Induction of β-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1
Induction of β-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1
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DOI：
10.2337/diabetes.53.1.149
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发表时间：
2004-01-01
期刊：
影响因子：
7.7
通讯作者：
Stewart, AF
中科院分区：
文献类型：
--
作者：
Cozar-Castellano, I;Takane, KK;Stewart, AF
差距-1/合成期（G（1）/S）细胞周期检查点的主要调节物是视网膜母细胞瘤蛋白（pRb），其部分受细胞周期蛋白依赖性激酶（cdk）-4和D细胞周期蛋白的活性调节。令人惊讶的是，考虑到β细胞复制对胰岛替代疗法的潜在重要性，尚未在β细胞中探索pRb的存在、磷酸化状态和功能。在这里，腺病毒表达cdk-4和细胞周期蛋白D，用于探索大鼠和人类pRb磷酸化和β细胞周期控制。pRb存在于大鼠和人类胰岛中，并且细胞周期蛋白D-1/cdk-4的过表达导致两种物种中pRb磷酸化显著增强。联合过表达cdk-4和细胞周期蛋白D-1导致[H-3]胸苷掺入增加三倍。使用胰岛素和溴脱氧尿苷免疫组织化学独立证实了这种增殖的增加，其中人β细胞复制率增加了10倍。Cdk-4或细胞周期蛋白D-1过表达对β细胞分化或功能没有不利影响。关键的细胞周期调节蛋白pRb可以利用细胞周期蛋白D-1/cdk-4来诱导人类和啮齿动物β细胞复制，增强复制而不会对功能或分化产生不利影响。这种方法将允许对β细胞、β细胞系和干细胞衍生的β细胞中调节细胞周期的细胞机制进行详细的分子研究。
The major regulator of the gap-1/synthesis phase (G(1)/S) cell cycle checkpoint is the retinoblastoma protein (pRb), and this is regulated in part by the activities of cyclin-dependent kinase (cdk)-4 and the D cyclins. Surprisingly, given the potential importance of beta-cell replication for islet replacement therapy, pRb presence, phosphorylation status, and function have not been explored in beta-cells. Here, adenoviruses expressing cdk-4 and cyclin D, were used to explore rat and human pRb phosphorylation and beta-cell cycle control. pRb is present in rat and human islets, and overexpression of cyclin D-1/cdk-4 led to strikingly enhanced pRb phosphorylation in both species. Combined overexpression of both cdk-4 and cyclin D-1 caused a threefold increase in [H-3]thymidine incorporation. This increase in proliferation was confirmed independently using insulin and bromodeoxyuridine immunohistochemistry, where human beta-cell replication rates were increased 10-fold. Cdk-4 or cyclin D-1 overexpression did not adversely effect beta-cell differentiation or function. The key cell cycle regulatory protein, pRb, can be harnessed to advantage using cyclin D-1/cdk-4 for the induction of human and rodent beta-cell replication, enhancing replication without adversely affecting function or differentiation. This approach will allow detailed molecular study of the cellular mechanisms regulating the cell cycle in beta-cells, beta-cell lines, and stem cell-derived beta-cells.