P21-activated kinases (PAK) are key effectors of the small GTPases Rac1 and Cdc42, as well as of Src family kinases. In particular, PAK1 has several well-documented roles, both kinase-dependent and kinase-independent, in cancer-related processes, such as cell proliferation, adhesion, and migration. However, PAK1 properties and functions have not been attributed to individual PAK1 isoforms: besides the full-length kinase (PAK1-full), a splicing variant lacking the exon 15 (PAK1Δ15) is annotated in protein databases. In addition, it is not clear if PAK1 and PAK2 are functionally overlapping. Using fluorescently tagged forms of human PAK1-full, PAK1Δ15, and PAK2, we analyzed their intracellular localization and mutual interactions. Effects of PAK inhibition (IPA-3, FRAX597) or depletion (siRNA) on cell-surface adhesion were monitored by real-time microimpedance measurement. Both PAK1Δ15 and PAK2, but not PAK1-full, were enriched in focal adhesions, indicating that the C-terminus might be important for PAK intracellular localization. Using coimmunoprecipitation, we documented direct interactions among the studied PAK group I members: PAK1 and PAK2 form homodimers, but all possible heterocomplexes were also detected. Interaction of PAK1Δ15 or PAK2 with PAK1-full was associated with extensive PAK1Δ15/PAK2 cleavage. The impedance measurements indicate, that PAK2 depletion slows down cell attachment to a surface, and that PAK1-full is involved in cell spreading. Altogether, our data suggest a complex interplay among different PAK group I members, which have non-redundant functions.

• MeSH
• Cell Adhesion genetics   physiology   MeSH
• Cell Line MeSH
• Exons genetics   MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• p21-Activated Kinases genetics   metabolism   MeSH
• Cell Movement genetics   physiology   MeSH
• Cell Proliferation genetics   physiology   MeSH
• Signal Transduction genetics   physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Interaction of leukemia blasts with the bone marrow extracellular matrix often results in protection of leukemia cells from chemotherapy and in persistence of the residual disease which is on the basis of subsequent relapses. The adhesion signaling pathways have been extensively studied in adherent cells as well as in mature haematopoietic cells, but the adhesion structures and signaling in haematopoietic stem and progenitor cells, either normal or malignant, are much less explored. We analyzed the interaction of leukemia cells with fibronectin (FN) using interference reflection microscopy, immunofluorescence, measurement of adherent cell fraction, real-time microimpedance measurement and live cell imaging. We found that leukemia cells form very dynamic adhesion structures similar to early stages of focal adhesions. In contrast to adherent cells, where Src family kinases (SFK) belong to important regulators of focal adhesion dynamics, we observed only minor effects of SFK inhibitor dasatinib on leukemia cell binding to FN. The relatively weak involvement of SFK in adhesion structure regulation might be associated with the lack of cytoskeletal mechanical tension in leukemia cells. On the other hand, active Lyn kinase was found to specifically localize to leukemia cell adhesion structures and a less firm cell attachment to FN was often associated with higher Lyn activity (this unexpectedly occurred also after cell treatment with the inhibitor SKI-1). Lyn thus may be important for signaling from integrin-associated complexes to other processes in leukemia cells.

• MeSH
• Cell Adhesion drug effects   physiology   MeSH
• Dasatinib pharmacology   MeSH
• Fibronectins metabolism   MeSH
• Focal Adhesions drug effects   metabolism   MeSH
• Focal Adhesion Protein-Tyrosine Kinases metabolism   MeSH
• Phosphorylation drug effects   MeSH
• Leukemia drug therapy   MeSH
• Humans MeSH
• src-Family Kinases drug effects   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Attachment of stem leukemic cells to the bone marrow extracellular matrix increases their resistance to chemotherapy and contributes to the disease persistence. In chronic myelogenous leukemia (CML), the activity of the fusion BCR-ABL kinase affects adhesion signaling. Using real-time monitoring of microimpedance, we studied in detail the kinetics of interaction of human CML cells (JURL-MK1, MOLM-7) and of control BCR-ABL-negative leukemia cells (HEL, JURKAT) with fibronectin-coated surface. The effect of two clinically used kinase inhibitors, imatinib (a relatively specific c-ABL inhibitor) and dasatinib (dual ABL/SRC family kinase inhibitor), on cell binding to fibronectin is described. Both imatinib and low-dose (several nM) dasatinib reinforced CML cell interaction with fibronectin while no significant change was induced in BCR-ABL-negative cells. On the other hand, clinically relevant doses of dasatinib (100 nM) had almost no effect in CML cells. The efficiency of the inhibitors in blocking the activity of BCR-ABL and SRC-family kinases was assessed from the extent of phosphorylation at autophosphorylation sites. In both CML cell lines, SRC kinases were found to be transactivated by BCR-ABL. In the intracellular context, EC50 for BCR-ABL inhibition was in subnanomolar range for dasatinib and in submicromolar one for imatinib. EC50 for direct inhibition of LYN kinase was found to be about 20 nM for dasatinib and more than 10 µM for imatinib. Cells pretreated with 100 nM dasatinib were still able to bind to fibronectin and SRC kinases are thus not necessary for the formation of cell-matrix contacts. However, a minimal activity of SRC kinases might be required to mediate the increase in cell adhesivity induced by BCR-ABL inhibition. Indeed, active (autophosphorylated) LYN was found to localize in cell adhesive structures which were visualized using interference reflection microscopy.

• MeSH
• Cell Adhesion drug effects   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology   MeSH
• Dasatinib pharmacology   MeSH
• Fibronectins metabolism   MeSH
• Phosphorylation drug effects   MeSH
• Imatinib Mesylate pharmacology   MeSH
• Kinetics MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Antineoplastic Agents pharmacology   MeSH
• src-Family Kinases metabolism   MeSH
• Protein Transport drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

P21-activated kinases (PAKs) are involved in the regulation of multiple processes including cell proliferation, adhesion and migration. However, the current knowledge about their function is mainly based on results obtained in adherent cell types. We investigated the effect of group I PAK inhibition using the compound IPA-3 in a variety of human leukemic cell lines (JURL-MK1, MOLM-7, K562, CML-T1, HL-60, Karpas-299, Jurkat, HEL) as well as in primary blood cells. IPA-3 induced cell death with EC50 ranging from 5 to more than 20 μM. Similar range was found for IPA-3-mediated dephosphorylation of a known PAK downstream effector, cofilin. The cell death was associated with caspase-3 activation, PARP cleavage and apoptotic DNA fragmentation. In parallel, 20 μM IPA-3 treatment induced rapid and marked decrease of the cell adhesivity to fibronectin. Per contra, partial reduction of PAK activity using lower dose IPA-3 or siRNA resulted in a slight increase in the cell adhesivity. The changes in the cell adhesivity were also studied using real-time microimpedance measurement and by interference reflection microscopy. Significant differences in the intracellular IPA-3 level among various cell lines were observed indicating that an active mechanism is involved in IPA-3 transport.

• MeSH
• Apoptosis drug effects   MeSH
• Cell Adhesion drug effects   MeSH
• Disulfides metabolism   pharmacology   MeSH
• Actin Depolymerizing Factors chemistry   metabolism   MeSH
• Fibronectins metabolism   MeSH
• Phosphorylation drug effects   MeSH
• Hematopoiesis drug effects   MeSH
• Protein Kinase Inhibitors metabolism   pharmacology   MeSH
• Intracellular Space drug effects   metabolism   MeSH
• Blood Cells cytology   drug effects   MeSH
• Leukemia pathology   MeSH
• Humans MeSH
• Lymphoma pathology   MeSH
• RNA, Small Interfering genetics   MeSH
• Naphthols metabolism   pharmacology   MeSH
• p21-Activated Kinases antagonists & inhibitors   deficiency   genetics   MeSH
• Cell Proliferation drug effects   MeSH
• Gene Expression Regulation drug effects   MeSH
• Serine metabolism   MeSH
• Gene Silencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Real-time cell analysis (RTCA) system based on measurement of electrical microimpedance has been introduced to monitor adherent cell cultures. We describe its use for real-time analysis of hematopoietic cell adhesion to bone marrow stroma proteins. Cells growing in suspension do not generate any significant change in the microimpedance signal until the surface with embedded microelectrodes is coated with a cell-binding protein. We show that in this case, the microimpedance signal specifically reflects cell binding to the coated surface. The optimized method was used to monitor the effect of two histone deacetylase inhibitors, suberoylanilide hydroxamic acid (SAHA) and tubastatin A, on JURL-MK1 cell adhesion to cell-binding fragment of fibronectin (FNF). Both compounds were used in non-toxic concentrations and induced an increase in the cell adhesivity. The kinetics of this increase was markedly slower for SAHA although tubulin hyperacetylation occurred rapidly for any of the two drugs. The strengthening of cell binding to FNF was paralleled with a decrease of Lyn kinase activity monitored using an anti-phospho-Src family antibody. The inhibition of Src kinase activity with PP2 accordingly enhanced JURL-MK1 cell interaction with FNF. Actin filaments were present at the proximity of the plasma membrane and in numerous membrane protrusions. In some cells, F-actin formed clusters at membrane regions interacting with the coated surface and these clusters colocalized with active Lyn kinase. Our results indicate that the role of Src kinases in the regulation of hematopoetic cell adhesion signaling is similar to that of c-Src in adherent cells.

• MeSH
• Acetylation drug effects   MeSH
• Actins metabolism   MeSH
• Cell Adhesion drug effects   MeSH
• Electric Impedance MeSH
• Fibronectins metabolism   MeSH
• Hematopoietic Stem Cells metabolism   MeSH
• Histone Deacetylases metabolism   MeSH
• Indoles pharmacology   MeSH
• Histone Deacetylase Inhibitors pharmacology   MeSH
• Jurkat Cells MeSH
• Hydroxamic Acids pharmacology   MeSH
• Humans MeSH
• Cell Communication MeSH
• Cell Movement MeSH
• Signal Transduction MeSH
• src-Family Kinases metabolism   MeSH
• Tubulin drug effects   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH