Kim W H, Kang K H, Kim M Y, Choi K H
Department of Life Science, College of Natural Sciences, Chung-Ang University, Seoul, South Korea.
Biochem Cell Biol. 2000;78(2):127-35.
Ceramide is known to induce pRb (retinoblastoma gene product) dephosphorylation through the activation of ceramide-activated protein phosphatase (CAPP) during G1 arrest, but other molecular mechanisms linked to regulation of pRb dephosphorylation during ceramide-induced G1 arrest are poorly understood. In this paper, we investigated whether p21, a cdk (cyclin-dependent kinase) inhibitor, is involved in the induction of pRb dephosphorylation during ceramide-induced G1 arrest. In SK-Hep-1 cells, the addition of ceramide resulted in pRb dephosphorylation and G1 arrest. The activity of cdk2 was inhibited in response to ceramide during this process. p21 protein and mRNA were remarkably induced, while the protein level of p53, known as a transcriptional activator of p21, was not elevated at the same condition. p21 induction was also observed in the Hep3B cells lacking a functional p53 after exposure to ceramide. Although p21 is induced in ceramide-treated Hep3B cells, Hep3B cells do not induce G1 arrest, because Hep3B cells are deficient in a functional pRb protein. To confirm that pRb is a critical target for the induction of G1 arrest by inhibiting cdk2 activity through p53-independent p21, pRb-expressing vector was transfected into Hep3B cells. After treatment with ceramide, pRb-expressing cells (pRb+/+), but not pRb-/- cells, were arrested in G1 phase. In pRb+/+ cells, ceramide-mediated G1 arrest was accompanied by the accumulation of hypophosphorylated pRb and p21 associated with cdk2. Together, these results suggest that p21, induced through p53-independent pathway, participates in the induction of pRb dephosphorylation by inhibiting cdk2 activity during ceramide-mediated G1 arrest in hepatocarcinoma cells.
已知神经酰胺在G1期阻滞过程中通过激活神经酰胺激活的蛋白磷酸酶（CAPP）诱导视网膜母细胞瘤基因产物（pRb）去磷酸化，但对于神经酰胺诱导的G1期阻滞过程中与pRb去磷酸化调节相关的其他分子机制了解甚少。在本文中，我们研究了细胞周期蛋白依赖性激酶（cdk）抑制剂p21是否参与神经酰胺诱导的G1期阻滞过程中pRb的去磷酸化。在SK-Hep-1细胞中，添加神经酰胺导致pRb去磷酸化和G1期阻滞。在此过程中，cdk2的活性因神经酰胺而受到抑制。p21蛋白和mRNA显著诱导，而作为p21转录激活因子的p53蛋白水平在相同条件下并未升高。在暴露于神经酰胺后，缺乏功能性p53的Hep3B细胞中也观察到了p21的诱导。尽管在神经酰胺处理的Hep3B细胞中诱导了p21，但Hep3B细胞并未诱导G1期阻滞，因为Hep3B细胞缺乏功能性pRb蛋白。为了证实pRb是通过不依赖p53的p21抑制cdk2活性诱导G1期阻滞的关键靶点，将表达pRb的载体转染到Hep3B细胞中。用神经酰胺处理后，表达pRb的细胞（pRb+/+）而非pRb-/-细胞在G1期停滞。在pRb+/+细胞中，神经酰胺介导的G1期阻滞伴随着与cdk2相关的低磷酸化pRb和p21的积累。总之，这些结果表明，通过不依赖p53的途径诱导的p21在肝癌细胞神经酰胺介导的G1期阻滞过程中通过抑制cdk2活性参与pRb去磷酸化的诱导。
