Phillips D C, Hunt J T, Moneypenny C G, Maclean K H, McKenzie P P, Harris L C, Houghton J A
Division of Molecular Therapeutics, Department of Oncology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA.
Cell Death Differ. 2007 Oct;14(10):1780-91. doi: 10.1038/sj.cdd.4402198. Epub 2007 Jul 13.
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.
鞘脂神经酰胺负责多种生化和细胞反应，包括在调节细胞周期进程中可能发挥的作用。在此，我们描述了通过外源应用C(6)-神经酰胺或暴露于鞘磷脂酶实现的神经酰胺积累，可诱导横纹肌肉瘤（RMS）细胞系发生G(2)期阻滞。利用RMS细胞系RD，我们发现这种G(2)期阻滞需要快速诱导p21(Cip1/Waf1)，且与DNA损伤无关。随后在较晚时间点（48小时）发生凋亡。Bcl-2过表达可阻止凋亡，但允许p21(Cip1/Waf1)蛋白表达维持升高以及G(2)期阻滞反应稳定。用环己酰亚胺（CHX）抑制p21(Cip1/Waf1)蛋白合成或用小干扰RNA（siRNA）使p21(Cip1/Waf1)沉默，可阻止神经酰胺介导的G(2)期阻滞和晚期凋亡诱导。此外，鉴于最近发现小鼠双微体2（MDM2）通过将这种细胞周期蛋白依赖性激酶抑制剂呈递给蛋白酶体进行降解来控制p21(Cip1/Waf1)表达，过表达MDM2的RD细胞消除了神经酰胺介导的p21(Cip1/Waf1)诱导、G(2)期阻滞和随后的晚期凋亡。总体而言，这些数据进一步支持了神经酰胺积累可调节细胞周期进程的观点。此外，这些观察结果突出了MDM2表达和蛋白酶体活性是细胞对神经酰胺积累反应的关键决定因素。
