Summary
The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170). The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells. Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 × 11 nm IMPs not found in wild type cells. The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane. Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells. However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.
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Abbreviations
- E face:
-
external fracture face
- DNR:
-
daunorubicin
- MDR:
-
Multidrug resistance
- IC50 :
-
50% growth inhibitory concentration of drug compared with drug free control
- IMP:
-
Intramembranous particle
- kD:
-
kiloDalton
- MW:
-
Molecular weight
- P face:
-
protoplasmic fracture face
- PBS:
-
Phosphate buffered saline
- SDS-PAGE:
-
Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis
- VER:
-
Verapamil
References
Arsenault AL, Ling V, Kartner N (1988) Altered plasma membrane ultrastructure in multidrug-resistant cells. Biochim Biophys Acta 938:315–321
Beck WT (1987) The cell biology of multiple drug resistance. Biochem Pharmacol 36:2879–2888
Breathnach AS, Gross M, Martin B, Stolinski C (1976) A comparison of membrane fracture faces of fixed and unfixed glycerinated tissue. J Cell Sci 21:437–448
Cano-Gauci DF, Riordan JR (1987) Action of calcium antagonists on multidrug resistant cells. Specific cytotoxicity independent of increased cancer drug accumulation. Biochem Pharmacol 36:2115–2123
Chen C, Chin JE, Ueda K, Clark DP, Pastan I, Gottesman MM, Roninson IB (1986) Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells. Cell 47:381–389
Cornwell MM, Safa AR, Felsted RL, Gottesman MM, Pastan I (1986) Membrane vesicles from multidrug-resistant human cancer cells contain a specific 150to 170-kDa protein detected by photoaffinity labeling. Proc Natl Acad Sci USA 83:3847–3850
Danø K (1971) Development of resistance to daunomycin (NSC-82151) in Ehrlich ascites tumor. Cancer Chemother Rep 55:133–141
Diddens H, Gekeler V, Neumann M, Niethammer D (1987) Characterization of actinomycin-D-resistant CHO cell lines exhibiting a multidrug-resistance phenotype and amplified DNA sequences. Int J Cancer 40:635–642
Friche E, Skovsgaard T, Nissen N (1987) Effect of verapamil on daunorubicin accumulation in Ehrlich ascites tumor cells. Cancer Chemother Pharmacol 19:35–39
Gerlach JH, Kartner N, Bell DR, Ling V (1986) Multidrug resistance. Cancer Surveys 5:25–46
Hinz UG, Welinder KG (1987) The light harvesting complex of photosystem II in barley. Structure and chlorophyll organization. Carlsberg Res Commun 52:39–54
Juliano RL, Ling V (1976) A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 455:152–162
Kartner N, Evernden-Porelle D, Bardley G, Ling V (1985) Detecton of P-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies. Nature 316:820–823
Kessel D, Bosmann HB (1970) On the characteristics of actinomycin D resistance in L5178Y cells. Cancer Res 30:2695–2701
Kessel D, Romine M (1985) Transport phenomena associated with anthracycline resistance. Proc Am Assoc Cancer Res 26:322
Montaudon D, Vrignaud P, Londos-Galgiardi D, Robert J (1986) Fluorescence anisotropy of cell membranes of doxorubicin-sensitive and -resistant rodent tumoral cells. Cancer Res 46:5602–5605
Oth D, Bégin M, Bischoff P, Leroux JY, Mercier G, Bruneau C (1987) Induction by adriamycin and mitomycin C, of modifications in lipid composition, size distribution, membrane fluidity and permeability of cultured RDM4 cells. Biochim Biophys Acta 900:198–208
Poste G, Papahadjopoulos D, Jacobsen K, Vail WJ (1975) Local anaesthetics increase susceptibility of untransformed cells to agglutination by concanavalin A. Nature 253:552–554
Ramu A, Glaubiger D, Magrath IT, Joshi A (1983) Plasma membrane lipid structural order in doxorubicin-sensitive and -resistant P388 cells. Cancer Res 43:5533–5537
Riordan JR, Ling V (1979) Purification of P-glycoprotein from plasma membrane vesicles of Chinese hamster ovary cell mutants with reduced colchicine permeability. J Biol Chem 254:12701–12705
Rintoul DA, Center MS (1984) Involvement of plasma membrane structural order in adriamycin resistance in Chinese hamster lung cells. Cancer Res 44:4978–4980
Sehested M, Skovsgaard T, van Deurs B, Winther-Nielsen H (1987) Increased plasma membrane traffic in daunorubicin resistant P388 leukemic cells. Effect of daunorubicin and verapamil. Br J Cancer 56:747–751
Sehested M, Skovsgaard T (1988) Lectin staining patterns of plasma membranes of daunorubicin and vincristine resistant Ehrlich ascites tumour cells. Virchows Arch Cell Pathol 54:366–370
Simpson DJ (1979) Freeze-fracture studies on barley plastid membranes III. Location of the light-harvesting chlorophyll-protein. Carlsberg Res Commun 44:305–336
Skovsgaard T (1978) Mechanism of resistance to daunorubicin in Ehrlich ascites tumor cells. Cancer Res 38:1785–1791
Skovsgaard T, Friche E (1983) Circumvention of resistance to daunorubicin. In: Hansen HH (ed) Anthracyclines and cancer therapy. Excerpta Medica, Amsterdam, pp 39–48
Sleytr UB, Robards AW (1982) Understanding the artefact problem in freeze-fracture replication: a review. J Microsc 126:101–122
Supino R, Prosperi E, Formelli F, Mariani M, Parmiani G (1986) Characterization of a doxorubicin-resistant murine melanoma line: Studies on cross-resistance and its circumvention. Br J Cancer 54:33–42
Tritton TR, Yee G (1982) The anticancer agent adriamycin can be actively cytotoxic without entering cells. Science 217:248–250
Tsuruo T, Iida H, Tsukagoshi S, Sakurai Y (1981) Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res 41:1967–1972
Wheeler C, Rader R, Kessel D (1982) Membrane alterations associated with progressive adriamycin resistance. Biochem Pharmacol 31:2691–2693
Wright LC, Dyne M, Holmes KT, Mountford CE (1985) Phospholipid and ether linked phospholipid content alter with cellular resistance to vinblastine. Biochem Biophys Res Commun 133:539–545
Yanovich S, Preston L (1984) Effects of verapamil on daunomycin cellular retention and cytotoxicity in P388 leukemic cells. Cancer Res 44:1743–1747
Yu J, Branton D (1976) Reconstitution of intramembrane particles in recombinants of erythrocyte protein Band 3 and lipid: Effects of spectrin-actin association. Proc Natl Acad Sci USA 73:3891–3895
van Zoelen EJJ, Verkleij AJ, Zwaal RFA, Deenen LLM (1978) Incorporation and asymmetric orientation of glycophorin in reconstituted protein-containing vesicles. Eur J Biochem 86:539–546
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Sehested, M., Simpson, D., Skovsgaard, T. et al. Freeze-fracture study of plasma membranes in wild type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells. Virchows Archiv B Cell Pathol 56, 327–335 (1988). https://doi.org/10.1007/BF02890034
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DOI: https://doi.org/10.1007/BF02890034