12th Congress of the European Hematology ... - Haematologica

12 th Congress of the European Hematology Association
identified as a new component of cell-cell junctions that controls αcatenin
recruitment and is able to interact with α-catenin, ARF1 and
ARF6, important proteins in the cytoskeleton assembly and adherent
junctions. Cell-to-cell adhesion is one of the major factors that restrict
cellular invasion and metastasis in malignant diseases. FAK is a nonreceptor
tyrosine kinase, and coordinates signals from integrins,
cytokines, growth factor receptors, and oncogenes. In AML cells, FAK
has been found to be overexpressed and associated with enhanced blast
migration, increased cellularity, and poor prognosis. Aims. In the attempt
to verify the role of ARHGAP21 in leukemogenesis, we aimed to verify
the expression level of ARHGAP21 mRNA and protein in normal
hematopoietic cells and in leukemia cells. In addition, we attempted to
verify a possible interaction of ARHGAP21 with proteins involved in cell
adhesion, as Focal adhesion kinase (FAK). Methods. Marrow aspirates
were obtained from 5 normal donors, 37 patients with Acute Myeloid
Leukemia (AML) and 9 patients with Acute Lymphoblast Leukemia
(ALL). The National Ethical Committee Board approved the study and
informed-written consent was obtained from all patients and donors.
Total cells were submitted to RNA extraction and the expression level
of mRNA was detected by real time RT-PCR. The relative quantification
value of gene expression was calculated using 2'DDCT. Leukemia cell
lines (Jurkat, Molt-4, K562, HL60) and normal peripheral blood mononuclear
cells (PBMC) obtained from 5 normal donors were submitted to
immunoprecipitation and Western blotting analysis to detected protein
expression and protein interactions. Pull down assays using three different
FAK-GST fusion proteins: C-terminal (residues 687-1054), FERM
domain (residues 60-349) and catalytic domain (residues 390-696) were
performed to confirm the interaction between ARHGAP21 and FAK.
Results. ARHGAP21 mRNA expression was found to be significantly
higher in AML and ALL samples when compared with normal
hematopoietic cells (medians: AML: 3.01 vs 0.25, p