A pilot study in two French medical schools for teaching histology using virtual microscopy.

PURPOSE: Glass slides and standard microscopes associated to a brief review of the lectures with projection slides were used during practical training in histology and histopathology for many years. Today it is necessary to develop new tools to improve teaching, and to face a lower number of teachers, as well the increase of the microscope maintenance costs. The goal of this study was to evaluate the feasibility of virtual slide implantation in several medical schools, the feedback from students, and to develop the interest in microscopic histology. METHODS: We used virtual slides generated by the Samba 2050 system produced by Samba technologies. A collection of all organs for histology training was realized and overviewed by three MD, PhD. A questionnaire was distributed in middle of the year to evaluate the feedback. RESULTS: The feedback of the students is highly positive. Students works faster, on better resources, interactivity between students is increased, and the fact that this is a new modality of teaching raises the students' interest. CONCLUSION: Today the teaching program in two French medical schools (Lyon and Grenoble) include virtual slides alone or in addition to microscopic glass slide examination to teach histology or pathology.

Effects of permeability transition inhibition and decrease in cytochrome c content on doxorubicin toxicity in K562 cells.

As mitochondria play a key role in the commitment to cell death, we have investigated the mitochondrial consequences of resistance to doxorubicin (DOX) in K562 cells. We found that the permeability transition pore (PTP) inhibitor cyclosporine A (CsA) failed to inhibit PTP opening in the resistant clone. Moreover, the Ca2+ loading capacity in the resistant clone was identical to that observed in the parent cells in the presence of CsA, suggesting that the PTP was already inhibited in a CsA-like manner in the resistant cells. In agreement with this proposal, the mitochondrial target of CsA cyclophilin D (CyD) decreased by half in the resistant cells. The levels of adenine nucleotide translocator, voltage anion-dependent channel, Bax, Bcl-2, Bcl-xL, AIF and Smac/Diablo, were similar in both cell lines, whereas cytochrome c content was divided by three in the resistant cells. Since P-glycoprotein inhibition did not restore DOX toxicity in the resistant cells, while DOX-induced cell death in the parent cells was prevented by either PTP inhibition or siRNA-induced decrease in cytochrome c content, we conclude that the inhibition of PTP opening and the decrease in cytochrome c content participate in the mechanism that makes K562 cells resistant to DOX.

Optimization of PKH67 labeling conditions for proliferation monitoring in daunorubicin-treated leukemic cells.

Daunorubicin (DNR) is commonly used to treat acute myeloid leukemia (AML). The aim of this study was to determine whether PKH67 dye dilution could be used for differential proliferation monitoring in chemosensitive (K562S) and chemoresistant (K562R) leukemic sublines after drug treatment. Cells were labeled with PKH67 and treated for 2 h with a sublethal dose of DNR or vincristine either immediately or after 3 h in fresh medium. Viability (TOTO-3 exclusion) and DNR uptake (total cellular DNR fluorescence) were assessed by flow cytometry, and nuclear DNR accumulation was determined by confocal laser microspectrofluorimetry. Immediate DNR treatment led to enhanced DNR uptake and decreased viability in PKH67-labeled K562S, whereas no excess toxicity was seen if DNR treatment was delayed for 3 h. Treatment with vehicle control (Diluent C) gave similar results. In contrast, PKH67 labeling had no effect on K562S viability after vincristine treatment. For K562R, DNR uptake measured at 120 min was unaltered by prior exposure to PKH67 or vehicle, but viability was again significantly reduced after immediate DNR treatment. As with K562S, delaying DNR treatment for 3 h normalized viability in K562R. The excess DNR toxicity seen for PKH67-labeled K562S appears to be drug related, since it is not seen with vincristine, and may be due to the daunosamine sugar moiety present in DNR. However, with the addition of a 3-h incubation in fresh medium prior to drug exposure, PKH67 dye dilution can be used for proliferation monitoring of both K562S and K562R cells after treatment with DNR.

Fluorescence-based assessment of LRP activity: a comparative study.

BACKGROUND: Broad resistance to anticancer drugs is a major cause of failure in cancer treatment. The Lung Resistance-related Protein (LRP) is a protein associated with drug resistance, which is involved in nucleo-cytoplasmic transport and is known to predict a poor response to chemotherapy in acute myeloid leukaemia. The only method allowing the detection of LRP activity is based on radio-labelled daunorubicin incorporation. Our goal was to develop a fluorescence-based assay to analyse LRP function. MATERIALS AND METHODS: We used human colon carcinoma cell lines treated with sodium butyrate (NaB) in order to induce LRP expression. Daunorubicin efflux in isolated nuclei was measured by flow cytometry, the localization and quantification of Daunorubicin analysed by confocal laser scanning microscopy (CLSM) and the diffusion coefficient of this drug estimated by Fluorescence Correlation Spectrometry (FCS). RESULTS: According to the method using [14C] Doxorubicin cells incubated with NaB displayed an efflux of Daunorubicin out of isolated nuclei demonstrated by flow cytometry or CLSM. The FCS method was able to evaluate kinetics of Daunorubicin molecules in nucleus and cytoplasm and showed a higher dispersion of Daunorubicin kinetics with cells previously NaB-treated. This argument is in favour of an increase of nucleo-cytoplasmic exchange. CONCLUSION: Using CLSM we showed that LRP was able to modify anticancer drug repartition in the cells. LRP activity assessment needs either isolated nuclei if flow cytometry is employed, or FCS, and only a few cells may be analysed.

A flow cytometric assay for simultaneous assessment of drug efflux, proliferation, and apoptosis.

BACKGROUND: Proliferative status and multidrug resistance play a key role in determining cell response to chemotherapy. There is a need to develop multiple labeling method that allows simultaneous assessment of multidrug resistant (MDR) phenotype, proliferative status, apoptosis related changes in mitochondrial potential, in chemosensitive and chemoresistant tumor cell populations. METHODS: A three-color labeling was performed using Hoechst 33342 (DNA), JC1 (mitochondrial potential), and a far red fluorescent membrane intercalating dye: PTIR271 (proliferation). RESULTS: Combined staining of DNA and mitochondrial potential allows identification of subpopulations expressing and MDR phenotype mediated by P-glycoprotein (Pgp), and, in Pgp negative subpopulations, identification of apoptotic cells and evaluation of cell cycle status in viable cells. Addition of a far red fluorescent membrane intercalating dye, PTIR271, allows simultaneous monitoring of cell division status by dye dilution in both drug sensitive and drug resistant populations. CONCLUSION: This triple labeling is an interesting method to study the proliferation status of drug sensitivity and drug resistance in viable tumor cells.

Rotenone inhibits the mitochondrial permeability transition-induced cell death in U937 and KB cells.

The permeability transition pore (PTP) is a mitochondrial inner membrane Ca(2+)-sensitive channel that plays a key role in different models of cell death. Because functional links between the PTP and the respiratory chain complex I have been reported, we have investigated the effects of rotenone on PTP regulation in U937 and KB cells. We show that rotenone was more potent than cyclosporin A at inhibiting Ca(2+)-induced PTP opening in digitonin-permeabilized cells energized with succinate. Consistent with PTP regulation by electron flux through complex I, the effect of rotenone persisted after oxidation of pyridine nucleotides by duroquinone. tert-butyl hydroperoxide induced PTP opening in intact cells (as shown by mitochondrial permeabilization to calcein and cobalt), as well as cytochrome c release and cell death. All these events were prevented by rotenone or cyclosporin A. These data demonstrate that respiratory chain complex I plays a key role in PTP regulation in vivo and confirm the importance of PTP opening in the commitment to cell death.

The mitochondrial consequences of uncoupling intact cells depend on the nature of the exogenous substrate.

In isolated mitochondria the consequences of oxidative phosphorylation uncoupling are well defined, whereas in intact cells various effects have been described. Uncoupling liver cells with 2,4-dinitrophenol (DNP) in the presence of dihydroxyacetone (DHA) and ethanol results in a marked decrease in mitochondrial transmembrane electrical potential (DeltaPsi), ATP/ADP ratios and gluconeogenesis (as an ATP-utilizing process), whereas the increased oxidation rate is limited and transient. Conversely, when DHA is associated with octanoate or proline, DNP addition results in a very large and sustained increase in oxidation rate, whereas the decreases in DeltaPsi, ATP/ADP ratios and gluconeogenesis are significantly less when compared with DHA and ethanol. Hence significant energy wastage (high oxidation rate) by uncoupling is achieved only with substrates that are directly oxidized in the mitochondrial matrix. Conversely in the presence of substrates that are first oxidized in the cytosol, uncoupling results in a profound decrease in mitochondrial DeltaPsi and ATP synthesis, whereas energy wastage is very limited.

Innocuousness and intracellular distribution of PKH67: a fluorescent probe for cell proliferation assessment.

PKH dyes were initially developed by Horan et al. to provide appropriate probes for in vitro and in vivo cell tracking. It has been reported for many cell types that PKH bind irreversibly to the cell membrane without significantly affecting cell growth. Thus, these probes provide an opportunity for long-term cell monitoring and the identification of cells of interest among a heterogeneous cell population. An important feature is that upon cell division, the probe is partitioned equally between each daughter cell, making it possible to quantify tell fluorescence by flow cytometry. In this situation. the flow cytometric study of PKH67 characteristics shows that this probe does not affect the main cell-functions such as viability or proliferation. Moreover, the intracellular distribution of PKH67 is demonstrated by following its kinetics of internalization by confocal microscopy. These results present PKH67 as a probe suitable for dynamic analysis of cell proliferation as well as the study of intracellular localization and membrane recycling mechanisms.

Nucleus labeling or membrane labeling for studying the proliferation of drug treated cells?

Proliferation and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukemia (AML). This study compared cell cycle analysis (nuclear labeling) with cell division analysis (membrane labeling, PKH67) for studying the proliferation of cells cultured with daunorubicin (DNR) and/or Cytarabine (Ara-C), drugs commonly used in AML treatment. PKHs are a family of lipophilic, fluorescent membrane intercalating dyes. When labeled cells divide, the resulting daughter cells receive half the label, reducing fluorescence intensity to one-half that of the parent cells. DNR has the disadvantage of overlapping the spectrum of propidium iodide (PI), which is the most commonly used marker of membrane integrity. In this study, necrosis was evaluated using TOTO-3, a marker of nucleic acids emitting fluorescence above 645 nm and which incorporates cells with disrupted membranes. Comparison of cell cycle analysis with cell division for studying proliferation revealed that PKH67 was a marker of choice for analyzing the mitotic process in cells cultured with drugs.

Quantitative study of dynamic behavior of cell monolayers during in vitro wound healing by optical flow analysis.

BACKGROUND: In vitro wound healing assays are experimental models commonly used to analyze cell behavior during the migration process. A new approach is proposed for the quantification of cell motility based on an optical flow method. METHODS: We assumed that cell-population dynamics can be defined by an a priori affine-motion model. Identified model parameters are used as motion descriptors quantifying both elementary and complex cell movements, either at the wound margins or within the cell monolayer. RESULTS: When compared with the estimation of cell motility calculated from wound area temporal variation, it allows a more detailed and precise characterization of cell population movements. Comparative analysis of normal and cancerous cell lines revealed that typical measured velocities were about 2 microm/h and 7 microm/h for L929 and HeLa cells, respectively, at the beginning of the wound closure. The quantification of the effect of Hoechst 33342 on cell dynamics showed a similar behavior for control and stained cells within 20 h after wound scratching, but then a decreased velocity of stained cells. CONCLUSIONS: The results demonstrate that this approach can be used to gain new insights into the dynamic changes induced by the extracellular environment and by anticancer drugs.

Response of chemosensitive and chemoresistant leukemic cell lines to drug therapy: simultaneous assessment of proliferation, apoptosis, and necrosis.

BACKGROUND: The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy. Commonly-used DNA analysis methods cannot study both parameters simultaneously. A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively. METHODS: To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance. RESULTS: We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity. Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug. CONCLUSIONS: Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients.

Optimized fluorescent probe combinations for evaluation of proliferation and necrosis in anthracycline-treated leukaemic cell lines.

Proliferation and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukaemia (AML). Anthracyclines such as daunorubicin (DNR) are typically used to treat AML and can induce drug resistance. The goal of the studies described here was to select a combination of fluorescent probes that could be used in combination with flow cytometry to monitor cell proliferation vs. cell death/necrosis as a function of anthracycline uptake. Propidium iodide (PI), the most commonly used marker of membrane integrity, cannot be used to evaluate necrosis in DNR-containing cells because of spectral overlap. A membrane integrity probe compatible with the use of a dye dilution method using PKH67 to study cell proliferation was also selected. The results show that DAPI and Cascade Blue (CB), like PI, were able to detect necrotic cells when no DNR was present, although CB gave less resolution between viable and necrotic cells than PI or DAPI. In the presence of DNR, DAPI cannot be used owing to the fluorescence quenching by DNR. However, it was found that a combination of DNR, CB, and PKH67 allows simultaneous identification of chemoresistant cells, based on reduced DNR accumulation, necrotic cells based on CB incorporation, and proliferating cells based on partitioning of PKH67 fluorescence between daughter cells. It was also found that unless a marker of necrosis is used in combination with the dye dilution assay, a moderate decrease of fluorescence as a result of necrosis may be incorrectly interpreted as proliferation.

Chromatin texture analysis in living cells.

In recent years, there has been an increasing interest in applications of fluorescence measurements to studies on many physiological mechanisms in living cells. However, few studies have taken advantage of DNA quantification by fluorometry for dynamic assessment of chromatin organization. This type of approach involves both optimal conditions for DNA staining and the use of image cytometry. In this context, this report describes the application of an internal grey-level segmentation method for the assessment of real time modifications of chromatin organization in living cells. These developments are based on a specific, stoichiometric method for nuclear DNA content measurement. Preliminary data obtained from Hela cells suggests the possibility of following variations of nuclear texture (heterogeneity, granularity, condensation, radial distribution) related to the cell cycle progression of cells that are maintained alive.

Characterization of cell deformation and migration using a parametric estimation of image motion.

This paper deals with the spatio-temporal analysis of two-dimensional deformation and motion of cells from time series of digitized video images. A parametric motion approach based on an affine model has been proposed for the quantitative characterization of cellular movements in different experimental areas of cellular biology including spontaneous cell deformation, cell mitosis, individual cell migration and collective migration of cell populations as cell monolayer. The accuracy and robustness of the affine model parameter estimation, which is based on a multiresolution algorithm, has been established from synthesized image sequences. A major interest of our approach is to follow with time the evolution of a few number of parameters characteristic of cellular motion and deformation. From the time-varying eigenvalues of the affine model square matrix, a precise quantification of the cell pseudopodial activity, as well as of cell division has been performed. For migrating cells, the motion quantification confirms that cell body deformation has a leading role in controlling nucleus displacement, the nucleus itself undergoing a larger rotational motion. At the cell population level, image motion analysis of in vitro wound healing experiments quantifies the heterogeneous cell populations dynamics.

A society of goal-oriented agents for the analysis of living cells.

This paper presents a new model for the segmentation and analysis of living cells. A multi-agent model has been developed for this application. It is based on a generic agent model, which is composed of different behaviors: perception, interaction and reproduction. The agent is further specialized to accomplish a specific goal. Different goals are defined from the different components of the cell images. The specialization specifies the parameters of the behaviors for the achievement of the agent's goal. From these goal-oriented agents, a society is defined, and it evolves dynamically as the agents are created and deleted. An internal manager is integrated in the agent to control the behavior's execution. It makes use of an event-driven scheme to manage the behavior priorities. The present design is mainly oriented toward image segmentation, however, it includes some features on tracking and motion analysis.

Coexpression of multidrug resistance involve proteins: a flow cytometric analysis.

Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.

2,4 Dinitrophenol-uncoupling effect on delta psi in living hepatocytes depends on reducing-equivalent supply.

Mitochondrial uncouplers, such as 2,4 dinitrophenol (DNP), increase the cellular respiration by decreasing mitochondrial membrane potential (delta psi). We show that this respiratory effect can be transient or even prevented in isolated liver cells depending on the exogenous substrate used (dihydroxyacetone vs. octanoate or proline). Moreover the decrease in ATP/ADP ratio induced by DNP is partially restored by addition of octanoate or proline. By using rhodamine 123 (Rh123) monitored by flow cytometry in living hepatocytes, we were able to follow in time delta psi in such DNP-uncoupled cells incubated with various substrates. The ability of this method to evaluate delta psi changes was assessed by using myxothiazol (3.6 microM), an inhibitor of the b-c1 complex of the respiratory chain which decreased delta psi (65%), or oligomycin (6 microg/ml), an inhibitor of the F0F1-ATPase which increased it (50%). Although DNP induced a dose-dependent decrease of delta psi, we found that octanoate or proline addition prevented such effect. We propose that octanoate or proline may counteract the uncoupling effect of DNP by providing a high supply of reducing equivalents to the respiratory chain.

Flow cytometric analysis of DNA content of living and fixed cells: a comparative study using various fixatives.

The majority of studies dealing with DNA analyses are made on fixed cells. In this context, the efficiency as fixatives of ethanol, methanol, acetone, Carnoy, Boehm-Sprenger and aldehydes was determined using two different DNA fluorescent probes, Hoechst 33342 and propidium iodide. The purpose of our study was to find the fixative that would provide the best results with respect to the following parameters: aggregates, cell size and granularity, and DNA staining analysis. Using murine fibroblasts, we found that 68% ethanol, 85% methanol and aldehydes did not increase aggregate formation, whereas Carnoy, acetone or Boehm-Sprenger fixatives did. The results show that aldehydes seem to alter cell size least. All fixatives induce an increase in cell granularity, which is very pronounced with alcohols, but aldehydes alter morphology less than alcohols. We observed that the fixatives giving the best resolution with Hoechst 33342 staining lead to a lower measurement variability than with propidium iodide staining. This study leads us to conclude that 68% ethanol and 85% methanol can be considered as appropriate fixatives for flow cytometry studies of DNA content.

Usefulness of PKHs for studying cell proliferation.

Classical methods for proliferative assessment (such as tritiated thymidine or bromodeoxyuridine (BrdUrd) incorporation) need sample fixation. As an alternative, we have evaluated the use of a dye dilution method using PKH26 to determine the rate and extent of proliferation in cell lines. Flow cytometric analysis associated with modelling software makes it possible to estimate the number of cells having undergone different numbers of cell divisions by sampling the cell population at varying times post-labelling. Two major questions were addressed in these studies. (i) Does PKH26 give a stable and reproducible labelling? (ii) Does labelling with PKH26 alter cellular proliferation characteristics? We conclude that the methods developed here provide a simpler, more complete means for assessment of cell proliferation in patients with hematological malignancies.

PKH26 probe in the study of the proliferation of chemoresistant leukemic sublines.

Proliferative status and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukemia. Although classical methods for proliferative assessment such as tritiated thymidine or BrdUrd incorporation, are correlated with treatment outcome, they are time consuming and difficult to standardize. As an alternative, we have evaluated the use of a dye dilution method using PKH26 to determine rate and extent proliferation in drug sensitive and resistant cell lines. When cells labelled with this fluorescent membrane intercalating dye divide, each resulting daughter cell receives half of the dye. Using flow cytometric analysis, it is possible to estimate the number of cells having undergone different numbers of cell divisions. Four different questions were addressed in these studies: a) does PKH26 give stable and reproducible labelling? b) does labelling with PKH26 alter cellular proliferation characteristics? c) is PKH26 a substrate for PGP and MRP? d) does PKH26 labelling alter PGP expression and/or PGP activity? We found that PKH26 labelling is stable, reproducible and has no effect on cell proliferation. It does not modify PGP activity or expression, nor does it appear to be a substrate for PGP or MRP, since the rate of decrease in fluorescence intensity is similar for sensitive and resistant cells which are proliferating at the same rate. Using the dye dilution method, it is possible to simultaneously assess PGP, proliferative status, and level of PGP expression. We conclude that the methods developed here provide a simpler, more complete means for assessment of the effects of the drug therapy on sensitive and resistant cell populations in patients with hematologic malignancies.