Abstract
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.
Highlights
Group I p21-activated kinases (PAKs) are implicated in a wide range of cellular processes including cell proliferation, apoptosis, migration and adhesion to the extracellular matrix [1,2]
The effects of IPA-3 were studied in a panel of human cell lines derived from chronic myelogenous leukemia (JURL-MK1, MOLM-7, K562, CML-T1), acute myeloid leukemia (HL-60), acute lymphoblastic leukemia (JURKAT), erythroleukemia (HEL) and anaplastic large cell lymphoma (Karpas-299), as well as in human peripheral blood mononuclear cells (PBMC)
IPA-3 treatment reduced the number of viable cells and induced cell death with EC50 ranging from 5 to more than 20 mM (Figure 1 A shows examples for CML-T1 and JURL-MK1 cells, similar results were obtained for other cell lines, see Figure S1)
Summary
Group I p21-activated kinases (PAKs) are implicated in a wide range of cellular processes including cell proliferation, apoptosis, migration and adhesion to the extracellular matrix [1,2]. PAK1 was identified as a major mediator of resistance to phosphoinositide 3-kinase inhibitors in lymphoma cell lines [15]. Attempts to develop a specific small molecule PAK inhibitor resulted in the discovery of IPA-3, an allosteric inhibitor of group I PAK activation [16,17,18] which is suitable for studies of PAK functions its properties preclude its use in the clinical practice. We have previously reported that IPA-3 treatment of human leukemic JURL-MK1 cells reduced their ability to bind to fibronectin, one of the major components of the bone marrow extracellular matrix [19] and we have noted IPA-3 toxicity for hematopoietic cells. We thoroughly investigated the effects of PAK inhibition in a panel of human leukemia/lymphoma cell lines as well as in normal primary blood cells
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