and assessment of its functional consequence in human WASp-deficient lymphocytes remain difficult. In this study, we generated Nalm-6 cells in which the WAS protein gene (WASP) was disrupted by homologous recombination-based gene targeting and a cell-permeable form of recombinant WASp for functional study. The WASP⁻/⁻ cells showed impaired adhesive capacity and polarization to plate-bound anti-CD47 mAb, anti-CD9 mAb, or to fibronectin. The defective morphological changes were accompanied by impaired intracellular signaling. In addition, the WASp-deficient cells displayed augmented apoptosis induced by CD24 cross-linking. A recombinant fusion protein composed of Hph-1 cell-permeable peptide and WASp prepared in Escherichia coli. Hph-1-WASp was efficiently transduced and expressed in WASP⁻/⁻ Nalm-6 cells in a dose-dependent manner. The wild-type WASp, but not the mutant restored adhesion capacity, spreading morphology, and cytoskeletal reorganization. Additionally, the recombinant protein was successfully transduced into normal lymphocytes. These findings suggest that gene-disrupted model cell lines and cell-permeable recombinant proteins may serve as important tools for the detailed analysis of intracellular molecules involved in PID.

PMID:
22311461
DOI:
10.1007/s12185-012-1013-1
[Indexed for MEDLINE]
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