Monitoring of ovarian cancer cell invasion in real time with frequency-dependent impedance measurement.

The conventional approach to assessing cancer invasion is primarily for end-point analysis, which does not provide temporal information on the invasion process or any information on the interactions between invading cells and the underlying adherent cells. To alleviate these limitations, the present study exploited electric cell-substrate impedance sensing (ECIS) to monitor the invasion of ovarian cancer cells (SKOV-3) through an adherent monolayer of human umbilical vein endothelial cells (HUVECs). Impedance was measured at 4 kHz of AC voltage or was measured as a function of AC frequency (25 Hz to 60 kHz). By measuring impedance at 4-kHz AC, we found that the invasion of SKOV-3 cells through the HUVEC monolayer was manifested as a rapid decrease in transendothelial electrical resistance in real time. The invasion was augmented in the presence of hepatocyte growth factor (HGF). The enhancing effect of HGF was attenuated by c-Met inhibitor (SU11274). By measuring the frequency-dependent impedance of SKOV-3 cells over time, we found that HGF-enhanced SKOV-3 cell invasion was accomplished with reduced junctional resistance (Rb), increased average cell-substrate separation (h), and increased micromotion. SU11274 attenuated the effects of HGF on Rb, h, and micromotion in the SKOV-3 monolayer. SU11274 also increased the barrier function of the HUVEC monolayer by increasing Rb and decreasing h In conclusion, this study demonstrated an improved method for monitoring and studying the interactions between cancer cells and the underlying adherent cells during invasion in real time. Alterations in cellular biophysical properties (Rb, h) associated with cancer transendothelial invasion were detected.

Spatially controlled expression of the Drosophila pseudouridine synthase RluA-1.

Pseudouridine (Ψ) synthases function in the formation of Ψ, the most abundant of the modified RNA residues. All Ψ synthases in E. coli are classified into one of five families according to their sequences. Among them, members of the RluA Ψ synthase family catalyze certain Ψ formations in ribosomal RNA. RluA family members are required for ribosomal assembly and bacterial growth. None of the RluA in multicellular organisms has been studied. In the Drosophila peripheral nervous system, multiple dendritic (MD) neurons are recognized by their dendritic arbors. MD neurons can also be identified by using the enhancer trap line E7-2-36, which expresses the lacZ gene in MD neurons. Here, we show that the P-element of E7-2-36 inserts into the Drosophila RluA-1 gene. RluA-1 is homologous to E. coli RluA family members and is evolutionarily conserved in multicellular organisms. In situ hybridization and immunocytochemistry revealed that RluA-1 is expressed in MD neurons. We investigated the RluA-1 enhancer responsible for MD expression and found that the membrane-tethered green fluorescent protein driven by RluA-1-GAL4 was expressed in the dendritic arbors of MD neurons, confirming that RluA-1 is indeed expressed in MD neurons. Thus, the expression of RluA-1 is spatially controlled during development.

Use of electric cell-substrate impedance sensing to assess in vitro cytotoxicity.

In vitro assessment of cytotoxicity based on electrochemical impedance spectroscopy (EIS) needs more quantitative methods to analyze the alteration of cell morphology and motility, and hence the potential risk to human health. Here, we applied electric cell-substrate impedance sensing (ECIS) to evaluate dose-dependent responses of human umbilical vein endothelial cells exposed to cytochalasin B. To detect subtle changes in cell morphology, the frequency-dependent impedance data of the cell monolayer were measured and analyzed with a theoretical cell-electrode model. To detect the alternation of cell micromotion in response to cytochalasin B challenge, time-series impedance fluctuations of cell-covered electrodes were monitored and the values of power spectrum, variance, and variance of the increments were calculated to verify the difference. While a dose-dependent relationship was generally observed from the overall resistance of the cell monolayer, the analysis of frequency-dependent impedance and impedance fluctuations distinguished cytochalasin B levels as low as 0.1 microM. Our results show that cytochalasin B causes a decrease of junctional resistance between cells, an increase of membrane capacitance, and the reduction in micromotion.

Activation of P2X(7) receptors decreases glutamate uptake and glutamine synthetase activity in RBA-2 astrocytes via distinct mechanisms.

Glutamate clearance by astrocytes is critical for controlling excitatory neurotransmission and ATP is an important mediator for neuron-astrocyte interaction. However, the effect of ATP on glutamate clearance has never been examined. Here we report that treatment of RBA-2 cells, a type-2-like astrocyte cell line, with ATP and the P2X(7) receptor selective agonist 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) decreased the Na+-dependent [3H]glutamate uptake within minutes. Mechanistic studies revealed that the decreases were augmented by removal of extracellular Mg2+ or Ca2+, and was restored by P2X7 selective antagonist , periodate-oxidized 2',3'-dialdehyde ATP (oATP), indicating that the decreases were mediated through P2X(7) receptors. Furthermore, stimulation of P2X7 receptors for 2 h inhibited both activity and protein expression of glutamine synthetase (GS), and oATP abolished the inhibition. In addition, removal of extracellular Ca(2+) and inhibition of protein kinase C (PKC) restored the ATP-decreased GS expression but failed to restore the P2X(7)-decreased [3H]glutamate uptake. Therefore, P2X7-mediated intracellular signals play a role in the down-regulation of GS activity/expression. Activation of P2X7 receptors stimulated increases in intracellular Na+ concentration ([Na+](i)) suggesting that the P2X(7)-induced increases in [Na+](i) may affect the local Na+ gradient and decrease the Na+-dependent [3H]glutamate uptake. These findings demonstrate that the P2X7-mediated decreases in glutamate uptake and glutamine synthesis were mediated through distinct mechanisms in these cells.

Endothelin-1 stimulated capacitative Ca2+ entry through ET(A) receptors of a rat brain-derived type-1 astrocyte cell line, IA-1g1.

The present study demonstrated that endotheline-1 (ET-1) stimulated a biphasic (transient and sustained) increase in [Ca(2+)](i) and signaling was blocked by BQ123 and inhibited by BQ788. RT-PCR analysis revealed that ET(A) was expressed more than ET(B) mRNA-suggesting that ET(A) is the major receptor. Simply reintroducing Ca(2+) in the buffer stimulated a sustained increase in [Ca(2+)](i) and the effect was inhibited by U73122, thapsigargin (TG), miconazole and SKF96365. When measured in Ca(2+)-free buffer, the ET-1-stimulated Ca(2+) transient decreased by 73% and the reintroduction of Ca(2+) induced a large sustained increase in [Ca(2+)](i). These effects were not affected by nifedipine, but were inhibited by miconazole and SKF96365-indicating that the sustained increase in [Ca(2+)](i) mediated by ET-1 was mostly due to capacitative Ca(2+) entry (CCE). The ET-1-induced CCE was inhibited by phorbol ester (PMA) but was enhanced by GF109203X; it was also enhanced by 8-bromo-cyclic AMP (8-Br-cAMP) but was inhibited by H89. Thus, protein kinase C (PKC) negatively regulated and cAMP-dependent protein kinase (PKA) positively regulated the ET-1-mediated CCE in these cells.