Biomedical subjects

C Dussert

Publications and source records attributed to C Dussert.

At least 19 recordsLinked to original sources

PubMed1999

Opioid agonists modify breast cancer cell proliferation by blocking cells to the G2/M phase of the cycle: involvement of cytoskeletal elements.

Opioids decrease cell proliferation in different systems including breast, prostate, lung, kidney, and intestine, through an interaction with opioid as well as other membrane-receptor systems (somatostatin, cholinergic), through an unidentified mechanism. Recently, we have reported an interaction of taxol with opioid membrane sites (BBRC 235, 201-204, 1997), and an involvement of opioids to the modification of actin cytoskeleton in renal OK cells (J Cell Biochem. [19981 70:60-69), indicating a possible action of the opioid effect. In the present work, we have examined the effect of two general opioid agonists (ethylketocyclazocine and etorphine) on the cell cycle, in human breast cancer T47D cells, as well as a possible modification of the cellular cytoskeleton under their action, in order to explain the antiproliferative effect of these agents. These two opioids produce a dose-dependent and reversible decrease of the proliferation of T47D cells, with a maximum attained at 10(-8) M. The addition of 10(-8) M of either opioid produced a significant increase of the number of cells arrested in the G2/M phase. Confocal laser microscopy revealed a modification of the actin and tubulin microfilaments, with a clear redistribution at the periphery of the cell, reversed by the addition of the general opioid antagonist diprenorphine. Furthermore, differences between the two opioids were obvious, attributed to the different receptor affinity of each agent. The observed redistribution of actin and tubulin cytoskeletal elements gives therefore a possible answer of the antiproliferative action of opioids. The modification of the cytoskeleton, directly involved to cell division, might provoke a "mechanical" obstacle, which could be the reason of the antiproliferative effect of these agonists. Furthermore, the observed tubulin-opioid interaction by opioids provides a possible explanation of the arrest at the G2/M phase of T47D cells under opioid treatment. Nevertheless, although the observed interaction of opioids with cytoskeletal elements gives a plausible answer of the antiproliferative effects of the agents, this might not be the only action of these agents in cell proliferation. Other, direct or indirect, genomic actions, which which remains to be elucidated, might be taken into consideration.

Actins↗

PubMed1997

Multifactorial comparative study of spatial point pattern analysis methods.

A way of studying cooperative behaviour of biological entities (proteins, cells, etc.) is by using topographical analysis: the quantification of the spatial patterns formed by the entities considered as points. Five methods of topographical analysis were compared in terms of discriminant power, stability of parameters, methodological bias and algorithms. We tested five methods (nearest neighbour distribution, radial distribution, Voronoï paving, quadrat count, minimal spanning tree graph) which generated nine parameters on four simulated models (random point process, hardcore model and two cluster models) and on experimental cellular models. The method which offers the best discrimination power and stability seems to be the minimal spanning tree graph edge length distribution.

Animals↗

PubMed1997

Distribution of estrogen receptor heterogeneity in growing MCF-7 cells measured by quantitative microscopy.

The existence of interactive subpopulations is a biological feature that can modulate the proliferation of tumor cells. The hormone-responsive breast cancer cell line MCF-7 has been described as heterogeneous in terms of density. In this study we describe a quantitative image analysis methodology that we developed for the in situ detection of different subpopulations in MCF-7 cell cultures. Using this technology, we demonstrate the heterogeneity of the MCF-7 cell line in terms of both nuclear size and estrogen-receptor content. Analysis of the organization (topography) of the different subpopulations in culture reveals a nonrandom distribution of cells. When studying the development of these cell subpopulations as a function of time of culture, we observe modifications of their topography associated with an increase of estrogen-receptor-expressing cells. Moreover, the use of cluster analysis allows study of the local organization of these subpopulations. These changes appear to be independent of cell proliferation.

Breast Neoplasms↗

PubMed1997

A minimal model for calcium signal generated by tyrosine kinase and G protein linked receptors; a stochastic computer simulation with CALSIM.

A software was designed to simulate the calcium signal following hormone or growth factor stimulation in epithelial cells. The software written in C runs on a PC under Windows environment. It is based on a Markov process where the dynamic of the system is characterised by phenomenological transition probabilities. Moreover a minimal model is proposed to analyse the role of plasma channels and IP3 receptors, together with the opposite action of the CaATPase pumps, in the cytosolic and endoplasmic reticulum (ER) calcium signal control. The simulation is applied on the calcium response following stimulation by carbacol (protein G coupled receptors) or epidermal growth factor (tyrosine kinase type receptors) in A431 epithelial cells. The experimental calcium signals can be grouped in three classes; a spike and a return to the basal level (signal A), a spike and a decrease to a plateau level (signal B) or a slow increase to a plateau (signal C). Epidermal growth factor induces signal A and B while carbacol gives signal B and C. When a 'pseudo' steady state is reached oscillations occur. Computer simulations show that signal A can result from the activation of IP3 receptors while signal C would result from the activation of the plasma channels; signal B appears as the additive contribution of both channels, while oscillations are compatible with a calcium induced calcium release mechanism. Simulations suggest that the calcium dynamic in the ER is a mirror of cytosolic calcium but that a simple way to produce similar calcium elevation in these two compartments is to activate plasma channels. Implications of such a mechanism is discussed.

Adenocarcinoma↗

PubMed1997

Topographical analysis of spatial patterns generated by a cellular automaton model of the proliferation of a cancer cell line in vitro.

A well-suited model to simulate cellular population dynamics is the two-dimensional cellular automaton model, which consists of a lattice of sites, the value ai,j of each site being updated in discrete time steps according to an identical deterministic rule depending on a neighbourhood of sites around it. A cellular automaton is described which mimics cell population proliferation by replacing the site values by the age and the cycle phase of cells. The model takes into account the size of the cells. It is used to simulate the proliferation of the human breast cancer cell line MCF-7 and the results of the simulation are compared with experimental data obtained from a light microscopic image analysis of the proliferation process. The initial configuration of the cellular automaton is obtained from the discretization of the results of the initial stage of the image processing. After each day of proliferation the pattern obtained from the simulation is compared to the experimental result of the corresponding image analysis. The comparison is made from a topographical point of view through the concept of the minimal spanning tree graph. The agreement between experiment and model is a good starting point to complex models such as cell proliferation under growth effectors or drugs.

Breast Neoplasms↗

PubMed1997

[Contribution of the topographical analysis to quantitative microscopy in cultured cell systems: application to the evaluation of hormone therapy].

Quantitative microscopy by image analysis allows not only to measure various parameters on each cell but also to consider the global population as a whole. In the hypothesis that cell position is reflecting the relational and dynamical structure of the system, spatial arrangement analysis may help to show up intercellular communication (interactions and control systems via contact or diffusible factors). We describe a topographical analysis method used to study these neighbour relationships, and thus the sociological behaviour of the cells. It is applied to the study of the effect of estrogenic and antiestrogenic treatments on a breast cancer cell line (MCF-7). It shows up that estrogens increase proliferation and induce an unusual topographical behaviour, notably in cell cycle phases: cells in S phases are very randomly distributed. It points out the role of estrogens on the cells neighbour relationships inducing the way to a permissive proliferation context. This effect is reversed by antiestrogenic treatment after a few days. Antiestrogenic treatment alone increases the proliferation constraint.

Breast Neoplasms↗

PubMed1996

Toward a new method to in situ study of apoptosis and its relations with cell cycle.

Apoptosis or programmed cell death plays a key role in many biological processes particularly in oncology. The detection of apoptotic cells is crucial for the study of the phenomenon itself and its relation with the proliferative cell cycle. A new method to detect apoptosis in situ in Feulgen stained cells was developed, based on multiparametric analysis (factorial and decisional analysis), using 15 densitometric and textural parameters measured on a SAMBA 200 cell image processor. Six reference files corresponding to G1, S, and G2 phases and to apoptotic cells derived from these cell cycle phases were constructed. The projection of these files in the factorial principal plane formed distinct clusters. Using the decisional discriminant analysis, it was possible to ascertain the state (apoptosis or proliferative) and the phase for each cell of a population. The correct classification rate of this analysis was 0.9962. Determining the cell cycle phase from which each apoptotic cell comes, we are able to study the relation between apoptosis and the proliferative cell cycle. Moreover, the detection in situ allows us to study cell-cell interactions.

Apoptosis↗

PubMed1996

Image cytometry and topographical analysis of proliferation of endothelial cells in vitro during Bartonella (Rochalimaea) infection.

Bartonella quintana and Bartonella henselae are clinically associated with proliferative neovascular lesions. The effect of Bartonella infection on human endothelial cells was evaluated in vitro by quantitative image analysis. Particular emphasis is placed on reporting the methodologies employed. Human umbilical vein endothelial cells were infected in vitro with the two Bartonella species. Cell proliferation (cell density), cell morphology (cell surface, form and elongation factors) and spatial reorganization (global topographical analysis and hierarchical cluster detection) were monitored over a 3-day period of infection. Firstly, infection stimulated endothelial cell proliferation. Secondly, infection induced obvious morphological changes; infected cells became larger, more elongated and spindle-shaped. Cytoskeletal reorganization was confirmed by staining of F actin. Thirdly, infection altered the spatial organization of cells within the monolayer; this could not have been due solely to the morphological modifications they experienced. This model demonstrates that Bartonella infection provoked endothelial cell proliferation, topographical rearrangements and morphological changes because of modifications of the cytoskeleton. These experimental findings provide a physiopathological explanation to the abnormal angiogenesis observed in bacillary angiomatosis.

Bartonella↗

PubMed1994

Agonist-antagonist activity of anti-estrogens in the human breast cancer cell line MCF-7: an hypothesis for the interaction with a site distinct from the estrogen binding site.

Non-steroidal anti-estrogens exhibit an extremely complex pharmacology because of their estrogenic and anti-estrogenic effects in different species. Recently, we have reported evidence for an immunochemical difference in the estrogen receptor (ER) when it is occupied with anti-estrogens as compared to estrogens (Martin et al., 1988). In this study, we have compared immunoreactivity of MCF-7 cell estrogen receptor when bound to anti-estrogen versus estrogen. We show that the occupation of ER with antiproliferative concentrations of various anti-estrogens leads to the appearance of additional antigenic determinants for the H222 monoclonal anti-estrogen receptor antibody. When performing ER immunoassay after sedimentation of estrogen receptors on sucrose gradients, we show that exposure of new epitopes induced by anti-estrogens can occur on a 4 s molecular form related to the 66 kDa monomeric estrogen receptor. Also, when ER are previously occupied by estradiol, the addition of low anti-estrogen concentrations, which are unable to displace estradiol from the estrogen receptor, leads to a significant increase of H222 epitopes. Our results led us to propose a molecular model for anti-estrogen-receptor interaction in which their dual agonist/antagonist activity may be due to the occupation of distinct binding sites on the estrogen receptor.

Binding, Competitive↗

PubMed1991

Estradiol and EGF requirements for cell-cycle progression of normal human mammary epithelial cells in culture.

The purpose of our study was to show the feasibility of accurately investigating the factors likely to control cell proliferation of normal human mammary epithelial (HME) cells, using a scanning cytometric method. The methodology was previously developed with the SAMBA 200 cell image processor to characterize in situ the cell-cycle phases of HME cells. Since various compartments constitute the mammary epithelium, cells obtained after reduction mammoplasty were cultured in a medium with a low calcium content (0.06 mM) to provide proliferating normal HME cells while maintaining their differentiation characteristics. Estradiol and EGF requirements for cell-cycle phase progression of these cells were examined. Then, we showed that 2 normal HME cell cultures displaying different phenotypic characteristics may differently progress through the cell cycle under the same hormonally defined conditions. Cell-cycle progression of the epithelial cells presenting luminal phenotype was induced only by sequential stimulation/pretreatment with estradiol followed by EGF treatment; this progression was enhanced when estradiol was maintained during EGF treatment. This definite order demonstrated that estradiol could have a permissive effect on EGF mitogenic activity. In contrast, epithelial cells likely to be localized in the basal position in the mammary gland showed the same proliferating activity whatever the estradiol and EGF treatment. These cells progressed profusely in cell cycle, independent of exogenous estradiol and EGF contribution, but they remained sensitive to estradiol regarding EGF-receptor detection. Our results suggest autonomous proliferation of these cells through an autocrine pathway, in the absence of negative regulators.

Adult↗

PubMed1991

Nuclear texture parameters as discriminant factors in cell cycle and drug sensitivity studies.

The authors have recently shown that cell cycle characteristics of in situ cell populations can be determined using the SAMBA 200 cell image processor by computing 15 densitometric and texture parameters on each Feulgen-stained nucleus and multiparametric analysis of data. The present paper displays the importance of chromatin pattern assessment and detection of conformational changes in DNA structure, based on nine nuclear texture parameters measured from the grey level cooccurrence and the run-length section matrices. Reference files were constructed by merging respective reference files (G0/G1, S, G2 and M) of MDA AG and MCF-7, two mammary epithelial cell lines presenting different morphological aspects and hormone responses, these files were found to be valid in the reclassification of any mammary epithelial cell in culture with a diploid or near diploid pattern. Moreover, the authors demonstrate that chromatin texture changes, following direct interaction of chemotherapeutic drugs with DNA, may be assessed owing to nuclear texture parameters. Consecutive to daunomycin addition (0.5 microgram/ml) and concomitant to the appearance of nuclear morphological alterations in MDA AG sensitive cells as viewed by microscopic observation, discriminant factorial analysis showed progressively increasing erroneous reclassification from 15 to 72 h of treatment. These experimental results were exploited with a kinetic mathematical model to quantify the daunomycin blocking effect: 20% in S phase and 80% in G2 phase. Interestingly, no textural change was observed on MDA A1 anthracycline resistant cells, indicating that these texture parameters may permit distinction of drug sensitive cells. This methodology 1) can be applied to test in vitro resistance-reversal molecules, 2) may be extended to other therapeutic agents giving rise to conformational changes in DNA structure, and 3) can be applied to cytopunctions or imprints of tumor biopsies with diploid-like DNA content to follow evolution of drug sensitivity or resistance during course of therapy.

Antibiotics, Antineoplastic↗

PubMed1991

Determination of oestrogen receptors: application of the Passing-Bablock linear regression technique for comparison of enzyme immunoassay and radioligand binding assay in 1841 breast cancer tumours.

To test the qualities of two assays in the same laboratory on the same tumours, a single-point dextran-coated charcoal radioligand binding assay (RLA-DCC) and the Abbott enzyme immunoassay (EIA) were used to perform oestrogen receptor determinations on cytosols from 1841 breast cancers over a 2-year period. Statistical analysis of the data was performed by the Passing-Bablock linear regression technique. The final regression curve between EIA (y) and RLA-DCC (x) yielded y = 1.187 x fmol/mg of protein. However, a high variability in this correlation was observed from 1986 to 1988. This variability could be explained by calibration problems in the immunoassay kits and changes in our technical team. The binding assay appears to be more sensitive to the technicians' experience than the immunoassay. Other technical points are discussed, particularly cytosol preparation and KCl presence or absence in the homogenisation buffer. Finally, the Passing-Bablock and the least squares regression procedures are compared. The conditions allowing optimal correlation and routine determination reliability are defined and the correlation variability is discussed.

Breast Neoplasms↗

PubMed1991

[Determination of estradiol receptors by immunoenzyme technique and by radioligand binding in 2134 breast tumors].

In order to test the qualities of the 2 assays, in the same laboratory and on the same tumors, a single-point dextran-coated charcoal radioligand binding assay (RLA-DCC) and Abbott enzyme immunoassay (EIA) were used for more than two years to perform estrogen receptor determinations on cytosols from 2,134 breast cancers. Statistical analysis of the data was performed according to the method of Passing-Bablock. The final regression curve between EIA (y) and RLA-DCC (x) was excellent y = 1.187 X fmol/mg of protein. However, from 1986 to 1988, a great variability was observed for this correlation. We report the study of this variability, which could be explained by several factors, especially calibration problems for the immunoassay kits and changes in our technical team. The binding assay appears to be more sensitive to the technicians' experience than the immunoassay. Technical points are discussed, particularly cytosol preparation and KCl presence or absence in the homogeneisation buffer. The conditions allowing for optimal correlation and routine determination fiability can therefore be defined.

Breast↗

PubMed1990

Abortive intermediates in transcription by wheat-germ RNA polymerase II. Dynamic aspects of enzyme/template interactions in selection of the enzyme synthetic mode.

At constant enzyme concentration and with the full set of nucleotide substrates dictated by template sequence, the chain-length distribution of polymeric product varies with template concentration in reactions catalysed by wheat-germ RNA polymerase II. Under the same conditions, but in the presence of a single ribonucleoside triphosphate, the rate of condensation of the triphosphate substrate to a dinucleotide primer also exhibits a complex dependence with the template concentration. This effect is observed using poly[d(A-T)] as a template. For both reactions there are two extreme types of behaviour in each of which transcription appears to involve a single enzyme synthetic mode, characterized by either a high (at low template concentration) or a low (at high template concentration) probability of releasing the transcripts. A strong correlation is found between these two pathways, such that conditions favouring the abortive release of trinucleotide products in the single-step addition reaction are associated with the synthesis of short-length RNA species in productive elongation, and reciprocally. A model previously developed by Papanicolaou, Lecomte & Ninio [(1986) J. Mol. Biol. 189, 435-448] to account for the kinetics of polymerization/excision ratios with Escherichia coli DNA polymerase I, and by Job, Soulié, Job & Shire [(1988) J. Theor. Biol. 134, 273-289] for kinetics of RNA-chain elongation by wheat-germ RNA polymerase II provides an explanation for the observed behaviour with the plant transcriptase. The basic requirement of this model is a slow equilibrium between two states of the polymerization complex with distinct probabilities of releasing the product. In the presence of Mn2+, and under conditions allowing the synthesis of poly[r(A-U)], one of these states is involved in the formation of oligonucleotides shorter than 15 bases, whereas the other catalyses the polymerization of chains longer than 40 bases.

DNA↗

PubMed1990

Spreading of B16 F1 cells on laminin and its proteolytic fragments P1 and E8: involvement of laminin carbohydrate chains.

The properties of EHS laminin and its proteolytic fragments E8 and P1 to promote spreading of B16 F1 murine melanoma cells were studied in short-term adhesion assays. The cells exhibited similar attachment rates but distinct spread morphologies on laminin, P1, and E8 fragments. The extent of spreading and the shape of the cells were quantitatively defined by two geometrical parameters: the surface and the form factor. These parameters were computed with an automatic image analyzer. Wheat germ agglutinin (WGA), applied to laminin-coated substrates, totally blocked cell spreading, but did not modify attachment percentages. Under similar conditions, WGA partially inhibited cell spreading on the E8 fragment and had no effect on the P1 fragment. In Western blot analysis, P1 fragment, contrary to laminin and E8, did not bind WGA. Laminin galactosylation and cell treatment with alpha-lactalbumin, which should prevent cell galactosyltransferase (GalTase) from binding to N-acetylglucosamine (GlcNAc) residues of the substrate, had no effect on the spreading ability of B16 F1 cells. The role of laminin N-linked carbohydrate chains in the induction of B16 F1 cell spreading was studied further after endoglycosidase F (Endo F) treatment of the substrates. The loss of carbohydrate chains was estimated by the reduction of iodinated lectin binding and by SDS-PAGE. Endo F treatment of laminin (85% of WGA binding inhibition) and E8 (40-50%) had no effect on cell spreading. In contrast, Endo F treatment of P1 fragment (85% of Con A binding inhibition) reduced both cell surface and form factor of B16 F1 cells. These results suggest that: (i) other spreading systems may act in concert with or in place of GalTase/GlcNAc interactions, (ii) the N-linked sugar chains of P1, which are not recognized by WGA, are involved in the spreading process of B16 F1 cells on this fragment, (iii) the epitopes of E8 fragment and E8 domain in laminin which are responsible for spreading are differently masked by WGA, (iv) the binding of WGA to laminin may impair cell spreading by steric hindrance.

Animals↗

PubMed1989

Molecular organization and clustering of cell-wall-bound enzymes as a source of kinetic apparent co-operativity.

When fixed charges and enzyme molecules are not homogeneously distributed in a matrix, the degree of organization of charges, of enzyme molecules and of charges with respect to enzyme molecules modulate the enzyme reaction rate. The overall reaction velocity of the bound enzyme system may be expressed in terms of monovariate moments of the charge density distribution and of the bivariate moments of the charge and enzyme density distributions. With respect to the situation where fixed charges and enzyme molecules are randomly distributed in the matrix, the molecular organization, as expressed by the monovariate and bivariate moments results in an increase or a decrease, of the overall reaction rate, as well as in the appearance of a kinetic cooperativity. The degree of spatial organization of objects may be expressed quantitatively through the concept of minimal spanning tree. This concept may thus be applied to the quantification of the degree of order that may exist in the bidimensional distribution of enzyme molecules in a charged matrix. Primary walls of isolated plant cells in sterile culture behave as a polyanion and contain different enzymes. The spatial distribution in sycamore cell walls of an acid phosphatase has been studied through the concept of minimal spanning tree and shown to be non-randomly distributed in the polyanionic matrix, but clustered in that matrix. This spatial organization results in a modulation of the reaction rate of the cell-wall-bound phosphatase reaction. Both the theoretical and experimental results presented in this study leave little doubt as to the validity of the idea that in situ the organization of fixed charges and enzyme molecules modulate the overall dynamics of enzyme reactions.

Acid Phosphatase↗

PubMed1989

Toward a new approach in tumor cell heterogeneity studies using the concept of order.

A new methodology was developed to study dynamic processes topographically in biological systems by means of a graph-theoretical method. It is based upon order parameters obtained from a minimal spanning tree analysis coupled with computer simulations. The method was used to analyse the heterogeneous behavior of two neoplastic cell lines after treatment with laminin. The laminin-induced cell detachment was quantitated and shown to be inversely related to cell population density and thus to cellular interactions. Our statistical analysis is a very powerful tool to obtain information from seemingly disorderly heterogeneous biological models.

Animals↗

PubMed1988

Quantization of directional properties in biological structures using the Minimal Spanning Tree.

A method that uses the Minimal Spanning Tree graph has previously been developed (Dussert et al., 1987, J. theor. Biol. 125, 317) in order to analyse the degree of order in biological structures. This graph is shown here to be very powerful in bringing out directional properties of biological structures which cannot be revealed by a simple visual examination. The method is illustrated by means of various computer simulations.

Computer Simulation↗