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Somatic Cell and Molecular Genetics

, Volume 20, Issue 6, pp 463–480 | Cite as

Subclonal heterogeneity of the multidrug resistance phenotype in a cell line expressing antisense MDR1 RNA

  • LeRoy A. Hanchett
  • Raymond M. Baker
  • Bruce J. Dolnick
Article

Abstract

A multidrug resistant (MDR) cell line was transfected with an antisense MDR1 expression vector and transfectant clones were analyzed for reversion of the MDR phenotype. Only one of 10 antisense-expressing transfectants showed a reduction in drug resistance, MDR1 mRNA and P-glycoprotein. Observations made using rhodamine-123, a fluorescent substrate for P-glycoprotein, revealed that dye retention in individual cells was highly variable within this antisense-expressing clone. Subpopulations were established from the original clone based on differences in rhodamine-123 retention. Rhodamine-123 retention varied inversely with levels of P-glycoprotein and MDR1 mRNA. All subpopulations expressed similar levels of antisense MDR1 RNA yet had dramatic differences in MDR1 mRNA levels. Analysis of vector integration site restriction fragment length polymorphisms confirmed that all populations originated from the same transfectant clone. Nuclear run-on analysis indicated that themdr1 gene is transcribed at the same rate in all populations, suggesting that the reduction in MDR1 mRNA is mediated posttranscriptionally. Cells with the greatest reduction in MDR1 mRNA accumulate distinct antisense RNA transcripts in the nuclear RNA fraction, suggesting that antisense effectiveness in this system is associated with a nuclear event or process. These results reveal that antisense RNA activity is not necessarily distributed equally within a clonal populations.

Keywords

Vector Integration Fluorescent Substrate Nuclear Event Original Clone MDR1 Expression 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Literature Cited

  1. 1.
    Dolnick, B.J. (1991).Cancer Invest. 9:185–194.Google Scholar
  2. 2.
    Euchi, Y., Itoh, T., and Tomizawa, J. (1991).Annu. Rev. Biochem. 60:631–652.Google Scholar
  3. 3.
    Melton, D.A. (1985).Proc. Natl. Acad. Sci. U.S.A. 82:144–148.Google Scholar
  4. 4.
    Wang, S., and Dolnick, B.J. (1993).Nucleic Acids Res. 21:4383–4391.Google Scholar
  5. 5.
    Woolf, T.M., Melton, D.A., and Jennings, C.G. (1992).Proc. Natl. Acad. Sci. U.S.A. 89:7305–7309.Google Scholar
  6. 6.
    Colman, A. (1990).J. Cell Sci. 97:399–409.Google Scholar
  7. 7.
    van der Krol, A.R., Mol, J.N., and Stuitje, A.R. (1988).Biotechniques 6:958–976.Google Scholar
  8. 8.
    Marcus-Sekura, C.J., Woerner, A.M., Shinozuka, K., Zon, G., and Quinnan, G., Jr. (1987).Nucleic Acids Res. 15:5749–5763.Google Scholar
  9. 9.
    Ueda, K., Cardarelli, C., Gottesman, M.M., and Pastan, I. (1987).Proc. Natl. Acad. Sci. U.S.A. 84:3004–3008.Google Scholar
  10. 10.
    van der Bliek, A.M. and Borst, P. (1989).Adv. Cancer Res. 52:165–203.Google Scholar
  11. 11.
    Gottesman, M.M. and Pastan, I. (1993).Annu. Rev. Biochem. 62:385–427.Google Scholar
  12. 12.
    Juranka, P.F., Zastawny, R.L., and Ling, V. (1989).FASEB J. 3:2583–2592.Google Scholar
  13. 13.
    Germann, U.A., Pastan, I., and Gottesman, M.M. (1993).Semin. Cell Biol. 4:63–76.Google Scholar
  14. 14.
    Neyfakh, A.A. (1988).Exp. Cell Res. 174:168–176.Google Scholar
  15. 15.
    Weaver, J.L., Pine, P.S., Aszalos, A., Schoenlein, P.V., Currier, S.J., Padmanabhan, R., and Gottesman, M. (1991).Exp. Cell Res. 196:323–329.Google Scholar
  16. 16.
    Efferth, T., Lohrke, H., and Volm, M. (1989).Anticancer Res. 9:1633–1638.Google Scholar
  17. 17.
    Ludescher, C., Thaler, J., Drach, D., Drach, J., Spitaler, M., Gattringer, C., Huber, H., and Hofmann, J. (1992).Br. J. Haematol. 82:161–168.Google Scholar
  18. 18.
    Chaudhary, P.M., and Roninson, I.B. (1991).Cell 56:85–94.Google Scholar
  19. 19.
    Spangrude, G.J., and Johnson, G.R. (1990).Proc. Natl. Acad. Sci. U.S.A. 87:7433–7437.Google Scholar
  20. 20.
    Gunning, P., Leavitt, J., Muscat, G., Ng, S.Y., and Kedes, L. (1987).Proc. Natl. Acad. Sci. U.S.A. 84:4831–4835.Google Scholar
  21. 21.
    Chen, C.J., Chin, J.E., Ueda, K., Clark, D.P., Pastan, I., Gottesman, M.M., and Roninson, I.B. (1986).Cell 47:381–389.Google Scholar
  22. 22.
    Sanger, F., Nicklen, S., and Coulsen, A.R. (1977).Proc. Natl. Acad. Sci. U.S.A. 74:5463–5467.Google Scholar
  23. 23.
    Akiyama, S., Fojo, A., Hanover, J.A., Pastan, I., and Gottesman, M.M. (1985).Somat. Cell Mol. Genet. 11:117–126.Google Scholar
  24. 24.
    Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrup, J.P., Ringold, G.M., and Danielsen, M. (1987).Proc. Natl. Acad. Sci. U.S.A. 84:7413–7417.Google Scholar
  25. 25.
    Chirgwin, J.J., Prbyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979).Biochemistry 18:5294–5299.Google Scholar
  26. 26.
    Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1993).Current Protocols in Molecular Biology, (John Wiley & Sons, New York).Google Scholar
  27. 27.
    Nevins, J.R. (1980).Methods Enzymol. 65:768–785.Google Scholar
  28. 28.
    Daar, I.O., and Maquat, L.E. (1988).Mol. Cell Biol. 8:802–813.Google Scholar
  29. 29.
    Lehrach, H., Diamond, D., Wozney, J.M., and Boedtker, H. (1977).Biochemistry 16:4743–4751.Google Scholar
  30. 30.
    Feinberg, A.P., and Vogelstein, B. (1983).Anal. Biochem. 132:6–13.Google Scholar
  31. 31.
    Gross-Bellard, M., Oudet, P., and Chambron, P. (1973).Eur. J. Biochem. 36:32–38.Google Scholar
  32. 32.
    Southern, E.M. (1975).J. Mol. Biol. 98:503–517.Google Scholar
  33. 33.
    Church, G.M., and Gilbert, W. (1984).Proc. Natl. Acad. Sci. U.S.A. 81:1991–1995.Google Scholar
  34. 34.
    Beck, E., Ludwig, G., Auerswald, E.A., Reiss, R., and Schaller, H. (1982).Gene 19:327–336.Google Scholar
  35. 35.
    Fredericks, W.J., Chen, Y.F., and Baker, R.M. (1991). InMolecular and Clinical Advances in Anticancer Drug Resistance, (Kluwer Academic Publishers, Boston), pp. 121–149.Google Scholar
  36. 36.
    Towbin, H., Staehlin, T., and Gordon, J. (1979).Proc. Natl. Acad. Sci. U.S.A. 76:4350–4354.Google Scholar
  37. 37.
    Baker, R.M., Fredericks, W.J., Chen, Y., Murawski, M.J., Meegan, R.L., Rustum, Y.M., Karakousis, C., and Piver, M.S. (1990). InDrug Resisance Mechanisms and Reversal, (John Libbey CIC, New York), pp. 167–188.Google Scholar
  38. 38.
    Kartner, N., Evernden, P.D., Bradley, G., and Ling, V. (1985).Nature 316:820–823.Google Scholar
  39. 39.
    O'Connor, J.L., and Wade, M.F. (1992).Biotechniques 12:238–243.Google Scholar
  40. 40.
    Flaspohler, J.A., and Milcarek, C. (1992).Biotechniques 13:68–72.Google Scholar
  41. 41.
    Beckler, G. (1992).Promega Notes 39:12–13.Google Scholar
  42. 42.
    Marzluff, W.F. (1978).Methods Cell Biol. 19:317–331.Google Scholar
  43. 43.
    Linial, M., Gunderson, N., and Groudine, M. (1985).Science 230:1126–1132.Google Scholar
  44. 44.
    Chin, K.V., Chauhan, S.S., Pastan, I., and Gottesman, M.M. (1990).Cell Growth Differ. 1:361–365.Google Scholar
  45. 45.
    Ueda, K., Clark, D.P., Chen, C.J., Roninson, I.B., Gottesman, M.M., and Pastan, I. (1987).J. Biol. Chem. 262:505–508.Google Scholar
  46. 46.
    Darzynkiewicz, L., Staiano-Coico, L., and Melamed, M.R. (1981).Proc. Natl. Acad. Sci. U.S.A. 78:2382–2387.Google Scholar
  47. 47.
    Dreyfuss, G., Matunis, M.J., Pinol-Rima, S., and Burd, C.G. (1993).Annu. Rev. Biochem. 62:289–321.Google Scholar
  48. 48.
    Stout, J.T., and Caskey, C.T. (1990).Somat. Cell Mol. Genet. 16:369–382.Google Scholar
  49. 49.
    Kim, S.K., and Wold, B.J. (1985).Cell 42:129–138.Google Scholar
  50. 50.
    Nellen, W., and Lichtenstein, C. (1993).Trends Biochem. Sci. 18:419–423.Google Scholar
  51. 51.
    Giebelhaus, D.H., Elb, D.W., and Moon, R.T. (1988).Cell 53:601–615.Google Scholar
  52. 52.
    Cornelissen, M., and Vandewiele, M. (1989).Nucleic Acids Res. 17:833–843.Google Scholar
  53. 53.
    Yokoyama, K., and Imamoto, F. (1987).Proc. Natl. Acad. Sci. U.S.A. 84:7363–7367.Google Scholar
  54. 54.
    Krystal, G.W., Armstrong, B.C., and Battey, J.F. (1990).Mol. Cell Biol. 10:4180–4191.Google Scholar
  55. 55.
    Crowley, T.E., Nellen, W., Gomer, R.H., and Firtel, R.A. (1985).Cell 43:633–641.Google Scholar
  56. 56.
    Nishikura, K., and Murray, J.M. (1987).Mol. Cell Biol. 7:639–649.Google Scholar
  57. 57.
    Kasid, U., Pfeifer, A., Brennan, T., Beckett, M., Weichselbaum, R.R., Dritschilo, A., and Mark, G.E. (1989).Science 243:1354–1356.Google Scholar
  58. 58.
    Meegan, J.M., and Marcus, P.I. (1989).Science 244:1089–1091.Google Scholar
  59. 59.
    Wagner, R.W., Smith, J.E., Cooperman, B.S., and Nishikura, K. (1989).Proc. Natl. Acad. Sci. U.S.A. 86:2647–2651.Google Scholar
  60. 60.
    Wagner, R.W., Yoo, C., Wrabetz, L., Kamholz, J., Buchhalter, J., Hassan, N., Khalili, K., Kim, S.U., Perussia, B., McMorris, F.A., and Nishikura, K. (1990).Mol. Cell Biol. 10:5586–5590.Google Scholar
  61. 61.
    Bass, B.L., and Weintraub, H. (1988).Cell 55:1089–1098.Google Scholar
  62. 62.
    Saccomanno, L., and Bass, B.L. (1994).Mol. Cell Biol. 14:5425–5432.Google Scholar
  63. 63.
    Munroe, S.H. (1988).EMBO J. 7:2523–2532.Google Scholar
  64. 64.
    Portmen, D.S., and Dreyfuss, G. (1994).EMBO J. 13:213–221.Google Scholar

Copyright information

© Plenum Publishing Corporation 1994

Authors and Affiliations

  • LeRoy A. Hanchett
    • 1
  • Raymond M. Baker
    • 1
  • Bruce J. Dolnick
    • 1
  1. 1.Department of Experimental Therapeutics, Grace Cancer Drug CenterRoswell Park Cancer InstituteBuffalo

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