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Downregulation of Skp2 and p27/Kip1 synergistically induces apoptosis in T98G glioblastoma cells

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Abstract

S-Phase kinase associated protein (Skp) 2 is an F-box protein required for substrate recognition of the SCFSkp2 ubiquitin ligase complex. Skp2 is often overexpressed in transformed cells and in various types of tumors. Downregulation or inhibition of Skp2 inhibits growth of breast cancer cells and small-cell lung carcinoma cells. We downregulated Skp2 in T98G glioblastoma cells using small interfering RNA (siRNA). Downregulation induced p27 and caused growth arrest and apoptosis. Downregulation of both Skp2 and p27 increased apoptosis synergistically. Cyclin E levels and cyclin E-CDK2 kinase activity increased dramatically when both Skp2 and p27 were downregulated. Coincidently, Bcl-2 but not Bcl-xL expression decreased, and caspase-3 was activated. Inhibition of cyclin E-CDK2 kinase activity by forced expression of p21 reversed these effects. Moreover, stable expression of Bcl-2 also abrogated apoptosis induced by downregulation of Skp2 and p27. We suggest that Skp2 in tumor cells suppresses apoptosis through Bcl-2 expression, potentially through regulation of cyclin E-CDK2 activity.

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Abbreviations

Br-dU :

5-Bromo-2-deoxyuridine

CDK :

Cyclin-dependent kinase

CMV :

Cytomegalovirus

DTT :

Dithiothreitol

FBS :

Fetal bovine serum

GFP :

Green fluorescent protein

PAGE :

Polyacrylamide gel electrophoresis

PI :

Propidium iodide

SDS :

Sodium dodecyl sulfate

siRNA :

Small interfering RNA

Skp :

S-phase kinase associated protein

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Acknowledgements

We gratefully thank Dr. Tetsu and Dr. Stokoe for critical advice. We also thank Dr. Evan and Dr. Gump for their reagents. We thank members of the UCSF Comprehensive Cancer Center Cytometry Core facilities for their helpful support. This work was supported by a grant from Daiichi Pharmaceuticals, Tokyo, Japan, for the Daiichi Cancer Research Program at UCSF.

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  1. Cancer Research Institute and Comprehensive Cancer Center, University of California, San Francisco, CA, 94143, USA

    Sang Hyun Lee & Frank McCormick

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  1. Sang Hyun Lee
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  2. Frank McCormick
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Correspondence to Frank McCormick.

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Lee, S.H., McCormick, F. Downregulation of Skp2 and p27/Kip1 synergistically induces apoptosis in T98G glioblastoma cells. J Mol Med 83, 296–307 (2005). https://doi.org/10.1007/s00109-004-0611-7

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