Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

Induction of endogenous Bcl-xS through the control of Bcl-x pre-mRNA splicing by antisense oligonucleotides

Abstract

Resistance to apoptosis, which plays an important role in tumors that are refractory to chemotherapy, is regulated by the ratio of antiapoptotic to proapoptotic proteins. By manipulating levels of these proteins, cells can become sensitized to undergo apoptosis in response to chemotherapeutic agents. Alternative splicing of the bcl-x gene gives rise to two proteins with antagonistic functions: Bcl-xL, a well-characterized antiapoptotic protein, and Bcl-xS, a proapoptotic protein. We show here that altering the ratio of Bcl-xL to Bcl-xS in the cell using an antisense oligonucleotide permitted cells to be sensitized to undergo apoptosis in response to ultraviolet B radiation and chemotherapeutic drug treatment. These results demonstrate the ability of a chemically modified oligonucleotide to alter splice site selection in an endogenous gene and illustrate a powerful tool to regulate cell survival.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Purchase on Springer Link

Instant access to full article PDF

Prices may be subject to local taxes which are calculated during checkout

Figure 1: (A) Illustration of the bcl-x gene and alternative splice products.
Figure 2: RPA analysis of oligonucleotide effects on Bcl-x splicing.
Figure 3: Effect of oligonucleotide concentration on Bcl-x expression levels.
Figure 4: Analysis of Bcl-x protein levels.
Figure 5: Physiological effect of Bcl-xS induction.

Similar content being viewed by others

References

  1. Raff, M. Cell suicide for beginners. Nature 396, 119– 122 (1998).

    Article  CAS  Google Scholar 

  2. White, E. Life, death, and the pursuit of apoptosis. Genes Dev. 10, 1–15 (1996).

    Article  CAS  Google Scholar 

  3. Jiang, Z-H. & Wu, J.Y. Alternative splicing and programmed cell death. Proc. Soc. Exp. Biol. Med. 220, 64–72 (1999).

    Article  CAS  Google Scholar 

  4. Boise, L.H. et al. Bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74, 597– 608 (1993).

    Article  CAS  Google Scholar 

  5. Yang, E. & Korsmeyer, S.J. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood 88, 386–401 (1996).

    CAS  Google Scholar 

  6. Kelekar, A. & Thompson, C.B. Bcl-2-family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol. 8 , 324–330 (1998).

    Article  CAS  Google Scholar 

  7. Gonzalez-Garcia, M. et al. Bcl-x is expressed in embryonic and postnatal neural tissues and functions to prevent neuronal cell death. Proc. Natl. Acad. Sci. USA 92, 4304–4308 (1995).

    Article  CAS  Google Scholar 

  8. Shiraiwa, N. et al. An additional form of rat Bcl-x, Bcl-xβ, generated by an unspliced RNA, promotes apoptosis in promyeloid cells. J. Biol. Chem. 271, 13258–13265 (1996).

    Article  CAS  Google Scholar 

  9. Ban, J., Eckhart, L., Weninger, W., Mildner, M. & Tschachler, E. Identification of a human cDNA encoding a novel Bcl-x isoform. Biochem. Biophys. Res. Commun. 248, 147–152 (1998).

    Article  CAS  Google Scholar 

  10. Reed, J.C. Regulation of apoptosis by bcl-2 family proteins and its role in cancer chemoresistance. Curr. Opin. Oncol. 7, 541– 546 (1995).

    Article  CAS  Google Scholar 

  11. Taylor, J.K. & Dean, N.M. Regulation of pre-mRNA splicing by antisense oligonucleotides. Curr. Opin. Drug Discovery Dev. 2, 147–151 (1999).

    CAS  Google Scholar 

  12. Kole, R. Modification of pre-mRNA splicing by antisense oligonucleotides. Acta Biochim Pol. 44, 231–238 (1997).

    CAS  PubMed  Google Scholar 

  13. Hodges, D. & Crooke, S.T. Inhibition of splicing of wild-type and mutated luciferase adenovirus pre-mRNAs by antisense oligonucleotides. Mol. Pharmacol. 48, 905– 918 (1995).

    CAS  PubMed  Google Scholar 

  14. Dominski, Z. & Kole, R. Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides. Proc. Natl. Acad. Sci. USA 90, 8673–8677 (1993).

    Article  CAS  Google Scholar 

  15. Sierakowska, H., Sambade, M.J., Agrawal, S. & Kole, R. Repair of thalassemic human β-globin mRNA in mammalian cells by antisense oligonucleotides. Proc. Natl. Acad. Sci. USA 93, 12840– 12844 (1996).

    Article  CAS  Google Scholar 

  16. Dunckley, M.G., Manoharan, M., Villiet, P., Eperon, I.C. & Dickson, G. Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides. Hum. Mol. Genet. 5, 1083– 1090 (1998).

    Article  Google Scholar 

  17. Dean, N.M. & Griffey, R.H. Identification and characterization of second-generation antisense oligonucleotides. Antisense Nucleic Acid Drug Dev. 7, 229–233 (1997).

    Article  CAS  Google Scholar 

  18. Taylor, J.K., Zhang, Q.Q., Monia, B.P., Marcusson, E.G. & Dean, N.M. Inhibition of Bcl-xL expression sensitizes normal human keratinocytes and epithelial cells to apoptotic stimuli. Oncogene 18, 4495–4504 (1999).

    Article  CAS  Google Scholar 

  19. Noble, J.R., Willetts, K.E., Mercer, W.E. & Reddel, R.R. Effects of exogenous wild-type p53 on a human lung carcinoma cell line with endogenous wild-type p53. Exp. Cell Res. 203, 297–304 (1992).

    Article  CAS  Google Scholar 

  20. Yu, K. et al. Ras-dependent apoptosis correlates with persistent activation of stress-activated protein kinases and induction of isoform(s) of Bcl-x. Cell Death Differ. 4, 745–755 (1997).

    Article  CAS  Google Scholar 

  21. Clarke, M.F. et al. A recombinant bcl-xS adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells. Proc. Natl. Acad. USA 92, 11024–11028 (1995).

    Article  CAS  Google Scholar 

  22. Sumantran, V.N., Ealovega, M.W., Nunez, G., Clarke, M.F. & Wicha, M.S. Overexpression of Bcl-xS sensitizes MCF-7 cells to chemotherapy-induced apoptosis. Cancer Res. 55, 2507–2510 (1995).

    CAS  PubMed  Google Scholar 

  23. Ealovega, M.W., McGinnis, P.K., Sumantran, V.N., Clarke, M.F. & Wicha, M.S. Bcl-xS gene therapy induces apoptosis of human mammary tumors in nude mice. Cancer Res. 56 , 1965–1969 (1996).

    CAS  PubMed  Google Scholar 

  24. Harris, A.L. & Hochhauser, D. Mechanisms of multidrug resistance in cancer treatment. Acta Oncol. 31, 205 –213 (1992).

    Article  CAS  Google Scholar 

  25. Minn, A.J., Boise, L.H. & Thompson, C.B. Bcl-xS antagonizes the protective effects of Bcl-xL. J. Biol. Chem. 271, 6306– 6312 (1996).

    Article  CAS  Google Scholar 

  26. Tao, W., Kurschner, C. & Morgan, J.I. Bcl-xS and Bad potentiate the death suppressing activities of Bcl-xL, Bcl-2, and A1 in yeast. J. Biol. Chem. 273 , 23704–23708 (1998).

    Article  CAS  Google Scholar 

  27. Ray, S. et al. Enforced expression of Bcl-XS induces differentiation and sensitizes chronic myelogenous leukemia-blast crisis K562 cells to 1-beta-D-arabinofuranosylcytosine-mediated differentiation and apoptosis. Cell Growth & Differ. 7, 1617–1623 (1996).

    CAS  Google Scholar 

  28. Pena, J.C., Fuchs, E. & Thompson, C.B. Bcl-x expression influences keratinocyte cell survival but not terminal differentiation. Cell Growth & Differ. 8, 619–629 (1997).

    CAS  Google Scholar 

  29. Fridman, J.S., Rehemtulla, A., Hofmann, A., Blau, H.M. & Maybaum, J. Expression of Bcl-XS alters cytokinetics and decreases clonogenic survival in K12 rat colon carcinoma cells. Oncogene 17, 2981–2991 (1998).

    Article  CAS  Google Scholar 

  30. Monia, B.P. et al. Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides. J. Biol. Chem. 267, 19954 –19962 (1992).

    CAS  PubMed  Google Scholar 

  31. Condon, T.P. & Bennett, C.F. Altered mRNA splicing and inhibition of E-selectin by an antisense oligonucleotide in human umbilical vein endothelial cells. J. Biol. Chem. 271, 30398– 30403 (1996).

    Article  CAS  Google Scholar 

  32. Schwandner, R., Wiegmann, K., Bernardo, K., Kreder, D. & Kronke, M. TNF receptor death domain-associated proteins TRADD and FADD signal activation of acid sphingomyelinase. J. Biol. Chem. 273, 5916–5922 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank Sue Freier, Todd Burckin, and Frank Bennett for advice with these studies.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Nicholas M. Dean.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Taylor, J., Zhang, Q., Wyatt, J. et al. Induction of endogenous Bcl-xS through the control of Bcl-x pre-mRNA splicing by antisense oligonucleotides. Nat Biotechnol 17, 1097–1100 (1999). https://doi.org/10.1038/15079

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/15079

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing