P53-mediated growth suppression in response to Nutlin-3 in cyclin D1-transformed cells occurs independently of p2l
P53-mediated growth suppression in response to Nutlin-3 in cyclin D1-transformed cells occurs independently of p2l
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DOI：
10.1158/0008-5472.can-07-0259
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发表时间：
2007-10-15
期刊：
影响因子：
11.2
通讯作者：
Jackson, Mark W.
中科院分区：
文献类型：
--
作者：
Kan, Charlene E.;Patton, John T.;Jackson, Mark W.
细胞周期蛋白D1与细胞周期蛋白依赖性激酶（CDK）的相互作用导致蛋白质的RB家族的过度磷酸化，从而灭活RB的肿瘤抑制功能。我们先前的发现表明，组成型细胞周期蛋白D1/CDK活性通过防止CDK抑制剂P21（p53的转录靶标）适当调节CDK活性，从而抑制了p53介导的基因抑制。为了研究细胞周期蛋白D1在驱动人类乳细胞转化中的作用，我们表达了一种组成型活性的细胞周期蛋白D1-CDK融合蛋白（D1/CDK）在永生的人类乳腺上皮细胞中。在存在野生型p53的情况下，表达D1/CDK的人类乳腺上皮细胞独立于锚固，与D1/CDK破坏下游p53信号传导的想法一致。使用此转换模型，我们检查了表达D1/CDK细胞对Nutlin-3的敏感性，Nutlin-3是激活p53的HDM2拮抗剂。令人惊讶的是，用Nutlin-3处理D1/CDK转化的细胞可阻止其锚固独立的生长。尽管存在高磷酸化的RB，但仍在表达D1/CDK的细胞中实现了Nutlin-3诱导的生长停滞，这意味着依赖P53的RB独立机制用于生长抑制。进一步的分析表明，在Nutlin-3稳定下，p53稳定稳定后，CDC2和Cyclin B1（关键细胞周期调节剂）稳定下调，与p53与Cdc2和Cyclin B1启动子之间的直接相互作用一致，从而导致通过甲基化对转录的抑制。与D1/CDK表达相反，p53的直接失活不会导致CDC2抑制，也没有细胞周期停滞。我们得出的结论是，由于特定p53靶基因的直接抑制，Nutlin-3诱导p53是具有组成型CDK信号传导的癌症中可行的治疗策略。
Interaction of cyclin D1 with cyclin-dependent kinases (CDK) results in the hyperphosphorylation of the RB family of proteins, thereby inactivating the tumor-suppressive function of RB. Our previous findings suggest that constitutive cyclin D1/CDK activity inhibits p53-mediated gene repression by preventing the appropriate regulation of CDK activity by the CDK inhibitor p21, a transcriptional target of p53. To study the role of cyclin D1 in driving human mammary cell transformation, we expressed a constitutively active cyclin D1-CDK fusion protein (D1/CDK) in immortalized human mammary epithelial cells. D1/CDK-expressing human mammary epithelial cells grew anchorage-independently in the presence of wild-type p53, consistent with the idea that D1/CDK disrupts downstream p53 signaling. Using this transformation model, we examined the sensitivity of the D1/CDK-expressing cells to Nutlin-3, an HDM2 antagonist that activates p53. Surprisingly, treatment of D1/CDK-transformed cells with Nutlin-3 prevented their anchorage-independent growth. The Nutlin-3-induced growth arrest was enforced in D1/CDK-expressing cells despite the presence of hyperphosphorylated RB implicating a p53-dependent, RB-independent mechanism for growth suppression. Further analysis identified that CDC2 and cyclin B1, key cell cycle regulators, were stably down-regulated following p53 stabilization by Nutlin-3, consistent with direct interaction between p53 and the CDC2 and cyclin B1 promoters, leading to the repression of transcription by methylation. In contrast to D1/CDK expression, direct inactivation of p53 resulted in no repression of CDC2 and no cell cycle arrest. We conclude that induction of p53 by Nutlin-3 is a viable therapeutic strategy in cancers with constitutive CDK signaling due to the direct repression of specific p53 target genes.