Expression of a proteasome regulatory subunit sensitizes HeLa cells to cell death by treatment with spindle poisons and the proteasome inhibitor MG132

Hiroshi Y Yamada* and Gary J Gorbsky
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation,
Mail Stop 48,  Oklahoma City, Oklahoma 73104, USA
*email: Hiroshi-yamada@omrf.ouhsc.edu

  Introduction.  Spindle poisons (e.g. Taxol, Vinblastin) inhibit mitotic spindle function, activate the spindle checkpoint (or metaphase checkpoint) and cause mitotic arrest. They are commonly used cancer chemotherapy drugs (Ref.1). Spindle checkpoint-deficient yeast cells are more sensitive to spindle poisons, since the loss of function allows mitotic cells to exit without proper chromosome segregation and thus causes chromosomal abnormalities in the daughter cells. By analogy we hypothesized that inactivation of the spindle checkpoint in mammalian cells might sensitize mammalian cells to spindle poisons, and enhance chemotherapeutic effects. Identification of additional genes whose products contribute to spindle checkpoint function in mammalian cells may aid in the development of novel cancer therapies to be used in combination with spindle poisons.

Methods.  To identify the genes that affect spindle checkpoint function, we developed a novel mammalian gene screening/cloning strategy. The screening/cloning is based on cell cycle-dependent change in cell morphology, and is a modified cycle cloning method (Ref.2). 

Results.  Through pilot screening we obtained several candidate inserts. We then generated HeLa cell lines in which the candidate gene fragments were integrated and constitutively expressed under a CMV promoter. Some of the integrants showed elevated sensitivity to spindle poisons (nocodazole and Taxol) and to the proteasome inhibitor MG132, suggesting mitotic progression might be affected in the integrants. These were not hypersensitive to a topoisomerase II inhibitor VM26 (teniposide).  Thus the sensitivity appeared specific to certain drugs. One of the drug-sensitive integrants was HeLa-SC3. Clone SC3 contains the C terminal region of  S8/p45/TRIP1 (S8 deltaN(1-87)). S8/p45/TRIP1 is an AAA(ATPase Associated with a variety of cellular Activities) family ATPase subunit of proteasome19S regulatory complex (Ref.3). 

Discussion. The proteasome is a downstream component of spindle checkpoint and drives mitotic anaphase progression by degrading polyubiquitylated mitotic regulatory proteins such as mitotic cyclins and securin. The alteration in S8/p45/TRIP1 expression may affect proteasome function and mitotic progression. Alternatively this protein has been reported to play a role in modulation of transcription (Ref.4, 5). It will be important to determine whether the increased drug sensitivity is caused by spindle checkpoint inactivation, or through some other pathway.

Through pilot screening we isolated a gene that affects cell survival under spindle poison challenge. Further screening may reveal a set of genes that is clinically useful to sensitize cancer cells to chemotherapy drugs such as spindle poisons and proteasome inhibitors.

Acknowledgement. This research was supported by a grant to G.J.G (National Institute of General Medical Science, RO1-GM50412), and by a postdoctoral training grant to H.Y.Y. (US Department of Defense (DOD), Breast Cancer Research Program).

References

1. Mollinedo, F. and Gajate, C. (2003) Apoptosis 5, 413-50.
2. Hannon, G.J., Sun, P., Carnero, A., Xie, L.Y., Maestro, R., Conklin, D.S. and Beach, D. (1999) Science. 283(5405):1129-30.
3. Voges, D., Zwickl, P. and Baumeister, W. (1999) Annu. Rev. Biochem. 68: 1015-1068
4. Ottosen, S., Francisco, J., Herrera, F.J. and Triezenberg, S.J. (2002)  Science. 296(5567):479-81.
5. Ferdous, A., Kodadek,T. and Johnston, S.A. (2002) Biochemistry.  41(42):12798-805.