Real-time monitoring of hematopoietic cell interaction with fibronectin fragment

Abstract

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.

Whole-cell biosensor for label-free detection of GPCR-mediated drug responses in personal cell lines

“…It is well known that the cell density and distribution on the electrodes can significantly influence impedance and experimental readout (Fang, 2011;Rocheville et al, 2013;Yu et al, 2006). In previous cases, fibronectin was often used to achieve cell adherence combined with increased cell densities with an optimal range of 60,000 to 45,000 cells/well (Gedge, 2010;Martinez-Serra et al, 2014;Obr et al, 2013;Schroder et al, 2011). Accordingly, we first tested various standard coating conditions and optimized cell density for impedance recordings in LCLs and found similar conditions to be optimal for LCLs.…”

“…Application of xCELLigence technology to suspension cells has been reported, however not for investigating GPCR signaling. Obr et al (2013) applied the technology to measure the effect of histone deacetylase inhibitors on hematopoietic cells. Martinez-Serra et al (2014) investigated the cytotoxic effect of antineoplastic agents on cells from hematological malignancies, which included the leukemia lymphoblast cell line K562.…”

xCELLigence system for real-time label-free monitoring of growth and viability of cell lines from hematological malignancies

“…For this reason, it was not useful in the study of cells derived from hematological malignancies 1,2,8–11. Recently, one report has described a system for the real-time measuring of cell adhesion in nonadherent cells 4. With this in mind, we have adapted the methodology described by others to xCELLigence for monitoring drug toxicity over hematologic cell lines.…”

“…Most leukemia or lymphoma cells usually grow in liquid media or suspension and they are unable to attach onto the cell culture wells’ surface, where the electrodes are located, so changes within the electric circuit cannot be properly measured. So far, with the xCELLigence technology, there has only been one report describing a system for measuring cell adhesion of cell lines derived from hematological malignancies 4. With this in mind, we have taken advantage of the strategy described by others,4–7 based on the addition of several coating substrates such as fibronectin, collagen, gelatin, and/or laminin.…”

Adhesion structures in leukemia cells and their regulation by Src family kinases

“…We reported previously that the microimpedance signal progressively increases for 1 to 1.5 h following leukemia cell addition to FN-coated wells, reflecting the cell attachment and spreading. 21 Figure 5A shows representative examples of extensive changes induced in adherent cells upon treatment with dasatinib or with cytochalasin D (cytD), an inhibitor of actin polymerization which releases the cytoskeletal tension and promotes disassembly of focal adhesions. Panels B-D show the effect of dasatinib, cytD and SKI-1 on OCI-AML3 cells, as a representative example of leukemia cell lines, and on HEL cells, an erythroleukemia cell line which is to some extent similar to adherent cells.…”