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. 1995 Aug 29;92(18):8338–8342. doi: 10.1073/pnas.92.18.8338

Antisense DNA downregulation of the ERBB2 oncogene measured by a flow cytometric assay.

J P Vaughn 1, J D Iglehart 1, S Demirdji 1, P Davis 1, L E Babiss 1, M H Caruthers 1, J R Marks 1
PMCID: PMC41152  PMID: 7667291

Abstract

A causal role has been inferred for ERBB2 overexpression in the etiology of breast cancer and other epithelial malignancies. The development of therapeutics that inhibit this tyrosine kinase cell surface receptor remains a high priority. This report describes the specific downregulation of ERBB2 protein and mRNA in the breast cancer cell line SK-BR-3 by using antisense DNA phosphorothioates. An approach was developed to examine antisense effects which allows simultaneous measurements of antisense dose and gene specific regulation on a per cell basis. A fluorescein isothiocyanate end-labeled tracer oligonucleotide was codelivered with antisense DNA followed by immunofluorescent staining for ERBB2 protein expression. Two-color flow cytometry measured the amount of both intracellular oligonucleotide and ERBB2 protein. In addition, populations of cells that received various doses of nucleic acids were physically separated and studied. In any given transfection, a 100-fold variation in oligonucleotide dosage was found. ERBB2 protein expression was reduced greater than 50%, but only in cells within a relatively narrow uptake range. Steady-state ERBB2 mRNA levels were selectively diminished, indicating a specific antisense effect. Cells receiving the optimal antisense dose were sorted and analyzed for cell cycle changes. After 2 days of ERBB2 suppression, breast cancer cells showed an accumulation in the G1 phase of the cell cycle.

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Selected References

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

  1. Akhtar S., Basu S., Wickstrom E., Juliano R. L. Interactions of antisense DNA oligonucleotide analogs with phospholipid membranes (liposomes). Nucleic Acids Res. 1991 Oct 25;19(20):5551–5559. doi: 10.1093/nar/19.20.5551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bacus S. S., Gudkov A. V., Zelnick C. R., Chin D., Stern R., Stancovski I., Peles E., Ben-Baruch N., Farbstein H., Lupu R. Neu differentiation factor (heregulin) induces expression of intercellular adhesion molecule 1: implications for mammary tumors. Cancer Res. 1993 Nov 1;53(21):5251–5261. [PubMed] [Google Scholar]
  3. Bacus S. S., Huberman E., Chin D., Kiguchi K., Simpson S., Lippman M., Lupu R. A ligand for the erbB-2 oncogene product (gp30) induces differentiation of human breast cancer cells. Cell Growth Differ. 1992 Jul;3(7):401–411. [PubMed] [Google Scholar]
  4. Bacus S. S., Stancovski I., Huberman E., Chin D., Hurwitz E., Mills G. B., Ullrich A., Sela M., Yarden Y. Tumor-inhibitory monoclonal antibodies to the HER-2/Neu receptor induce differentiation of human breast cancer cells. Cancer Res. 1992 May 1;52(9):2580–2589. [PubMed] [Google Scholar]
  5. Bacus S. S., Stancovski I., Huberman E., Chin D., Hurwitz E., Mills G. B., Ullrich A., Sela M., Yarden Y. Tumor-inhibitory monoclonal antibodies to the HER-2/Neu receptor induce differentiation of human breast cancer cells. Cancer Res. 1992 May 1;52(9):2580–2589. [PubMed] [Google Scholar]
  6. Barnes D. M., Lammie G. A., Millis R. R., Gullick W. L., Allen D. S., Altman D. G. An immunohistochemical evaluation of c-erbB-2 expression in human breast carcinoma. Br J Cancer. 1988 Oct;58(4):448–452. doi: 10.1038/bjc.1988.238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bennett C. F., Chiang M. Y., Chan H., Shoemaker J. E., Mirabelli C. K. Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol Pharmacol. 1992 Jun;41(6):1023–1033. [PubMed] [Google Scholar]
  8. Bouchard L., Lamarre L., Tremblay P. J., Jolicoeur P. Stochastic appearance of mammary tumors in transgenic mice carrying the MMTV/c-neu oncogene. Cell. 1989 Jun 16;57(6):931–936. doi: 10.1016/0092-8674(89)90331-0. [DOI] [PubMed] [Google Scholar]
  9. Cazenave C., Stein C. A., Loreau N., Thuong N. T., Neckers L. M., Subasinghe C., Hélène C., Cohen J. S., Toulmé J. J. Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodeoxynucleotides. Nucleic Acids Res. 1989 Jun 12;17(11):4255–4273. doi: 10.1093/nar/17.11.4255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  11. Culouscou J. M., Plowman G. D., Carlton G. W., Green J. M., Shoyab M. Characterization of a breast cancer cell differentiation factor that specifically activates the HER4/p180erbB4 receptor. J Biol Chem. 1993 Sep 5;268(25):18407–18410. [PubMed] [Google Scholar]
  12. Di Fiore P. P., Pierce J. H., Kraus M. H., Segatto O., King C. R., Aaronson S. A. erbB-2 is a potent oncogene when overexpressed in NIH/3T3 cells. Science. 1987 Jul 10;237(4811):178–182. doi: 10.1126/science.2885917. [DOI] [PubMed] [Google Scholar]
  13. Drebin J. A., Link V. C., Greene M. I. Monoclonal antibodies specific for the neu oncogene product directly mediate anti-tumor effects in vivo. Oncogene. 1988 Apr;2(4):387–394. [PubMed] [Google Scholar]
  14. Fisher T. L., Terhorst T., Cao X., Wagner R. W. Intracellular disposition and metabolism of fluorescently-labeled unmodified and modified oligonucleotides microinjected into mammalian cells. Nucleic Acids Res. 1993 Aug 11;21(16):3857–3865. doi: 10.1093/nar/21.16.3857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gao W. Y., Han F. S., Storm C., Egan W., Cheng Y. C. Phosphorothioate oligonucleotides are inhibitors of human DNA polymerases and RNase H: implications for antisense technology. Mol Pharmacol. 1992 Feb;41(2):223–229. [PubMed] [Google Scholar]
  16. Iglehart J. D., Kerns B. J., Huper G., Marks J. R. Maintenance of DNA content and erbB-2 alterations in intraductal and invasive phases of mammary cancer. Breast Cancer Res Treat. 1995 Jun;34(3):253–263. doi: 10.1007/BF00689717. [DOI] [PubMed] [Google Scholar]
  17. Iglehart J. D., Kraus M. H., Langton B. C., Huper G., Kerns B. J., Marks J. R. Increased erbB-2 gene copies and expression in multiple stages of breast cancer. Cancer Res. 1990 Oct 15;50(20):6701–6707. [PubMed] [Google Scholar]
  18. Juliano R. L., Akhtar S. Liposomes as a drug delivery system for antisense oligonucleotides. Antisense Res Dev. 1992 Summer;2(2):165–176. doi: 10.1089/ard.1992.2.165. [DOI] [PubMed] [Google Scholar]
  19. Kraus M. H., Popescu N. C., Amsbaugh S. C., King C. R. Overexpression of the EGF receptor-related proto-oncogene erbB-2 in human mammary tumor cell lines by different molecular mechanisms. EMBO J. 1987 Mar;6(3):605–610. doi: 10.1002/j.1460-2075.1987.tb04797.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lacroix H., Iglehart J. D., Skinner M. A., Kraus M. H. Overexpression of erbB-2 or EGF receptor proteins present in early stage mammary carcinoma is detected simultaneously in matched primary tumors and regional metastases. Oncogene. 1989 Feb;4(2):145–151. [PubMed] [Google Scholar]
  21. Leonetti J. P., Mechti N., Degols G., Gagnor C., Lebleu B. Intracellular distribution of microinjected antisense oligonucleotides. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2702–2706. doi: 10.1073/pnas.88.7.2702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lowe S. W., Ruley H. E., Jacks T., Housman D. E. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell. 1993 Sep 24;74(6):957–967. doi: 10.1016/0092-8674(93)90719-7. [DOI] [PubMed] [Google Scholar]
  23. Maier L. A., Xu F. J., Hester S., Boyer C. M., McKenzie S., Bruskin A. M., Argon Y., Bast R. C., Jr Requirements for the internalization of a murine monoclonal antibody directed against the HER-2/neu gene product c-erbB-2. Cancer Res. 1991 Oct 1;51(19):5361–5369. [PubMed] [Google Scholar]
  24. Peles E., Bacus S. S., Koski R. A., Lu H. S., Wen D., Ogden S. G., Levy R. B., Yarden Y. Isolation of the neu/HER-2 stimulatory ligand: a 44 kd glycoprotein that induces differentiation of mammary tumor cells. Cell. 1992 Apr 3;69(1):205–216. doi: 10.1016/0092-8674(92)90131-u. [DOI] [PubMed] [Google Scholar]
  25. Peles E., Yarden Y. Neu and its ligands: from an oncogene to neural factors. Bioessays. 1993 Dec;15(12):815–824. doi: 10.1002/bies.950151207. [DOI] [PubMed] [Google Scholar]
  26. Pierce J. H., Arnstein P., DiMarco E., Artrip J., Kraus M. H., Lonardo F., Di Fiore P. P., Aaronson S. A. Oncogenic potential of erbB-2 in human mammary epithelial cells. Oncogene. 1991 Jul;6(7):1189–1194. [PubMed] [Google Scholar]
  27. Press M. F., Cordon-Cardo C., Slamon D. J. Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene. 1990 Jul;5(7):953–962. [PubMed] [Google Scholar]
  28. Press M. F., Jones L. A., Godolphin W., Edwards C. L., Slamon D. J. HER-2/neu oncogene amplification and expression in breast and ovarian cancers. Prog Clin Biol Res. 1990;354A:209–221. [PubMed] [Google Scholar]
  29. Slamon D. J., Godolphin W., Jones L. A., Holt J. A., Wong S. G., Keith D. E., Levin W. J., Stuart S. G., Udove J., Ullrich A. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989 May 12;244(4905):707–712. doi: 10.1126/science.2470152. [DOI] [PubMed] [Google Scholar]
  30. Stancovski I., Hurwitz E., Leitner O., Ullrich A., Yarden Y., Sela M. Mechanistic aspects of the opposing effects of monoclonal antibodies to the ERBB2 receptor on tumor growth. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8691–8695. doi: 10.1073/pnas.88.19.8691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stein C. A., Cheng Y. C. Antisense oligonucleotides as therapeutic agents--is the bullet really magical? Science. 1993 Aug 20;261(5124):1004–1012. doi: 10.1126/science.8351515. [DOI] [PubMed] [Google Scholar]
  32. Thierry A. R., Dritschilo A. Intracellular availability of unmodified, phosphorothioated and liposomally encapsulated oligodeoxynucleotides for antisense activity. Nucleic Acids Res. 1992 Nov 11;20(21):5691–5698. doi: 10.1093/nar/20.21.5691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wagner R. W. Gene inhibition using antisense oligodeoxynucleotides. Nature. 1994 Nov 24;372(6504):333–335. doi: 10.1038/372333a0. [DOI] [PubMed] [Google Scholar]
  34. Wagner R. W., Matteucci M. D., Lewis J. G., Gutierrez A. J., Moulds C., Froehler B. C. Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines. Science. 1993 Jun 4;260(5113):1510–1513. doi: 10.1126/science.7684856. [DOI] [PubMed] [Google Scholar]
  35. van de Vijver M. J., Peterse J. L., Mooi W. J., Wisman P., Lomans J., Dalesio O., Nusse R. Neu-protein overexpression in breast cancer. Association with comedo-type ductal carcinoma in situ and limited prognostic value in stage II breast cancer. N Engl J Med. 1988 Nov 10;319(19):1239–1245. doi: 10.1056/NEJM198811103191902. [DOI] [PubMed] [Google Scholar]

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