Biol. Cell (1991), 73

POTENTIAL USE OF FLUORESCENT IN $1TU HYBRIDIZATION (FISH) FOR THE CONTROL OF THE

GENETIC STABILITY AND HOMOGENEITY OF CULTURED CELLS ." APPLICATION TO THE HUMAN

BREAST CANCER CELL LINE MCF-7 LEGER Isabeile. THOMAS Michael. RONOT XavieF

and BRUGAL Gerard Equtpe de Reconnaissance des Formes et Micmscopie Quantitative.

USR CNRS B690. Universit~ Joseph Fourier, F-Grenoble

Simultaneous visualization of nuclei and hybridization signals is a rapid and efficient method for counting the number of chromosomes of interest in each nucleus from a population of cells in culture and thus 8ires information on potential aneuploidy for the cell line under investigation.

We applied this procedure on MCF-7 breast.cancer stemline whose chromosome number has been determined to be ranged from hypertriploidy to hypotetraploidy. The nucleic acid probe that was used, pUC !.77, designates a clone of the plasmid vector pUC9 containing a 1.77 kb long human Eco R ! fragment as an insert. The insert represents a tandemly organized repetitive sequence in the region qh of chromosome I. The plasmid was nicktranslated with either biotin 11-dUTP or digoxigenin 11-dUTP. Following in situ hybridization, the hybridized probes were detected by immunofluorescence with avidin-fluorescein isothiocyanate (FITC) or anti-digoxigenin antibody linked to FITC. The nuclei were conterslained with propidium iodide, The slides were examined with a fluorescent microscope : at least 600 cells were counted per slide.

The results suggest the presence of a minor clone subpopulation with 6 chromosomes ! in a major population of tnsomtc MCF-7 cells for this chromosome.

This procedure, which does not require the preparation of metaphases, can be used as a substitute for whole cytogenetic analysis for laboratories without special competence. The future development of image analyser resident software will allow the automation of the counting phase, thus leading tc a routine procedure. Moreover. the attempts to develop in silu hybridization procedures compatible with a DNA quantification will give information on the cell cyO phase of each hybridized cell in the population.

USE OF NONYL A C R I D I N E O R A N G E AND RHODAMINE 123 TO ASSESS STRUCTURAL AND FUNCTIONAL MODIFICATIONS OF MITOCHONDRIA IN MULTIDRUG RESISTANT CELLS. DENIS-GAY Michelle, MAFTAH Abderrahman. ~ATINAU~

JULIEN Raymond. inszitut de biotechnologie, 123 Avenue Albert Thomas, 87060 LIMOGES C~dex.

The uptake of Rhodamine 123 (Rh 123) by living cells has been reported to be potential dependant and allows mitochondrial ability to synthetize ATP. The Nonyl Acridine Orange (NAO) binds specifically to cardiofipin and pennies an estimation of mitochondrial membrane content. Moreover, NAO allows to determine at sanu-adon the canJiolipin asymmetric distribution between the two leaflem of the inner mitochondrial membrane. By combined use of Rh 123 and NAO, structure and function disruptions in mitochondria of mulddrug resistant (MDR) cells have been esmbfished comparatively to sensitive cell types. We have used two cell lines resistant to adriamycin (ADM) or daunomycin (DNM) which insert in inner mitochondrial membrane where they induce some structural and functional perturbations. Also, it has been demonstrated that Rh 123 uptake in MDR cells is gready reduced and unsmbled. In support of these notions, we have selected MDR cells as cellular model. Our results on Rh 123 uptake are in good agreement with those of the literature. The ionophore effects are lower in MDR cell than in sensitive cells, in first approximation, data may be a consequence of mitochondrial activity perturbation in resistant cells. In ADM resistant cells, the cardiolipin content increases by $$% comparatively to sensitive ceils while it enhances by 8% in DNM resistant cells. This increase more imporumt in ADM resistant ceils may be due to the ADM affinity for cardiolipin why MDR cells have an ampliticadon of target drug. For ADM sensitive cells, about 49% of total cardiolipin was found to be locatedin the cytoplasmic face of the inner membrane, whereas the cardiolipin dislribudon in inner membrane is reversed in ADM resistant cells (80% of total cardiolipin in the cytoplasmic face). The results seem indicate the structural and functional permd~tions of mitochondria in MDR cells and may conuribute to the drug resistance phenomenon.

FLOW CYTOMETRIC CHARACTERIZATION OF 9 MULTIDRUG.RESISTANT CELL LINES

LEONCE Stdohane. MAROUILLAT Sylviane, PEREZ Valdrie, ANSTETT Monique, PIERRE Alain, and ATASSI Ohanem

Canedrologie exl~rbnenmle , [nslimt de Reckerckes SER V1ER I1 rue des moulineaux 92150 5URESNES, FRANCE

Overexpression of a membrane.associated glycoprotein of 170 Kd (P-gp), which by an ATP.dependent emux pump function, decreases the intracellular drug accumulation, is one of the major characteristics of cell lines displaying the multidrug.resistunce (MDR) phenotype, We have analysed by flow cytometry (FC~Vl) the P.gp expression in 4 rodent and $ human resistant cell lines (P388/ADR.I, P3881ADR.IO, P388/ADR.40, DC-3F/AD, KB-AI, K562/R, SI/tMDRI, Colo320DM.50, Colo320DM.500) labelled by 2 monoclonal antibodies, MP, KI6 and C"2 i .,. Accumulation of Adriamycin (ADR) in resistant and conespondin8 sensitive cells was also studied by FCM. Under our experimental conditions, the antibody MRKIS, in spite of an heterogeneous binding, appears to be more appropriate than C219 to detect low P.gp expression in human cells, Furthermore, a good correlation was observed between the P-gp overexpression of each cell line (relative to the parental sensitive cells) and the factor of resistance measured by the Microculture Tetrazolium Assay, These results and the lower ADR accumulation in resistant cells, suggest that the major mechanism of re,,;istance of these cells to ADR is the consequence of the P.gp overexpression lowering the intraceilular accumulation of the cytotoxic drug through an increased efflux. These cell lines are therefore interesting tools for the " vLq..y.j.L~ screening of new MDR modulatom.

NUCLEAR ALTERATIONS IN HUMAN DRUG- RESISTANT (MDR) CELLS.

~ , BROGLIO Christine, BOBICHON H~l~ne, HUBERT lsabelle, HENNEQUIN Elisabeth, DELVINCOURT

Chantal and JARDILLI~R Jean-Claude. GJ.B.$A., lnstitut Jean.Godinot , i, rue Gal Koenig and UFR

Pharmacie, $1, rue. Cognacq.Jay, F.S I IO0 Reims, France.

Resistance of rumor cells to chemotherapy is a serious clinical problem .The deten~inadon of quandtsdve morphological features linked to the drug-resistance phenomenon in cancer cells might be of interest since it could represent a method for resistant cell detection on conventional smears.

Recently, we showed that human K$62 cells resistant to Addamycin (ADM) displayed specific morphological changes as evaluated by image analysis (l).Tbese changes have been further confu'med by ulu'asu'uctural analysis of K562 cells,

In this work, the nuclear features of 3 human cell lines have been compared : erythroleukemic K$62 and mammary tumor MCF'/ceils, both resistant to ADM, iymphoblastoid CEM cells resistant to vinblasdn. All resistant cell lines exhibited mdri gene DNA amplification and RNA overexpression. Quantitative cytological analysis was performed on Feulgen stained smears using a SAMBA 200 cell image processor. Analyses of dam indicate that drug resistance induces very similar nuclear alterations in the cell lines tested (r=0.93; p<O.001). These nuclear changes appear better'Correlated with resistance index than with mdrl gene expression.

in conclusion, mulddrug resistance is associated with specific nuclear morphological changes in human tumor cells. These variations am neither linked to the cell origin, to the drug used for resistance induction, nor to the mdrl gene expression levels.

(l) DUFER L, AKEL! M.G., JEAHNESSON P., DESPLACES A. and JARDILLW:R J.C. Cytomelry, t989, 10, 37-43.

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