Ceramide-induced G2 arrest in rhabdomyosarcoma (RMS) cells requires p21Cip1/Waf1 induction and is prevented by MDM2 overexpression

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 C-6-ceramide or exposure to sphingomyelinase, induces a G(2) arrest in Rhabdomyosarcoma (RMS) cell lines. Utilizing the RMS cell line RD, we show that this G(2) 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 G(2) arrest response. Inhibition of p21(Cip1/Waf1) protein synthesis with cyclohexamide (CHX) or silencing of p21(Cip1/Waf1) with siRNA, prevented ceramide-mediated G(2) 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, G(2) 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.