The Role of Rb phosphorylation in proliferation and apoptosis of breast cancer ce

描述(申请人提供)：控制视网膜母细胞瘤(RB)磷酸化的分子被称为“RB途径”，包括D型细胞周期蛋白、细胞周期蛋白依赖性蛋白激酶(CDK)和细胞周期蛋白依赖性蛋白激酶抑制因子(CDKI)，以及RB本身在大多数癌症类型中有规律地被破坏 包括导致Rb蛋白过度磷酸化(过度磷酸化)的乳腺癌。众所周知，Rb的过度磷酸化可以促进细胞增殖和抑制细胞凋亡。抑制cdk活性的治疗方法已经开始在几种癌症类型的临床上显示出希望。虽然Rb可以在15个氨基酸残基上被磷酸化，但对于任何一个单独的磷酸化氨基酸对Rb在增殖和凋亡中的作用知之甚少。在这个项目中，我们建议利用三维培养的乳腺上皮细胞模型进行Rb在增殖和凋亡中的磷酸化的第一次研究。这个模型系统被用来概括乳腺上皮细胞调节细胞过程的生理背景。我们建议使用一种新的方法来激活在3D培养中生长的乳腺上皮细胞中Rb特异的磷酸酶活性，从而靶向Rb的过度磷酸化。通过这种方式，我们可以确定Rb的去磷酸化对这些细胞的增殖和凋亡的影响。此外，我们将对每个Rb磷酸化位点进行定点突变，以阻断每个位点的磷酸化或模拟磷酸化，并评估其对3D培养模型中生长的乳腺上皮细胞增殖和凋亡的影响。最后，我们将阐明Rb与受Rb去磷酸化调控的促凋亡蛋白Bak形成复合体在细胞凋亡中的功能意义。因此，拟议的研究将阐明特定的Rb磷酸化位点在乳腺癌增殖和凋亡调控中的作用，并将产生有用的信息，为未来针对癌症Rb磷酸化的治疗提供信息。

DESCRIPTION (provided by applicant): The molecules that control Retinoblastoma (Rb) phosphorylation called "the Rb pathway" consisting of D-type cyclins, cyclin dependent kinases (cdks) and cdk inhibitors (cdkis) as well as Rb itself are regularly disrupted in most cancer types including breast cancer leading to excessive phosphorylation (hyperphosphorylation) of the Rb protein. Hyperphosphorylation of Rb is known to promote proliferation and block apoptosis. Treatments that inhibit cdk activity have begun to exhibit promise in the clinic in several cancer types. Although Rb can be phosphorylated on 15 amino acid residues, very little is known about the contribution any individual phosphorylated amino acid has on the function of Rb in proliferation and apoptosis. In this project we propose to undertake the first investigation of Rb phosphorylation in proliferation and apoptosis utilizing a model of breast epithelial cells using three-dimensional cultures. This model system is employed to recapitulate the physiological context in which breast epithelial cells regulate cellular processes. We propose to target Rb hyper- phosphorylation by using a novel method of activating Rb-specific phosphatase activity in breast epithelial cells grown in 3D cultures. In this way we can determine the effect of dephosphorylation of Rb on proliferation and apoptosis in these cells. In addition, we will perform site directed mutagenesis of each Rb phosphorylation site to either alanine or glutamic acid to block phosphorylation or mimic phosphorylation at each site and evaluate the effects on proliferation and apoptosis in breast epithelial cells grown in the 3D culture model. Finally, we will clarify the functional significance of complex formation between Rb and the pro-apoptotic protein Bak that is regulated by Rb dephosphorylation in apoptosis. Thus, the proposed studies will elucidate the role of specific Rb phosphorylation sites involved in the regulation of proliferation and apoptosis in breast cancer and will yield useful information that could inform the development of future therapies that target Rb phosphorylation in cancer.