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. 1995 Sep;72(3):543–549. doi: 10.1038/bjc.1995.371

Functional detection of MDR1/P170 and MRP/P190-mediated multidrug resistance in tumour cells by flow cytometry.

N Feller 1, C M Kuiper 1, J Lankelma 1, J K Ruhdal 1, R J Scheper 1, H M Pinedo 1, H J Broxterman 1
PMCID: PMC2033905  PMID: 7669559

Abstract

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma membrane drug transporter P-glycoprotein (P-gp) or the recently discovered multidrug resistance-associated protein (MRP). In this study we investigated the specificity and sensitivity of the fluorescent probes rhodamine 123 (R123), daunorubicin (DNR) and calcein acetoxymethyl ester (calcein-AM) in order to detect the function of the drug transporters P-gp and MRP, using flow cytometry. The effects of modulators on the accumulation and retention of these probes were compared in several pairs of sensitive and P-gp- as well as MRP-overexpressing cell lines. R123, in combination with the modulator PSC833, provided the most sensitive test for detecting P-gp-mediated resistance. Moreover, in a 60 min drug accumulation assay R123 can be regarded as a P-gp-specific probe, since R123 is not very efficiently effluxed by MRP. In contrast to R123, a 60 min DNR or calcein-AM accumulation test could be used to detect MRP-mediated resistance. The MRP-specific modulator genistein could be used in combination with DNR, but not with calcein-AM. Vincristine (VCR) can be used to increase the cellular uptake of calcein-AM in MDR cells, but is not specific for MRP. Thus, although the combination of DNR with genistein appeared to be as sensitive as the combination of calcein-AM with VCR, the former may be used to probe specific MRP activity whereas the latter provides a combined (P-gp + MRP) functional MDR parameter. With these functional assays the role and relative importance of P-gp and MRP can be studied in, for example, haematological malignancies.

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Selected References

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  1. Bradley G., Juranka P. F., Ling V. Mechanism of multidrug resistance. Biochim Biophys Acta. 1988 Aug 3;948(1):87–128. doi: 10.1016/0304-419x(88)90006-6. [DOI] [PubMed] [Google Scholar]
  2. Burger H., Nooter K., Zaman G. J., Sonneveld P., van Wingerden K. E., Oostrum R. G., Stoter G. Expression of the multidrug resistance-associated protein (MRP) in acute and chronic leukemias. Leukemia. 1994 Jun;8(6):990–997. [PubMed] [Google Scholar]
  3. Center M. S. Mechanisms regulating cell resistance to adriamycin. Evidence that drug accumulation in resistant cells is modulated by phosphorylation of a plasma membrane glycoprotein. Biochem Pharmacol. 1985 May 1;34(9):1471–1476. doi: 10.1016/0006-2952(85)90686-0. [DOI] [PubMed] [Google Scholar]
  4. Cole S. P., Bhardwaj G., Gerlach J. H., Mackie J. E., Grant C. E., Almquist K. C., Stewart A. J., Kurz E. U., Duncan A. M., Deeley R. G. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science. 1992 Dec 4;258(5088):1650–1654. doi: 10.1126/science.1360704. [DOI] [PubMed] [Google Scholar]
  5. Endicott J. A., Ling V. The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu Rev Biochem. 1989;58:137–171. doi: 10.1146/annurev.bi.58.070189.001033. [DOI] [PubMed] [Google Scholar]
  6. Flens M. J., Izquierdo M. A., Scheffer G. L., Fritz J. M., Meijer C. J., Scheper R. J., Zaman G. J. Immunochemical detection of the multidrug resistance-associated protein MRP in human multidrug-resistant tumor cells by monoclonal antibodies. Cancer Res. 1994 Sep 1;54(17):4557–4563. [PubMed] [Google Scholar]
  7. Fojo A. T., Ueda K., Slamon D. J., Poplack D. G., Gottesman M. M., Pastan I. Expression of a multidrug-resistance gene in human tumors and tissues. Proc Natl Acad Sci U S A. 1987 Jan;84(1):265–269. doi: 10.1073/pnas.84.1.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Grant C. E., Valdimarsson G., Hipfner D. R., Almquist K. C., Cole S. P., Deeley R. G. Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs. Cancer Res. 1994 Jan 15;54(2):357–361. [PubMed] [Google Scholar]
  9. Grogan T. M., Spier C. M., Salmon S. E., Matzner M., Rybski J., Weinstein R. S., Scheper R. J., Dalton W. S. P-glycoprotein expression in human plasma cell myeloma: correlation with prior chemotherapy. Blood. 1993 Jan 15;81(2):490–495. [PubMed] [Google Scholar]
  10. Higgins C. F. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8:67–113. doi: 10.1146/annurev.cb.08.110192.000435. [DOI] [PubMed] [Google Scholar]
  11. Holló Z., Homolya L., Davis C. W., Sarkadi B. Calcein accumulation as a fluorometric functional assay of the multidrug transporter. Biochim Biophys Acta. 1994 May 11;1191(2):384–388. doi: 10.1016/0005-2736(94)90190-2. [DOI] [PubMed] [Google Scholar]
  12. Homolya L., Holló Z., Germann U. A., Pastan I., Gottesman M. M., Sarkadi B. Fluorescent cellular indicators are extruded by the multidrug resistance protein. J Biol Chem. 1993 Oct 15;268(29):21493–21496. [PubMed] [Google Scholar]
  13. Jedlitschky G., Leier I., Buchholz U., Center M., Keppler D. ATP-dependent transport of glutathione S-conjugates by the multidrug resistance-associated protein. Cancer Res. 1994 Sep 15;54(18):4833–4836. [PubMed] [Google Scholar]
  14. Kuss B. J., Deeley R. G., Cole S. P., Willman C. L., Kopecky K. J., Wolman S. R., Eyre H. J., Lane S. A., Nancarrow J. K., Whitmore S. A. Deletion of gene for multidrug resistance in acute myeloid leukaemia with inversion in chromosome 16: prognostic implications. Lancet. 1994 Jun 18;343(8912):1531–1534. doi: 10.1016/s0140-6736(94)92938-6. [DOI] [PubMed] [Google Scholar]
  15. Lampidis T. J., Munck J. N., Krishan A., Tapiero H. Reversal of resistance to rhodamine 123 in adriamycin-resistant Friend leukemia cells. Cancer Res. 1985 Jun;45(6):2626–2631. [PubMed] [Google Scholar]
  16. Lankelma J., Spoelstra E. C., Dekker H., Broxterman H. J. Evidence for daunomycin efflux from multidrug-resistant 2780AD human ovarian carcinoma cells against a concentration gradient. Biochim Biophys Acta. 1990 Dec 10;1055(3):217–222. doi: 10.1016/0167-4889(90)90035-c. [DOI] [PubMed] [Google Scholar]
  17. McGrath T., Latoud C., Arnold S. T., Safa A. R., Felsted R. L., Center M. S. Mechanisms of multidrug resistance in HL60 cells. Analysis of resistance associated membrane proteins and levels of mdr gene expression. Biochem Pharmacol. 1989 Oct 15;38(20):3611–3619. doi: 10.1016/0006-2952(89)90134-2. [DOI] [PubMed] [Google Scholar]
  18. Miller T. P., Grogan T. M., Dalton W. S., Spier C. M., Scheper R. J., Salmon S. E. P-glycoprotein expression in malignant lymphoma and reversal of clinical drug resistance with chemotherapy plus high-dose verapamil. J Clin Oncol. 1991 Jan;9(1):17–24. doi: 10.1200/JCO.1991.9.1.17. [DOI] [PubMed] [Google Scholar]
  19. Mülder H. S., Lankelma J., Dekker H., Broxterman H. J., Pinedo H. M. Daunorubicin efflux against a concentration gradient in non-P-glycoprotein multidrug-resistant lung-cancer cells. Int J Cancer. 1994 Oct 15;59(2):275–281. doi: 10.1002/ijc.2910590221. [DOI] [PubMed] [Google Scholar]
  20. Nooter K., Sonneveld P., Oostrum R., Herweijer H., Hagenbeek T., Valerio D. Overexpression of the mdr1 gene in blast cells from patients with acute myelocytic leukemia is associated with decreased anthracycline accumulation that can be restored by cyclosporin-A. Int J Cancer. 1990 Feb 15;45(2):263–268. doi: 10.1002/ijc.2910450210. [DOI] [PubMed] [Google Scholar]
  21. Ross D. D., Wooten P. J., Sridhara R., Ordóez J. V., Lee E. J., Schiffer C. A. Enhancement of daunorubicin accumulation, retention, and cytotoxicity by verapamil or cyclosporin A in blast cells from patients with previously untreated acute myeloid leukemia. Blood. 1993 Aug 15;82(4):1288–1299. [PubMed] [Google Scholar]
  22. Schuurhuis G. J., Broxterman H. J., Ossenkoppele G. J., Baak J. P., Eekman C. A., Kuiper C. M., Feller N., van Heijningen T. H., Klumper E., Pieters R. Functional multidrug resistance phenotype associated with combined overexpression of Pgp/MDR1 and MRP together with 1-beta-D-arabinofuranosylcytosine sensitivity may predict clinical response in acute myeloid leukemia. Clin Cancer Res. 1995 Jan;1(1):81–93. [PubMed] [Google Scholar]
  23. Schuurhuis G. J., Broxterman H. J., de Lange J. H., Pinedo H. M., van Heijningen T. H., Kuiper C. M., Scheffer G. L., Scheper R. J., van Kalken C. K., Baak J. P. Early multidrug resistance, defined by changes in intracellular doxorubicin distribution, independent of P-glycoprotein. Br J Cancer. 1991 Nov;64(5):857–861. doi: 10.1038/bjc.1991.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Spoelstra E. C., Westerhoff H. V., Dekker H., Lankelma J. Kinetics of daunorubicin transport by P-glycoprotein of intact cancer cells. Eur J Biochem. 1992 Jul 15;207(2):567–579. doi: 10.1111/j.1432-1033.1992.tb17083.x. [DOI] [PubMed] [Google Scholar]
  25. Twentyman P. R., Rhodes T., Rayner S. A comparison of rhodamine 123 accumulation and efflux in cells with P-glycoprotein-mediated and MRP-associated multidrug resistance phenotypes. Eur J Cancer. 1994;30A(9):1360–1369. doi: 10.1016/0959-8049(94)90187-2. [DOI] [PubMed] [Google Scholar]
  26. Van Acker K. L., Van Hove L. M., Boogaerts M. A. Evaluation of flow cytometry for multidrug resistance detection in low resistance K562 cells using daunorubicin and monoclonal antibodies. Cytometry. 1993 Oct;14(7):736–746. doi: 10.1002/cyto.990140706. [DOI] [PubMed] [Google Scholar]
  27. Versantvoort C. H., Broxterman H. J., Pinedo H. M., de Vries E. G., Feller N., Kuiper C. M., Lankelma J. Energy-dependent processes involved in reduced drug accumulation in multidrug-resistant human lung cancer cell lines without P-glycoprotein expression. Cancer Res. 1992 Jan 1;52(1):17–23. [PubMed] [Google Scholar]
  28. Versantvoort C. H., Schuurhuis G. J., Pinedo H. M., Eekman C. A., Kuiper C. M., Lankelma J., Broxterman H. J. Genistein modulates the decreased drug accumulation in non-P-glycoprotein mediated multidrug resistant tumour cells. Br J Cancer. 1993 Nov;68(5):939–946. doi: 10.1038/bjc.1993.458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Versantvoort C. H., Withoff S., Broxterman H. J., Kuiper C. M., Scheper R. J., Mulder N. H., de Vries E. G. Resistance-associated factors in human small-cell lung-carcinoma GLC4 sub-lines with increasing adriamycin resistance. Int J Cancer. 1995 May 4;61(3):375–380. doi: 10.1002/ijc.2910610317. [DOI] [PubMed] [Google Scholar]
  30. Zaman G. J., Flens M. J., van Leusden M. R., de Haas M., Mülder H. S., Lankelma J., Pinedo H. M., Scheper R. J., Baas F., Broxterman H. J. The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8822–8826. doi: 10.1073/pnas.91.19.8822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zijlstra J. G., de Vries E. G., Mulder N. H. Multifactorial drug resistance in an adriamycin-resistant human small cell lung carcinoma cell line. Cancer Res. 1987 Apr 1;47(7):1780–1784. [PubMed] [Google Scholar]
  32. van der Valk P., van Kalken C. K., Ketelaars H., Broxterman H. J., Scheffer G., Kuiper C. M., Tsuruo T., Lankelma J., Meijer C. J., Pinedo H. M. Distribution of multi-drug resistance-associated P-glycoprotein in normal and neoplastic human tissues. Analysis with 3 monoclonal antibodies recognizing different epitopes of the P-glycoprotein molecule. Ann Oncol. 1990;1(1):56–64. [PubMed] [Google Scholar]

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