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. 1992 Apr;65(4):538–544. doi: 10.1038/bjc.1992.111

P-glycoprotein overexpression cannot explain the complete doxorubicin-resistance phenotype in rat glioblastoma cell lines.

S Huet 1, B Schott 1, J Robert 1
PMCID: PMC1977580  PMID: 1348623

Abstract

We have associated pharmacological studies to a semi-quantitative evaluation of P-glycoprotein(s) expression, to establish if classical multidrug resistance (MDR) could account for the complete resistance phenotype exhibited by progressively doxorubicin-resistant rat glioblastoma cells. Three resistant variants (C6 0.001, C6 0.1 and C6 0.5) of the C6 glioblastoma cell line (C6 S) were selected by long-term culture in the presence of three concentrations of doxorubicin (0.001, 0.1 and 0.5 microgram.ml-1 respectively). The degree of doxorubicin resistance was respectively 7, 33 and 400, and all the cell variants were cross-resistant to m-AMSA, etoposide and vincristine. Doxorubicin incorporation was reduced similarly in all resistant cells, irrespective of the level of resistance. When exposed to their respective doxorubicin IC50, the 7-fold resistant cells had the same intracellular drug incorporation as the sensitive cells, whereas the 33-fold and 400-fold resistant cells could incorporate respectively 3.7 and 17 times more drug. The ratio of doxorubicin exposures required for 50% DNA synthesis inhibition and 50% growth inhibition was dependent on the degree of resistance; this ratio was 12.8 in C6 S, 11.6 in C6 0.001, 6.3 in C6 0.1 and 1.8 in C6 0.5. P-glycoprotein(s) overexpression was of the same magnitude as the resistance factor in variants C6 0.001 and C6 0.1, but was lower than resistance factor in variant C6 0.5. Reversal of drug incorporation by verapamil was complete in all resistant cell lines; however, reversal of doxorubicin cytotoxicity was complete only in the 7-fold resistant line and was only partial in the most resistant lines, which remained 10-fold and 20-fold resistant to doxorubicin. These results suggest that classical MDR was the first phenotype selected by doxorubicin in C6 0.001, whereas mechanism(s) of doxorubicin resistance other than classical MDR are added in the most resistant lines.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baas F., Jongsma A. P., Broxterman H. J., Arceci R. J., Housman D., Scheffer G. L., Riethorst A., van Groenigen M., Nieuwint A. W., Joenje H. Non-P-glycoprotein mediated mechanism for multidrug resistance precedes P-glycoprotein expression during in vitro selection for doxorubicin resistance in a human lung cancer cell line. Cancer Res. 1990 Sep 1;50(17):5392–5398. [PubMed] [Google Scholar]
  2. Beck W. T., Cirtain M. C., Danks M. K., Felsted R. L., Safa A. R., Wolverton J. S., Suttle D. P., Trent J. M. Pharmacological, molecular, and cytogenetic analysis of "atypical" multidrug-resistant human leukemic cells. Cancer Res. 1987 Oct 15;47(20):5455–5460. [PubMed] [Google Scholar]
  3. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Chang B. K., Brenner D. E., Gutman R. Dissociation of the verapamil-induced enhancement of doxorubicin's cytotoxicity from changes in cellular accumulation or retention of doxorubicin in pancreatic cancer cell lines. Anticancer Res. 1989 Mar-Apr;9(2):347–351. [PubMed] [Google Scholar]
  6. Danks M. K., Schmidt C. A., Cirtain M. C., Suttle D. P., Beck W. T. Altered catalytic activity of and DNA cleavage by DNA topoisomerase II from human leukemic cells selected for resistance to VM-26. Biochemistry. 1988 Nov 29;27(24):8861–8869. doi: 10.1021/bi00424a026. [DOI] [PubMed] [Google Scholar]
  7. Dano K. Active outward transport of daunomycin in resistant Ehrlich ascites tumor cells. Biochim Biophys Acta. 1973 Oct 25;323(3):466–483. doi: 10.1016/0005-2736(73)90191-0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Ganapathi R., Grabowski D. Differential effect of the calmodulin inhibitor trifluoperazine in modulating cellular accumulation, retention and cytotoxicity of doxorubicin in progressively doxorubicin-resistant L1210 mouse leukemia cells. Lack of correlation between cellular doxorubicin levels and expression of resistance. Biochem Pharmacol. 1988 Jan 15;37(2):185–193. doi: 10.1016/0006-2952(88)90716-2. [DOI] [PubMed] [Google Scholar]
  10. Gerlach J. H., Kartner N., Bell D. R., Ling V. Multidrug resistance. Cancer Surv. 1986;5(1):25–46. [PubMed] [Google Scholar]
  11. Huet S., Robert J. The reversal of doxorubicin resistance by verapamil is not due to an effect on calcium channels. Int J Cancer. 1988 Feb 15;41(2):283–286. doi: 10.1002/ijc.2910410220. [DOI] [PubMed] [Google Scholar]
  12. Juliano R. L., Ling V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta. 1976 Nov 11;455(1):152–162. doi: 10.1016/0005-2736(76)90160-7. [DOI] [PubMed] [Google Scholar]
  13. Keizer H. G., Schuurhuis G. J., Broxterman H. J., Lankelma J., Schoonen W. G., van Rijn J., Pinedo H. M., Joenje H. Correlation of multidrug resistance with decreased drug accumulation, altered subcellular drug distribution, and increased P-glycoprotein expression in cultured SW-1573 human lung tumor cells. Cancer Res. 1989 Jun 1;49(11):2988–2993. [PubMed] [Google Scholar]
  14. Kessel D., Wilberding C. Anthracycline resistance in P388 murine leukemia and its circumvention by calcium antagonists. Cancer Res. 1985 Apr;45(4):1687–1691. [PubMed] [Google Scholar]
  15. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Merry S., Fetherston C. A., Kaye S. B., Freshney R. I., Plumb J. A. Resistance of human glioma to adriamycin in vitro: the role of membrane transport and its circumvention with verapamil. Br J Cancer. 1986 Jan;53(1):129–135. doi: 10.1038/bjc.1986.18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Morrow C. S., Cowan K. H. Multidrug resistance associated with altered topoisomerase II activity--topoisomerases II as targets for rational drug design. J Natl Cancer Inst. 1990 Apr 18;82(8):638–639. doi: 10.1093/jnci/82.8.638. [DOI] [PubMed] [Google Scholar]
  19. Mukhopadhyay T., Kuo M. T. Expression of the P-glycoprotein gene in multidrug-resistant Chinese hamster ovary cells. Anticancer Res. 1989 May-Jun;9(3):575–578. [PubMed] [Google Scholar]
  20. Rogan A. M., Hamilton T. C., Young R. C., Klecker R. W., Jr, Ozols R. F. Reversal of adriamycin resistance by verapamil in human ovarian cancer. Science. 1984 Jun 1;224(4652):994–996. doi: 10.1126/science.6372095. [DOI] [PubMed] [Google Scholar]
  21. Safa A. R. Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7187–7191. doi: 10.1073/pnas.85.19.7187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schott B., Robert J. Comparative cytotoxicity, DNA synthesis inhibition and drug incorporation of eight anthracyclines in a model of doxorubicin-sensitive and -resistant rat glioblastoma cells. Biochem Pharmacol. 1989 Jan 1;38(1):167–172. doi: 10.1016/0006-2952(89)90164-0. [DOI] [PubMed] [Google Scholar]
  23. Schuurhuis G. J., Broxterman H. J., Cervantes A., van Heijningen T. H., de Lange J. H., Baak J. P., Pinedo H. M., Lankelma J. Quantitative determination of factors contributing to doxorubicin resistance in multidrug-resistant cells. J Natl Cancer Inst. 1989 Dec 20;81(24):1887–1892. doi: 10.1093/jnci/81.24.1887. [DOI] [PubMed] [Google Scholar]
  24. Singh S. V., Nair S., Ahmad H., Awasthi Y. C., Krishan A. Glutathione S-transferases and glutathione peroxidases in doxorubicin-resistant murine leukemic P388 cells. Biochem Pharmacol. 1989 Oct 15;38(20):3505–3510. doi: 10.1016/0006-2952(89)90121-4. [DOI] [PubMed] [Google Scholar]
  25. Skovsgaard T. Mechanisms of resistance to daunorubicin in Ehrlich ascites tumor cells. Cancer Res. 1978 Jun;38(6):1785–1791. [PubMed] [Google Scholar]
  26. Tsuruo T., Iida H., Tsukagoshi S., Sakurai Y. Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res. 1981 May;41(5):1967–1972. [PubMed] [Google Scholar]

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