Real-time Cell Electronic Sensing Research Articles

Microelectronic-sensing assay to detect presence of Verotoxins in human faecal samples

To develop a novel Vero cell assay that implements a real-time cell electronic sensing (RT-CES) system for the determination of the presence of verotoxin-producing Escherichia coli (VTEC). The assay overcomes the major drawbacks in conventional Vero cell assay, for example, labour-intensive and time-consuming. Cells were grown onto the surfaces of microelectronic sensors that are integrated into the bottom surfaces of the microtiter plate. Cellular viability was monitored in real-time and quantified based on changes in the sensor's electrical impedance. For cell viability measurement, the data generated on the RT-CES system correlated well with those obtained by the Vero cell assay for Verotoxins. To assess cytotoxicity, test cells growing on microelectronic sensors were treated with either supernatant from pure cultures, or stool samples. The specific neutralizing antibodies of VT1 and VT2 were used to identify specific toxins in the samples. The RT-CES assay provides a sensitive measurement comparable to conventional crystal violet assay. The assay has been successfully and specifically used to identify VTEC in human faecal samples. The RT-CES assay significantly shortens the testing time from 48 to 72 h required by the crystal violet assay to only 15 h with automated operation.

4-hexylresorcinol stimulates the differentiation of SCC-9 cells through the suppression of E2F2, E2F3 and Sp3 expression and the promotion of Sp1 expression

The dormancy-inducing factors of bacteria inhibit tumor cell growth. In the present study, we evaluated the antitumor effects of the dormancy-inducing factor 4-hexylresorcinol (4-HR) using real-time cell electronic sensing (RT-CES) in SCC-9 cells (tongue squamous cell carcinoma cells). Treatment with 4-HR suppressed the growth of SCC-9 cells in a dose-dependent manner. We used a DNA microarray to identify genes that showed a significant change in expression upon 4-HR administration in SCC-9 cells. Among the differentially expressed genes, the protein expression of several cell proliferation related factors, including E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, Sp1 and Sp3, were determined by western blot analyses. Treatment with 4-HR strongly suppressed E2F2 and slightly suppressed E2F3 but did not change the expression of E2F1, E2F4, E2F5 and E2F6 relative to no treatment. Furthermore, 4-HR increased Sp1 expression in a dose-dependent manner and decreased Sp3 expression. Therefore, the ratio of Sp1 to Sp3, an important driving force of epithelial cell differentiation, was drastically increased. Consistent with this observation, 4-HR increased the expression of the epithelial cell differentiation markers involucrin and keratin 10. Together, our results indicate that 4-HR induces the differentiation of SCC-9 via the modulation of the E2F-mediated signaling pathway.

Therapeutic targeting of oncogenic transforming growth factor-β1 signaling by antisense oligonucleotides in oral squamous cell carcinoma

The transforming growth factor-β1 (TGF-β1) signaling pathway is important in human oral squamous cell carcinoma (OSCC). Accordingly, the aims of this study were to evaluate the effect of antisense TGF-β1 oligonucleotides (ODNs) on OSCC in cell culture and in a xenograft model, as well as to evaluate any effects ODNs have on proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase-2 (MMP-2) expression in the xenograft model. We performed real-time cell electronic sensing (RT-CES) to determine the effect of antisense TGF-β1 ODNs on SCC-9cell growth. To examine the invivo effect of antisense TGF-β1 ODN therapy, SCC-9cells were grafted into nude mice. Antisense ODNs were injected into the mass daily. Tumor size, body weight and duration of survival were assessed daily. Specimens from the main mass were used for immunohistochemical staining to analyze PCNA and MMP-2 expression. Invitro treatment with antisense TGF-β1 ODNs decreased TGF-β1 expression and growth of SCC-9cells. In the xenograft model, the antisense TGF-β1 ODN group exhibited a significantly decreased tumor growth rate compared to the control, which received Dulbecco's modified Eagle's medium (DMEM) (P=0.022). However, mean survival time and body weights were not significantly different between the groups (P>0.05). Immunohistochemistry showed that tumors from animals that received antisense TGF-β1 ODNs had significantly lower expression levels of PCNA and MMP-2 compared to tumors from animals in the DMEM group (P<0.05). In conclusion, antisense TGF-β1 ODN therapy significantly inhibits tumor growth compared to controls, however, there are no significant differences between groups with respect to changes in body weight.

LBSO potentiate the effects of Hg in breast cancer cells

Breast cancer is the second major cause of death in women. Reports show that breast cancer cells have high level of glutathione. This research was done to characterize the kinetic signature and mechanism of cell death using the oxidative stress pathway when mercury (Hg) and L‐buthionine sulphoximine (LBSO, inhibits glutathione production) are presented to MCF‐7 cells. The hypothesis tested is that the potentiating effects of LBSO will be higher with Hg than with As. Production was inhibited by treating the MCF7 cell line with 2.5 mM (LBSO) and then exposed to mercury 5.4ppm Hg. The cytotoxic effect was studied in real time using Real Time Cell Electronic Sensing (RTCES). To determine the oxidative stress pathway used, mitochondrial transmembrane potential (&amp;#8710;øm), Reactive Oxygen Species (ROS), superoxide anion and Glutathione (GSH) levels were studied using Rhodamine123 fluorescent (Rho123), 2′,7′‐Dichlorodihydrofluorescein (H2DCFDA), Dihydroethidium (DHE), 5‐Chloromethylfluorescein diacetate (CMFDA) dyes respectively. Each sample, 10,000 events were collected using FACSCalibur flow cytometer. ROS and O2 • levels were expressed as mean fluorescence intensities (MFI), calculated with CellQuest. The results did not show a vast difference between Hg and LBSO and Hg alone. Results reveal that Hg might be a better candidate for cancer therapy compared to As due to As high toxicity.

Recognition of chemical compounds in contaminated water using time-dependent multiple dose cellular responses

An early determination of toxicant compounds of water contaminations can gain critical time to protect citizens’ health and save substantial amounts of medical costs. To determine toxins in real time, a multi-dose classification algorithm using cellular state variable identification (CSVID) is developed in this paper. First, the dynamic cytotoxicity response profiles of living cells are measured using a real-time cell electronic sensing (RT-CES) system. Changes in cell number expressed as cell index (CI) are recorded on-line as time series. Then CSVID, which reflects the cell killing, cell lysis and certain cellular pathological changes, is extracted from those dynamic cellular responses. Finally, a support vector machine (SVM) algorithm based on CSVID is employed to classify chemical compounds and determine their analogous cellular response pathway. In order to increase the classification accuracy, a majority vote of the class labels is also proposed. Several validation studies demonstrate that CSVID-based classification algorithm has great potential in distinguishing the cytotoxicity response of the cells in the presence of toxins.

Dynamic Investigation of Sensitivity and Action Mechanism of Antitumor Drug

Dynamic responses of A549 cells to epirubicin (EPI), daunorubicin (DNR), cisplatin (DDP) and carboplatin (CBP) were monitored by real-time cell electronic sensing (RT-CES) system. A new defined parameter, the detachment duration, was defined to quantitatively characterize drug-cell action mechanism. For DNR, there were two different linear dependent regions of the detachment duration. The rate constant of DNR in lower concentration region (20.4±2.0 h/μM) is much larger than that in higher concentration region (3.2±0.1 h/μM). Moreover, the rate constant of DNR in lower concentration region is close to that of EPI in higher concentration region (17.5±0.5 h/μM). Similar observation occurred in the case of DDP and CBP treatment, and the rate constant is (2.8±0.4)×10 h/μM and (4.6±1.3)×10 h/μM respectively. Taken together, A549 cells are more sensitive to DNR than EPI and to DDP than CBP. This study provides new insight into cell-drug interaction. RT-CES system may play important role in future drug screening, mechanism exploration and clinical therapy.

New bis(thiosemicarbazonate) gold(III) complexes inhibit HIV replication at cytostatic concentrations: Potential for incorporation into virostatic cocktails

Four bis(thiosemicarbazonate)gold(III) complexes (1–4) with a general formula [Au(L)]Cl {L = L1, glyoxal-bis(N 4-methylthiosemicarbazone); L2, glyoxal-bis(N 4-ethylthiosemicarbazone); L3, diacetyl-bis(N 4-methylthiosemicarbazone); L4, diacetyl-bis(N 4-ethylthiosemicarbazone)} were synthesised and screened for activity against the human immunodeficiency virus (HIV). Complexes 1–4 were characterised using 1H-NMR and IR spectroscopy; and their purity established by micronanalysis. Complex 3 inhibited viral infection of TZM-bl cells by 98% (IC 50 = 6.8 ± 0.6 μM) at a non toxic concentration of 12.5 μM while complex 4 inhibited infection of these cells by 72 and 98% (IC 50 = 5.3 ± 0.4 μM) at concentrations of 6.25 and 12.5 μM respectively. The mechanism of inhibition of infection in TZM-bl cells is presumably as a result of the cytostatic or anti-proliferative activity that was observed for complex 4 in real time cell electronic sensing (RT-CES) and carboxyflourescein succinimidyl ester (CFSE) analysis. Treatment of T lymphocytes from HIV infected individuals with 4 decreased CD4+ T cell expression (p = 0.0049) as demonstrated by multi-parametric flow cytometry without suppressing cytokine production. None of the ligands (L1–L4) demonstrated anti-viral activity, supporting the importance of metal (gold) complexation in these potential drugs. Complexes 3 and 4 were shown to have ideal lipophilicity values that were similar when shake flask (0.97 ± 0.5 and 2.42 ± 0.6) and in silico prediction (0.8 and 1.5) methods were compared. The activity and drug-like properties of complexes 3 and 4 suggests that these novel metal-based compounds could be combined with virus inhibitory drugs to work as cytostatic agents in the emerging class of anti-HIV drugs known as virostatics.

Bioactivity of the Conjugation of Green-Emitting CdTe Quantum Dots with a Carborane Complex

In this report, we describe the effect of conjugating o-carborane-C(1)C(2)-dicarboxylic acid (o-C2B10H10-C2O4H2, denoted as Cbac2) to cadmium telluride quantum dots (CdTe QDs) capped with cysteamine on the photophysics and cytotoxicity of the QDs. Cbac2 quenches the fluorescence intensity and induces a red shift of the fluorescence emission peak. Meanwhile, studies with a real time cell electronic sensing (RT-CES) system and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay indicate that the combination of the carborane carboxylic acid derivative Cbac2 with relevant QDs can efficiently improve the inhibition efficiency for target cancer cells when compared with a single ligand or the CdTe QDs alone. This study raises the possibility for the labeling of the important pharmacophore with QDs and the design of new promising anticancer agents containing the carborane pharmacophores for cancer therapy.

Data-based modeling and prediction of cytotoxicity induced by contaminants in water resources

Abstract This paper is concerned with dynamic modeling, prediction and analysis of cell cytotoxicity induced by water contaminants. A real-time cell electronic sensing (RT-CES) system has been used for continuously monitoring dynamic cytotoxicity responses of living cells. Cells are grown onto the surfaces of the microelectronic sensors. Changes in cell number expressed as cell index (CI) have been recorded on-line as time series. The CI data are used to develop dynamic prediction models for cell cytotoxicity process. We consider support vector regression (SVR) algorithm to implement data-based system identification for dynamic modeling and prediction of cytotoxicity. Through several validation studies, multi-step-ahead predictions are calculated and compared with the actual CI obtained from experiments. It is shown that SVR-based dynamic modeling has great potential in predicting the cytotoxicity response of the cells in the presence of toxicant.

New Potential Anticancer Agent of Carborane Derivatives: Selective Cellular Interaction and Activity of Ferrocene-Substituted Dithio-o-carborane Conjugates

The large diversity of structures and unique bonding modes of organometallic complexes make them possible to act as promising candidate therapeutic agents. In this study, the new type of ferrocene-substituted dithio-o-carborane conjugates (FcSB1, FcSB2, and FcSBCO) has been synthesized, and their in vitro antineoplastic activities have been explored by means of the electrochemical study, the real time cell electronic sensing (RT-CES) system, and biological assays. The conjugate-cell interactions were first monitored by electrochemistry, and the results show different cell uptake efficiency for FcSB1, FcSB2, and FcSBCO toward target cells. Both the highly hydrophobic ferrocenyl and carboranyl groups render the conjugates able to rapidly enter cells and exert acute cytotoxicity after 4 h incubation in serum-free media. However, FcSB1, FcSB2, and FcSBCO display different inhibition efficiencies toward SMMC-7721 and HepG2 cancer cells via the G(0)/G(1) arrest mechanism in a physiological environment. The anticancer activity is in the order FcSB2 > FcSB1 > FcSBCO, which is parallel to the order of the redox potentials of the ferrocenyl groups in the three complexes. In particular, FcSB1 and FcSB2 display a potent selective inhibition effect on the proliferation of the cancer cell lines SMMC-7721 and HepG2, but almost no effect on the normal cell line, the human embryonic lung fibroblast (HELF) cells. Thus, these results may provide some clues for use of the ferrocene-carborane conjugates in developing anticancer drugs.

Cellular impedance measurement as a new tool for poxvirus titration, antibody neutralization testing and evaluation of antiviral substances

Impedance-based biosensing known as real-time cell electronic sensing (RT-CES) belongs to an emerging technology for analyzing the status of cells in vitro. In the present study protocols were developed for an RT-CES-based system (xCELLigence™, Roche Applied Science, ACEA Biosciences Inc.) to supplement conventional techniques in pox virology. First, proliferation of cells susceptible to orthopoxviruses was monitored. For virus titration cells were infected with vaccinia virus and cell status, represented by the dimensionless impedance-based cell index (CI), was monitored. A virus-dose dependent decrease in electrical impedance could be shown. Calculation of calibration curves at a suitable CI covering a dynamic range of 4 log enabled the quantification of virus titers in unknown samples. Similarly, antiviral effects could be determined as shown for anti-poxviral agents ST-246 and Cidofovir. Published values for the in vitro concentration that inhibited virus replication by 50% (IC 50) could be confirmed while cytotoxicity in effective concentrations was excluded in long-term incubation experiments. Finally, an RT-CES-based virus neutralization test was established. Various poxvirus-specific antibodies were examined for their neutralizing activity and a calculation mode for the neutralizing antibody titer was introduced. In summary, the presented RT-CES-based methods outmatch end-point assays by observing the cell population throughout the entire experiment while workload and time to result are reduced.

Cytotoxicity and Cell Cycle Perturbation of Arsenic and Mercury Exposure on MCF‐7 cells

Breast cancer is one of the leading causes of death in women in the United States. There are several factors that are known to contribute to the onset of this disease. We hypothesize that environmental exposure to heavy metals, such as mercury and arsenic, is a contributing factor. In this study, the cytotoxicity of As and Hg was studied in exponentially growing and asynchronous MCF‐7 human breast cancer cells. Cytoxicity was determined with real time cell electronic sensing (Rt‐CES). LC50 of each metal and the combination of metals was determined after 24, 48, 72, and 96 hours. The results show that the LC50 of As decreased tenfold from 24 hours through 96 hours of exposure: 1.8 x10‐3 to 1.8 x10‐4 mg/ml. The LC50 for Hg exposure showed a relatively slighter change: 7.2x10‐2 to 4.5 x10‐2 mg/ml. The LC50 for the mixture decreased from 3.3 x10‐4 mg/ml after 24 hours exposure to 3.0x10‐5mg/ml after 96 hours. Cell cycle analysis revealed significant differences in the percentage of cells in S and G2 phases in all samples, while the percentage of cells in G1 phase did not show any significant difference. As and Hg, individually and in combination, cause marked perturbation of the cell cycle kinetics of MCF‐7 human breast cancer cells by arresting the transit of cells at S phase. Further studies are needed to determine the mechanisms leading to the observed perturbations. Supported by NIH R25GM063775.

&lt;I&gt;In Vitro&lt;/I&gt; Study of Silica Nanoparticle-Induced Cytotoxicity Based on Real-Time Cell Electronic Sensing System

The biological fate and potential toxicity of nanoparticles have become hot topics since the use of nanoparticles was extended to the biomedical and biotechnological field. In this paper, an in vitro study of silica (SiO2) nanoparticle-induced cytotoxicity was presented mainly by real-time cell electronic sensing (RT-CES) system, which could be used for label-free, dynamic measurement of cell responses to cytotoxicants. The cytotoxicity of both 20-nm silica nanoparticles and microscale SiO2 powder in cultured RAW264.7 cells at dosage levels from 31.25 to 8000 microg/ml was evaluated comparatively. The experimental results showed that the cell index (CI) decrease induced by SiO2 nanoparticles appeared earlier than that by the microscale SiO2 powder at the same dose. The nanoparticles in the dose of 500 micdrog/ml had the toxic effect by the Cell Index Curves of RT-CES, while the microscale SiO2 powders in the same dose did not show obvious effect. The above experimental results were compared with the experiments of a tetrazolium compound-based colorimetric method (MTT assay) and LDH activity in medium. The results showed that the RT-CES assay displayed equal sensitivity and correlation to MTT assay. LDH activity caused by SiO2 nanoparticles at dose of 250 and 500 microg/ml was higher than that by microscale SiO2 particles, which indicated that the exposure to SiO2 nanoparticles could result in a dose-dependent cytotoxicity in cultured RAW264.7 cells due to cell membrane injury. The RT-CES system allows a realtime, continuous monitoring and quantitative recording of the whole assay process and provides a new insight into the cell-toxicant interaction.

Probing the Dynamic Effect of Cys-CdTe Quantum Dots toward Cancer Cells in Vitro

The application of quantum dots (QDs) in various biomedical areas requires detailed studies of their toxicity. We report a new strategy for probing the biocompatibility of these nanocrystals, namely, a dynamic investigation of cellular uptake images, cell growth curves, metabolic activity changes, and apoptosis aspects of cadmium telluride QDs capped with cysteamine (Cys-CdTe QDs) on human hepatocellular carcinoma SMMC-7721 cells. We used a real-time cell electronic sensing (RT-CES) system in combination with fluorescence microscopy, 3-(4,5-dimethyl-thiazol-zyl)-2,5-diphenyltetrazolium bromide assay, and flow cytometry (FCM) analysis. As observed from fluorescence images and RT-CES system results, Cys-CdTe QDs can readily bind on the cell plasma membrane and then enter into the cancer cell, causing decreased adherence of cancer cells during the initial 6-12 h, while the metabolic activity apparently decreased. After 24 h, the metabolic activity of the cancer cells was significantly reduced, with continued reduction in metabolic activity observed at even longer incubation times. Moreover, FCM observation and DNA fragmentation analysis clearly indicate apoptosis-related phenomena when SMMC-7721 cells were treated with the Cys-CdTe QDs. Thus, our study reveals details of the cellular aging and death process induced by Cys-CdTe QDs.

Clinical outcome in children with recurrent neuroblastoma treated with ABT‐751 and effect of ABT‐751 on proliferation of neuroblastoma cell lines and on tubulin polymerization in vitro

ABT-751, an orally bioavailable sulfonamide, binds beta-tubulin to inhibit microtubule polymerization. We described response and event-free survival (EFS) in children with neuroblastoma and other solid tumors receiving ABT-751, assessed in vitro cytotoxicity of ABT-751 and evaluated the effect of ABT-751 on tubulin polymerization in peripheral blood mononuclear cells (PBMC) and pediatric tumor cell lines. Patients with neuroblastoma (n = 50) or other solid tumors (n = 26) enrolled on the ABT-751 pediatric phase I and pilot trials were reviewed. The sulforhodamine B (SRB) and ACEA Real-Time Cell Electronic Sensing (RT-CES) assays were used to determine the in vitro cytotoxicity. Pharmacodynamic effects on tubulin polymerization/depolymerization were assessed by Western blot and confocal microscopy using antibodies specific for post-translational modifications of polymerized tubulin. Forty-five patients with neuroblastoma were evaluated for anti-tumor response. No complete or partial responses were documented. The median EFS was 9.3 weeks for children with neuroblastoma and 3.3 weeks for children other solid tumors (P < 0.0001). The ABT-751 IC(50) was 0.6-2.6 mcM in neuroblastoma and 0.7-4.6 mcM in other solid tumor cell lines. Following drug exposure, polymerized tubulin decreased in a concentration- and time-dependent manner in cell lines. In children treated with ABT-751, the EFS is longer in children with neuroblastoma as compared to other diagnoses. In vitro, ABT-751 was cytotoxic at concentrations tolerable in children. Effects of ABT-751 on polymerization and microtubule structure were time- and dose-dependent but not dependent on tumor type.

Synergistic Effect of Functionalized Nickel Nanoparticles and Quercetin on Inhibition of the SMMC-7721 Cells Proliferation.

The effect of functionalized nickel (Ni) nanoparticles capped with positively charged tetraheptylammonium on cellular uptake of drug quercetin into hepatocellular carcinoma cells (SMMC-7721) has been explored in this study via microscopy and electrochemical characterization as well as MTT assay. Meanwhile, the influence of Ni nanoparticles and/or quercetin on cell proliferation has been further evaluated by the real-time cell electronic sensing (RT-CES) study. Our observations indicate that Ni nanoparticles could efficiently improve the permeability of cancer cell membrane, and remarkably enhance the accumulation of quercetin in SMMC-7721 cells, suggesting that Ni nanoparticles and quercetin would facilitate the synergistic effect on inhibiting proliferation of cancer cells.

A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals

A cell-microelectronic sensing technique is developed for profiling chemical cytotoxicity and is used to study different cytotoxic effects of the same class chemicals using nitrosamines as examples. This technique uses three human cell lines (T24 bladder, HepG2 liver, and A549 lung carcinoma cells) and Chinese hamster ovary (CHO-K1) cells in parallel as the living components of the sensors of a real-time cell electronic sensing (RT-CES) method for dynamic monitoring of chemical toxicity. The RT-CES technique measures changes in the impedance of individual microelectronic wells that is correlated linearly with changes in cell numbers during t log phase of cell growth, thus allowing determination of cytotoxicity. Four nitrosamines, N-nitrosodimethylamine (NDMA), N-nitrosodiphenylamine (NDPhA), N-nitrosopiperidine (NPip), and N-nitrosopyrrolidine (NPyr), were examined and unique cytotoxicity profiles were detected for each nitrosamine. In vitro cytotoxicity values (IC 50) for NDPhA (ranging from 0.6 to 1.9 mM) were significantly lower than the IC 50 values for the well-known carcinogen NDMA (15–95 mM) in all four cell lines. T24 cells were the most sensitive to nitrosamine exposure among the four cell lines tested (T24 > CHO > A549 > HepG2), suggesting that T24 may serve as a new sensitive model for cytotoxicity screening. Cell staining results confirmed that administration of the IC 50 concentration from the RT-CES experiments inhibited cell growth by 50% compared to the controls, indicating that the RT-CES method provides reliable measures of IC 50. Staining and cell-cycle analysis confirmed that NDPhA caused cell-cycle arrest at the G0/G1 phase, whereas NDMA did not disrupt the cell cycle but induced cell death, thus explaining the different cytotoxicity profiles detected by the RT-CES method. The parallel cytotoxicity profiling of nitrosamines on the four cell lines by the RT-CES method led to the discovery of the unique cytotoxicity of NDPhA causing cell-cycle arrest. This study demonstrates a new approach to comprehensive testing of chemical toxicity.

Suppressive action of pituitary adenylate cyclase activating polypeptide (PACAP) on proliferation of immature mouse Leydig cell line TM3 cells

We aimed in the current study to understand the participation of PACAP in stage-specific Leydig and Sertoli cell functions. For this purpose, clonal cell lines TM3 (Leydig) and TM4 (Sertoli) cells, derived from the testis of immature BALB/c mice, were used. PACAP-specific receptors were detected in TM3 cells, but not in TM4 cells, which were characterized as PAC1 (type I) receptors. Stimulation of cAMP accumulation and testosterone secretion were observed in TM3 cells during 1-2 h treatment with PACAP38 (10(-10)-10(-7) M) or PACAP27 (10(-11)-10(-7) M). After around 10 h treatment with 10(-11)-10(-7) M PACAP38 or PACAP27, proliferation of TM3 cells was suppressed in time- and dose-dependent manners, which was confirmed by real-time cell electronic sensing (RT-CES) system and phase-contrast microscopy. At 6 h after the addition of PACAP38, the percent cell population in G(2)/M phases increased significantly, while that in S phase showed significant decrease with little change in G(0)/G(1) phases. The results revealed that PACAP exerts, in addition to early stimulatory effect on cAMP formation-steroidogenesis, sustained suppressive effect on cell proliferation in TM3 cells by controlling progression of the cell cycle. The suppressive action of PACAP on proliferation in TM3 cells supports the stage-specific participation of the peptide in differentiation of immature mouse Leydig cells.

Cell-electronic sensing of particle-induced cellular responses

We report a new technique for the continuous and real-time measurement of microparticle-induced cellular responses using a real-time cell-electronic sensing (RT-CES) technology. The method involves the use of microelectrode-embedded microwells seeded with one of two lung cancer carcinoma cell lines (A549 and SK-MES-1), allowing for continuous measurements of impedance. The change in impedance that is automatically converted to the cell index is linearly correlated with the numbers of the seeding cells during the log phase, providing quantitative measurement of cytotoxicity. After 24 h of initial incubation in 96 microwells, the cultures are treated with microparticles, and changes in the cell index are monitored in real time. Multiple data, including dose response curves, IC(50) (a concentration inhibiting 50% cell growth), and cell-specific and particulate-specific cell responses, are obtained from a single set of experiments. SK-MES-1 cells consistently showed more severe effects and lower IC(50) values than A549 cells when they were treated with quartz particle suspensions. The different effects detected using the RT-CES technique were related to morphological change and apoptosis, supported by the scanning electronic microscopy and flow cytometry results. The method is further used to test the cytotoxicity of two PM(10) standard reference materials of urban air dust and diesel particulates, demonstrating the potential application of this new technique for biomonitoring of air particulates.

Role of Promyelocytic Leukemia Zinc Finger Protein in Proliferation of Cultured Human Corneal Endothelial Cells

To review the role of promyelocytic leukemia zinc finger (PLZF), a transcriptional repressor and negative regulator of cell cycling, in the proliferation of cultured human corneal endothelial cells (HCECs). The expression pattern of PLZF mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative PCR in HCECs and normal human corneal epithelia. The effect of cell-cell contact on expression of the PLZF gene was studied after incubation of the cultured HCECs in EDTA. The proliferation rate of cultured HCECs was assayed by a real-time electronic sensing (RT-CES) system, and DNA microarray analysis was performed to find the PLZF-regulating genes in cultured HCECs infected with LacZ- and PLZF-carrying adenoviruses (Ad-LacZ, Ad-PLZF). PLZF mRNA was expressed in HCECs in vivo and in completely confluent HCECs but not in subconfluent HCECs in vitro. Real-time PCR showed that the expression of PLZF mRNA was decreased by approximately 20-fold when incubated with EDTA and returned to a normal level as the cell-cell contact reformed. Cell proliferation assay by the RT-CES system showed that infection of cultured HCECs with Ad-PLZF inhibited proliferation. These findings suggest that PLZF plays an important role in the suppression of proliferation of HCECs.