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E V A L U A T I O N OF A M E T H O D F OR D E T E C T I O N OF C E L L S W I T H R E D U C E D D R U G R E T E N T I O N IN SOLID T U M O U R S

CAI~PENTLER Yves, GOKISSE Marie-Claude and DESOIZE Bernard GIBSA, laboratoire de Pharmacologie, Institut J Godinot, BP 171, Reims, F

A method for detection of cells with reduced drug retention was evaluated in solid tumours. After a one hour incubation with daunorubicin (DNR.), the right angle scatter (P,.AS), forward angle scatter (FAS) and specific fluorescence (Flue) were measured in sensitive and resistant cells ; only Flue was related qualitatively, but not quantitavely, to resistance. Various incubation conditions were examined. When the pH of the incubation medium increased, the DNR. retention increased in sensitive and resistant cells. In contrast, when the cell concentration increased, the DNR retention decreased. Using sensitive and resistant cell lines, a proportion of resistant cells lower than 10 % can be detected in a mixture. To analyse cells from solid turnouts, the cells, were dissociated by repeated fine needle aspirations. Tumours from 22 patients have been processed with this technique ; 8 samples were classified as "S" (sensitive), 2 as "R" (resistant), and 12 as "I" (intermediate).

Further experiments were run to study and improve the method. Another method of detection of dead cells was tested. The inlra-assay variability of the teclmique was found to be less than l0 %. When the study was pertbrmed with different fragments of the same tumour, the variation, corresponding to the tumour heterogeneity, rose to 21 to 36 % The inter- assay reproducibility was too high, so a variant of this technique has been adapted, using, verapamil or cyclosporin A, which are able to block DNK efflux ; this new method allows tumour cells to be used as their own controls.

F L O W A N D I M A G E C Y T O M E T R Y , M o n t p e l l i e r , D e c e m b e r 1992

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ANALYSIS OF CARDIOLIPIN ORGANIZATION IN INNER MITOCHONDRIAL MEMBRANE OF

ADRIAMYCIN RESISTANT CELLS.

DENIS-GAY Michelle, RATINAUD Marie-Halone, JULIEN Raymond. Institut de Biotechnologie, 123 Avenue Albert Thomas 87060 Limoges cedex, France.

Several enzymes of inner milochondrial membrane are involved in ATP production. Their optimal activities depend on the presence and the organization of cardiolipin within the membrane.

In the present work, the amount and the transverse distribution of cardiolipin in the inner mitochondrial membrane were determined in sensitive and adriamycin (ADM) resistant K562 cells. These measurements were performed with 10-N Nonyl Acridine Orange (NAO), a fluorescent dye known to bind specifically to cardiolipin.

The results indicate that cardiolipin amount increases by 26% in ADM resistant cells comparatively to sensitive cells. Moreover, this phospholipid has been found to be equally distributed between the two leaflets of .the inner milochondrial membrane of sensitive cells. In ADM resistant cells, cardiolipin becomes widely located in the cytoplasmic monolayer (81% of total cardiolipin). The same asymmetrical distribution is also obtained when ATP synthesis by mitochondria of sensitive cells is inhibited by oligomycin.

These results would suggest that large modifications of the organization of cardiolipin and maybe o! the other lipids occur in the inner mitochondrial membrane of cells which became ADM resistant.

M O D U L A T I O N O F T H E G L U T A T I I I O N E P O O L S P E C I F I C A L L Y IN MU G L U T A T H I O N E S - T R A N S F E R A S E O V E R P R O D U C I N G C E L L S : A P P L I C A T I O N T O C E L L SORTING AFTER M O N O C H L O R O B I M A N E STAINING ORSINI-LUNEL C~cile LOUISE Anne, BUTTIN G~rard, ROBERT DE SAINT VINCENT BIuno Unite de G~ndtiqae Somatique, Institut Pasteur, 25, rue du Docteur Rott~, 75724 Paris Cddex 15

Glutathione S-transferases (GST) are a complex superfamily of enzymes involved in the detoxification of many electrophile chemicals including anticancer drugs. Alteration in the pattern of expression of certain members of the GST family is a common trait of cancer cells and has been implicated in natural or acquired drug resistance. We have developed a method to sort cells on the basis of their mu GST content after staining with mbnochlorobimane (MCBM). MCBM is a specific substrate of the GST and its conjugation with glutathione (GSH) gives a highly fluorescent compound. It has been used for the determination of GSH levels in many tissues including HIV-infected T cells (1). Our experimental system is a cell line of Chinese hamster fibroblasts (GMA32) and its derivative, HC474, overproducing the mu family of GST due to the amplification of the corresponding gene (2). Exposure of cells to MCBM induced a rapid GSH depletion in both cell lines and cells reached a fluorescence level reflecting their GSH level rather than their level of GST activity. This fluorescence decreased slowly and disappeared after 4 hours. Fluorescence was initially concentrated in one peak reflecting the GSH content of the ceils and then, with time, shifted to a second peak of very low fluorescence, probably corresponding to non-conjugated MCBM bound to hydrophobic membranes. Exposing cells to trans-stilbene oxide (TSO), a mu GST-specific substrate (3), did not alter the GSH pool in GMA32 but induced a complete GSH depletion in less than 2 hours in HC474. MCBM staining of such TSO-treated cells gave a fluorescent population of low expressing mu GST cells easy to son on a background of non-fluorescent overproducing mu GST cells. Minor modifications of the TSO treatment allowed the son of GST overexpressing cells on a background of low expressing cells. Similar methods should be applicable to sorting cells overexpressing other GSTs as long as specific substrates are available. (I) STAAL, FJ.T. et al, (1992). Aids Research and Human Retroviruses, 8, 305-311 (2) ROBERT DE SAINT VINCENT, B., HYRIEN, O., DEBATISSE, M., BU"I~I'IN, G. (1990). Eur. J. Biochem., 193, 19-24 (3) SEIDEGARD. J., GUTHENBERG, C., PERO, R.W., MANNERVIK, B. (1987). Biechem. J., 246, 783-785

Q U A N T I T A T I V E NUCLEAR A L T E R A T I O N S IN M U R I N E M U L T I D R U G - R E S I S T A N T FRIEND C E L L SUBLINES :

C O R R E L A T I O N S WITH RESISTANCE LEVEL AND MDR GENE E X P R E S S I O N .

BROGLIO Christine, GORISSE Marie-Claude, DELVINCOURT Chantal, DEVIE-HUBERT Isabelle, CARPENTIER Yves, DESOIZE Bernard and

DUFER Jean. GIBSA, Institut Jean-Godinot, 1, rue Gal Koenig and UFR Pharmacie,

51, rue Cognacq-Jay, 51100 Reims, France.

Resistance of tumor cells to chemotherapy is a serious clinical problem and the detection of drug-resistant cells on conventional smears might be of panicular interest. Recently, we showed that human erythroleukemic human and murine cells resistant to Doxorubicin (DOX) display specific morphological changes as evaluated by image analysis (I). In this work, the nuclear features of 4 murine Friend cell sublines selected for their different capabilities to resist DOX were studied. The Resistance Indexes to DOX and Daunorubicin (DNR) were computed by measurement of IC50 of drugs. "mdr" gene DNA amplification was assessed by Southern and slot blot analyses. Quantitative cytological analysis was performed on Feulgen stained smears using a SAMBA 200 cell image processor. According to the resistance indexes to DOX and DNR, two main mechanisms of resistance seem to be involved : one predominant in weakly resistant cells (major resistance to DNR) and one predominant in highly resistant cells (major resistance to DOX). These two phenomena are associated with different nuclear changes as assessed by principal component analysis of all the cytological data obtained in the different cell populations. Moreover, within these two subgroups, the variations of some nuclear features correlate with mdr gene amplification and resistance index to DNR but not to resitance index to DOX (e.g., nuclear area,...), while other features do the converse (e.g., DNA index,...).

In conclusion, mechanisms of resistance within "typical mdr" murine Friend cell sublines selected by the same drug (DOX) are not unique and result in different cell variants. At the morphological level, nuclear changes observed in these resistant cells appear to depend on the degree of expression of these muhifactorial mechanisms in the variants.

(I) DUFER J., BROGLIO C., OKIEMY M.G., DESOIZE B., CARPENTIER Y. ;llltl JARDILLIER J.C. (1990). In "Advances in Analytical Cellular Pathology", G. BURGER, M. OBERHOLZER and G.P. VOOIJS, eds., Excerpta Mcdit'a. Amsterdam, 311-312.