Abstract
Human cell lines derived from three epithelial carcinomas (CaSki, HeLa, SiHa), one B lymphoma (BL60), one promyelocytic (HL60), one monocytic (U937) leukemia, one chronic myelogenous leukemia (sensitive K562S; multichemoresistant K562R) and normal human skin fibroblasts were compared for their capacity of staining with rhodamine 123 (Rh 123) and their kinetics of dye exclusion. Cells were exposed for 30 min to 10 μg/ml of Rh 123 in culture medium; fluorescence intensity was measured by flow cytometry immediately or 1, 2, 3 and 4 h after staining. The highest fluorescence intensity was observed in carcinoma cell lines; there was no incorporation in multichemoresistant K562R cells. Exclusion of Rh 123 was evaluated from 0 to 4 h, both by flow cytometry and by fluorimetry. Fluorescence intensity measured by flow cytometry decreased slightly in carcinoma and leukemia cells and rapidly in fibroblasts. In all cell lines Rh 123 exclusion was inhibited by 40 μmol/L verapamil and 5 mmol/L probenecid. Thus, incorporation and exclusion of Rh 123 allows distinction between normal and tumoral cells; moreover, inhibition of exclusion by verapamil and probenecid favors the involvement of active cell membrane mechanisms in the exclusion process.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- PBS:
-
phosphate-buffered saline
- Rh 123:
-
rhodamine 123
References
Ames GFL. Bacterial periplasmic transport systems: structure, mechanism, and evolution. Annu Rev Biochem. 1986a;55:397–425.
Ames GFL. The basis of multidrug resistance in mammalian cells: homology with bacterial transport. Cell. 1986b;47:323–4.
Bernal SD, Lampidis TJ, McIsaac RM, Chen LB. Anticarcinoma activityin vivo of Rh 123, a mitochondrial-specific dye. Science. 1983;222:169–72.
Cole SPC, Bhardwaj G, Gerlach JH et al. Overexpression of a transporter gene in a multidrug-resistant human lung cancer line. Science. 1992;258:1650–4.
Cunningham RF, Israili ZH, Dayton PG. Clinical pharmacokinetics of probenecid. Clin Pharmacol. 1981;6:135–51.
Darzynkiewicz Z, Staiano-Coico L, Melamed MR. Increased mitochondrial uptake of rhodamine 123 during lymphcyte stimulation. Proc Natl Acad Sci USA. 1981;78:2383–7.
Darzynkiewicz Z, Traganos F, Staiano-Coico L, Kapuscinki J, Melamed MR. Interaction of Rh 123 with living cells studied by flow cytometry. Cancer Res 1982;42:799–806.
Dordal MS. Flow-cytometric analysis of multidrug resistance in cancer. Clin Immunol. 1993;13:86–9.
Efferth T, Löhrke H, Volm M. Reciprocal correlation between expression of P-glycoprotein and accumulation of Rh 123 in human tumors. Anticancer Res. 1989;9:1633–8.
Evenson DP, Darzynkiewicz Z, Melamed MR. Simultaneous measurement by flow cytometry of sperm cell viability and mitochondrial membrane potential related to cell mobility. J Histochem Cytochem. 1982;30:279–80.
Ford JM, Hait WN. Pharmacology of drugs that alter multidrug resistance in cancer. Pharmacol Rev. 1990;42:155–99.
Goldstein SD, Korczac LB. Status of mitochondria in living fibroblasts during growth and senescence in vitro: use of the laser dye rhodamine 123. J Cell Biol. 1981;91:392–8.
Horio M, Gottesman MM, Pastan I. ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells. Proc Natl Acad Sci USA. 1988;85:3580–4.
Hyde SC, Emsley P, Hartshorn MJ et al. Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature. 1990;346:362–5.
Johnson LV, Walsh ML, Chen LB. Localization of mitochondria in living cells with Rh 123. Proc Natl Acad Sci USA. 1980;77:990–4.
Johnson LV, Walsh ML, Bockus BJ, Chen LB. Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. J Cell Biol. 1981;88:526–35.
Juan G, Cavazzoni M, Saez GT, O'Connor JE. A fast kinetic method for assessing mitochondrial membrane potential in isolated hepatocytes with Rh 123 and flow cytometry. Cytometry. 1994;15:335–42.
Kinnaly LW, Tedeschi H, Maloff BL. Use of dyes to estimate the electrical potential of the mitochondrial membrane. Biochemistry. 1978;17:3419–28.
Lampidis TJ, Bernal SD, Summerhayes IC, Chen LB. Selective toxicity of Rh 123 in carcinoma cellsin vitro. Cancer Res. 1983;43:716–20.
Lee KS, Tsien RW. Mechanism of calcium channel blockade by verapamil, D600, diltiazem and nitrendipine in single dialysed heart cells. Nature. 1983;302:790–4.
Leyland-Jones B, Dalton W, Fisher GA, Sikic BI Reversal of multidrug resistance to cancer chemotherapy. Cancer (Supplement). 1993;72:3484–8.
Lizard G, Chignol MC, Chardonnet Y, Schmitt D Accumulation and retention of Rh 123 in carcinoma cells. Anal Cell Pathol. 1994;6:200.
Maynadie M, Lizard G, Solary E, Parmeland C, Carli PM. Intérêt de la rhodamine 123 pour la détection de faibles proportions de cellules multichimiorésistantes. Bull Cancer. 1994;81: 409–13.
Pastan I, Gottesman M. Multiple-drug resistance in human cancer. N Engl J Med. 1987;316:1388–92.
Peters D, Branscomb, E, Dean P et al. The LLNL high speed sorter: design features, operational characteristics and biological utility. Cytometry. 1985;6:290–301.
Ratinaud MH Leprat P, Julien R.In situ flow cytometry analysis of nonyl acridine orange stained mitochondria from splenocytes. Cytometry. 1988;9:206–12.
Ronot X, Benel L, Adolphe M, Mounolou JC, Mitochondrial analysis in living cells. Biol Cell. 1986;57:1–8.
Shapiro HM, Natale PJ, Kamenstsky LA. Estimation of membrane potentials of individual lymphocytes by flow cytometry. Proc Natl Acad Sci USA. 1979;76:5728–30.
Shapiro HM Practical flow cytometry. 2nd edn. New York: Alan R Liss; 1988.
Steinberg TH, Newman AS, Swanson JA, Silverstein SC. Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix. J Cell Biol. 1987;105:2695–702.
Summerhayes IC, Lampidis TJ, Bernal SD et al. Unusual retention of rhodamine 123 by mitochondria in muscle and carcinoma cells. Proc Natl Acad Sci USA. 1982;79:5292–6.
Thalhammer T, Gessi A, Braakman I, Graf A. Separation of hepatocytes of different acinar zones by flow cytometry. Cytometry. 1989;10:772–8.
Udomsakdi C, Lansdorp M, Hogge DE, Reid DS, Eaves AC, Eaves CJ. Characterization of primitive hematopoietic cells in normal human peripheral blood. Blood. 1992;80:2513–21.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lizard, G., Chignol, M.C., Chardonnet, Y. et al. Active cell membrane mechanisms involved in the exclusion of RH 123 allow distinction between normal and tumoral cells. Cell Biol Toxicol 10, 399–406 (1994). https://doi.org/10.1007/BF00755789
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00755789