Spinedi A, Amendola A, Di Bartolomeo S, Piacentini M
Department of Biology, University of Rome Tor Vergata, Italy.
Biochem Biophys Res Commun. 1998 Feb 24;243(3):852-7. doi: 10.1006/bbrc.1998.8184.
Recent evidence suggests that untimely retinoblastoma protein (RB) dephosphorylation and/or proteolytic degradation might provide key events down-stream cysteine protease (caspase) activation in apoptosis induction. We have dealt with this issue by studying apoptosis induced by N-hexanoylsphingosine (C6-Cer) in CHP-100 human neuroepithelioma cells, maintained in complete growth medium. We report that C6-Cer-induced apoptosis occurred predominantly in G1/S phases of the cycle and was associated with RB dephosphorylation, in the setting of negligible Bcl-2 expression. Apoptosis was also associated with poly(ADP-ribose) polymerase (PARP) cleavage, thus indicating activation of CPP32/Yama/apopain (caspase-3); however, while the tripeptide caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone was able to prevent both C6-Cer-induced PARP cleavage and apoptosis, it was ineffective in preventing RB dephosphorylation. Moreover proteolytic RB cleavage occurred only to a marginal extent after C6-Cer treatment. These results indicate that apoptosis induced by ceramide in CHP-100 cells is caspase-mediated, but RB post-translational modification does not provide a key step, downstream caspase activation, in apoptosis execution.
最近的证据表明，视网膜母细胞瘤蛋白（RB）去磷酸化和/或蛋白水解降解时机不当可能是凋亡诱导过程中半胱氨酸蛋白酶（caspase）激活下游的关键事件。我们通过研究在完全生长培养基中培养的CHP-100人神经上皮瘤细胞中N-己酰鞘氨醇（C6-神经酰胺）诱导的凋亡来处理这个问题。我们报告，在Bcl-2表达可忽略不计的情况下，C6-神经酰胺诱导的凋亡主要发生在细胞周期的G1/S期，并且与RB去磷酸化有关。凋亡还与聚（ADP-核糖）聚合酶（PARP）裂解有关，从而表明CPP32/Yama/apopain（caspase-3）被激活；然而，虽然三肽caspase抑制剂Z-缬氨酸-丙氨酸-DL-天冬氨酸-氟甲基酮能够阻止C6-神经酰胺诱导的PARP裂解和凋亡，但它在阻止RB去磷酸化方面无效。此外，C6-神经酰胺处理后仅在边缘程度上发生RB的蛋白水解裂解。这些结果表明，神经酰胺在CHP-100细胞中诱导的凋亡是由caspase介导的，但RB的翻译后修饰在凋亡执行过程中并非caspase激活下游的关键步骤。
