Division of Nephrology, Department of Internal Medicine and Cancer Center, University of California-Davis, Davis, California 95616, USA.
J Urol. 2010 Nov;184(5):2143-9. doi: 10.1016/j.juro.2010.06.088. Epub 2010 Sep 20.
We evaluated the effect of roscovitine (Sigma-Aldrich®), a pharmacological inhibitor of cyclin dependent kinase, on renal cell carcinoma cell lines in vitro.
We exposed several renal cell carcinoma cell lines to roscovitine and examined apoptotic signaling pathways using immunoblotting and immunohistochemistry.
As expected, roscovitine caused dose and time dependent inhibition of cyclin dependent kinase 2 autophosphorylation, and of cyclin dependent kinase mediated Pol II phosphorylation in the ACHN (p53-wt) and 786-O (p53 inactive) renal cell carcinoma cell lines (ATCC®). Roscovitine also induced apoptosis in each cell line within a narrow concentration range (about 10 μg/ml). Apoptosis induction was more efficient in ACHN than in 786-O cells and at least partly due to p53 activity. In ACHN cells roscovitine induced apoptosis was associated with p21 induction, and decreased Akt1, XIAP and phospho-Rb expression. These changes also depended on p53 and were not present (p21) or showed a different dose pattern (Akt1, XIAP and phospho-Rb) in 786-O cells. Partial restoration of roscovitine induced apoptosis in 786-O cells by the Mdm-2 inhibitor nutlin-3 (Sigma-Aldrich) suggests that the inactivating mutation of VHL in these cells and its destabilizing effect on p53 are responsible for the decreased sensitivity to apoptosis.
Our data extend previous studies documenting the pro-apoptotic effect of roscovitine and to our knowledge show for the first time that this activity is restricted to a narrow dose range in renal cell carcinoma cells and partly depends on p53. Thus, roscovitine is a novel potential chemotherapy in a subset of patients with renal cell carcinoma if a narrow therapeutic window is used. These data also provide insight into the role of VHL mutation and p53 in the renal cell carcinoma response to therapeutic cyclin dependent kinase manipulation.
我们评估了细胞周期蛋白依赖性激酶的药理学抑制剂罗司维亭（Sigma-Aldrich®）对体外肾癌细胞系的影响。
我们将几种肾癌细胞系暴露于罗司维亭中，并使用免疫印迹和免疫组织化学检查凋亡信号通路。
正如预期的那样，罗司维亭导致 ACHN（p53-wt）和 786-O（p53 失活）肾癌细胞系（ATCC®）中环依赖性激酶 2 自身磷酸化以及环依赖性激酶介导的 Pol II 磷酸化的剂量和时间依赖性抑制。罗司维亭也在每个细胞系的狭窄浓度范围内诱导凋亡（约 10 μg/ml）。在 ACHN 细胞中，凋亡诱导比在 786-O 细胞中更有效，至少部分归因于 p53 活性。在 ACHN 细胞中，罗司维亭诱导的凋亡与 p21 的诱导有关，并降低 Akt1、XIAP 和磷酸化-Rb 的表达。这些变化也依赖于 p53，并且在 786-O 细胞中不存在（p21）或显示不同的剂量模式（Akt1、XIAP 和磷酸化-Rb）。在 786-O 细胞中，通过 Mdm-2 抑制剂 nutlin-3（Sigma-Aldrich）部分恢复罗司维亭诱导的凋亡表明，这些细胞中 VHL 的失活突变及其对 p53 的稳定作用是导致对凋亡敏感性降低的原因。
我们的数据扩展了先前记录罗司维亭促凋亡作用的研究，并据我们所知，首次表明这种活性仅限于肾癌细胞系的狭窄剂量范围，部分取决于 p53。因此，如果使用狭窄的治疗窗口，罗司维亭是肾细胞癌患者的一种新的潜在化疗药物。这些数据还为 VHL 突变和 p53 在肾细胞癌对治疗性细胞周期蛋白依赖性激酶作用的反应中的作用提供了深入了解。
