1. ¹Division of Molecular Therapeutics, Department of Oncology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA
2. ²Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA

Correspondence: JA Houghton, Current address:Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue/NB4-59, Cleveland, OH 44195, USA. Tel: +216 445 9652; Fax: +216 444 3164; E-mail: houghtj@ccf.org

Received 21 November 2006; Revised 28 May 2007; Accepted 7 June 2007; Published online 13 July 2007.

Abstract

The sphingoplipid ceramide is responsible for a diverse range of biochemical and cellular responses including a putative role in modulating cell cycle progression. Herein, we describe that an accumulation of ceramide, achieved through the exogenous application of C₆-ceramide or exposure to sphingomyelinase, induces a G₂ arrest in Rhabdomyosarcoma (RMS) cell lines. Utilizing the RMS cell line RD, we show that this G₂ arrest required the rapid induction of p21^Cip1/Waf1 independent of DNA damage. This was followed at later time points (48 h) by the commitment to apoptosis. Apoptosis was prevented by Bcl-2 overexpression, but permitted the maintenance of elevated p21^Cip1/Waf1 protein expression and the stabilization of the G₂ arrest response. Inhibition of p21^Cip1/Waf1 protein synthesis with cyclohexamide (CHX) or silencing of p21^Cip1/Waf1 with siRNA, prevented ceramide-mediated G₂ arrest and the late induction of apoptosis. Further, adopting the recent discovery that murine double minute 2 (MDM2) controls p21^Cip1/Waf1 expression by presenting this CDK inhibitor to the proteasome for degradation, RD cells overexpressing MDM2 abrogated ceramide-mediated p21^Cip1/Waf1 induction, G₂ arrest and the late ensuing apoptosis. Collectively, these data further support the notion that ceramide accumulation can modulate cell cycle progression. Additionally, these observations highlight MDM2 expression and proteasomal activity as key determinants of the cellular response to ceramide accumulation.