Skip to main content

Cell-based expression cloning for identification of polypeptides that hypersensitize mammalian cells to mitotic arrest

Abstract

Microtubule inhibitors such as Vinblastine and Paclitaxel are chemotherapy agents that activate the mitotic spindle checkpoint, arresting cells in mitosis and leading to cell death. The pathways that connect mitotic arrest to cell death are not well characterized. We developed a mammalian cell-based cDNA cloning method to isolate proteins and protein fragments whose expression inhibits colony formation in the presence of microtubule inhibitors. Understanding how these proteins impact cellular responses to microtubule drugs will lead to better understanding of the biochemical pathways connecting mitotic arrest and cell death in mammalian cells and may provide novel targets that can enhance microtubule inhibitor-mediated chemotherapy.

References

  1. Bhalla KN. Microtubule-targeted anticancer agents and apoptosis. Oncogene 2003; 22:9075–9086.

    Article  PubMed  CAS  Google Scholar 

  2. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer 2004; 4:253–265.

    Article  PubMed  CAS  Google Scholar 

  3. Mollinedo F, Gajate C. Microtubules, microtubuleinterfering agents and apoptosis. Apoptosis 2003; 8:413–450.

    Article  PubMed  CAS  Google Scholar 

  4. Blagosklonny MV, Fojo T. Molecular effects of paclitaxel: myths and reality (a critical review). Int J Cancer 1999; 83(2):151–156.

    Article  PubMed  CAS  Google Scholar 

  5. Bharadwaj R, Yu H. The spindle checkpoint, aneuploidy, and cancer. Oncogene 2004; 23:2016–2027.

    Article  PubMed  CAS  Google Scholar 

  6. Cleveland DW, Mao Y, Sullivan KF. Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling. Cell 2003; 112:407–421.

    Article  PubMed  CAS  Google Scholar 

  7. Gorbsky GJ. The mitotic spindle checkpoint. Curr Biol 2001; 11:R1001–1004.

    Article  PubMed  CAS  Google Scholar 

  8. Weaver BA, Cleveland DW. Decoding the links between mitosis, cancer, and chemotherapy: The mitotic checkpoint, adaptation, and cell death. Cancer Cell 2005; 8(1):7–12.

    Article  PubMed  CAS  Google Scholar 

  9. Tao W, South VJ, Zhang Y, Davide JP, Farrell L, Kohl NE, Sepp-Lorenzino L, Lobell RB. Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage. Cancer Cell 2005; 8(1):49–59.

    Article  PubMed  CAS  Google Scholar 

  10. Michel L, Diaz-Rodriguez E, Narayan G, Hernando E, Murty VV, Benezra R. Complete loss of the tumor suppressor MAD2 causes premature cyclin B degradation and mitotic failure in human somatic cells. Proc Natl Acad Sci USA 2004; 101:4459–4464.

    Article  PubMed  CAS  Google Scholar 

  11. Michel LS, Liberal V, Chatterjee A, Kirchwegger R, Pasche B, Gerald W, Dobles M, Sorger PK, Murty VV, Benezra R. MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature 2001; 409:355–359.

    Article  PubMed  CAS  Google Scholar 

  12. Kops GJ, Foltz DR, Cleveland DW. Lethality to human cancer cells through massive chromosome loss by inhibition of the mitotic checkpoint. Proc Natl Acad Sci USA 2004; 101:8699–8704.

    Article  PubMed  CAS  Google Scholar 

  13. Lee EA, Keutmann MK, Dowling ML, Harris E, Chan G, Kao GD. Inactivation of the mitotic checkpoint as a determinant of the efficacy of microtubule-targeted drugs in killing human cancer cells. Mol Cancer Ther 2004; 3:661–669.

    PubMed  CAS  Google Scholar 

  14. Kienitz A, Vogel C, Morales I, Muller R, Bastians H. Partial downregulation of MAD1 causes spindle checkpoint inactivation and aneuploidy, but does not confer resistance towards taxol. Oncogene 2005; 24(26):4301–4310.

    Article  PubMed  CAS  Google Scholar 

  15. Baker DJ, Jeganathan KB, Malureanu L, Perez-Terzic C, Terzic A, van Deursen JM. Early aging-associated phenotypes in Bub3/Rae1 haploinsufficient mice. J Cell Biol 2006; 172(4):529–540.

    Article  PubMed  CAS  Google Scholar 

  16. Baker DJ, Jeganathan KB, Cameron JD, Thompson M, Juneja S, Kopecka A, Kumar R, Jenkins RB, de Groen PC, Roche P, van Deursen JM. BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice. Nat Genet 2004; 36(7):744–749.

    Article  PubMed  CAS  Google Scholar 

  17. Baek KH, Shin HJ, Jeong SJ, Park JW, McKeon F, Lee CW, Kim CM. Caspases-dependent cleavage of mitotic checkpoint proteins in response to microtubule inhibitor. Oncol Res 2005; 15(3):161–168.

    PubMed  CAS  Google Scholar 

  18. Kim M, Murphy K, Liu F, Parker SE, Dowling ML, Baff W, Kao GD. Caspase-mediated specific cleavage of BubR1 is a determinant of mitotic progression. Mol Cell Biol 2005; 25(21):9232–9248.

    Article  PubMed  CAS  Google Scholar 

  19. Hannon GJ, Sun P, Carnero A, Xie LY, Maestro R, Conklin DS, Beach D. MaRX: an approach to genetics in mammalian cells. Science 1999; 283(5405):1129–1130.

    Article  PubMed  CAS  Google Scholar 

  20. Maestro R, Dei Tos AP, Hamamori Y, Krasnokutsky S, Sartorelli V, Kedes L, Doglioni C, Beach DH, Hannon GJ. Twist is a potential oncogene that inhibits apoptosis. Genes Dev 1999; 13(17):2207–2217.

    Article  PubMed  CAS  Google Scholar 

  21. Sun P, Dong P, Dai K, Hannon GJ, Beach D. p53- independent role of MDM2 in TGF-beta1 resistance. Science 1998; 282(5397):2270–2272.

    Article  PubMed  CAS  Google Scholar 

  22. Yamada HY, Gorbsky GJ. Inhibition of TRIP1/S8/hSUG1, a component of the human 19S proteasome, enhances mitotic apoptosis induced by spindle poisons. Molecular Cancer Therapeutics 2006; 5(1):29–38.

    Article  PubMed  CAS  Google Scholar 

  23. Mayer TU, Kapoor TM, Haggarty SJ, King RW, Schreiber SL, Mitchison TJ. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 1999; 286(5441):971–974.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Hiroshi Y. Yamada.

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and Permissions

About this article

Cite this article

Yamada, H.Y., Gorbsky, G.J. Cell-based expression cloning for identification of polypeptides that hypersensitize mammalian cells to mitotic arrest. Biol. Proced. Online 8, 36–43 (2006). https://doi.org/10.1251/bpo116

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1251/bpo116

Indexing terms

  • Microtubules
  • Mitosis
  • Cell Death
  • Nocodazole