BIOLOGICAL Function of the Retinoblastoma Gene Product

我们研究的长期目标是了解视网膜母细胞瘤肿瘤抑制蛋白（RB）的生物学功能。在目前的资助期间，我们已经获得了支持RB作为细胞增殖和细胞死亡抑制剂的结果。RB的这种双重作用允许与长期存活偶联的终末分化，例如，肌肉和神经元。基因突变、病毒癌蛋白或磷酸化导致RB失活与肿瘤发生有关。我们推断，RB的失活必须由细胞凋亡缺陷来补充，从而导致肿瘤的发展。拟议的研究旨在确定由RB调节的遗传程序，以建立终末生长停滞和抗凋亡。我们感兴趣的假设，RB可能会调节一个单一的“转录组”，以阻止分化细胞的促有丝分裂和凋亡刺激。拟议的研究是基于我们创建的两个有趣的RB突变体。点突变体N757 F可以抑制S期进入，但不能在肌肉分化期间建立抗有丝分裂原的生长停滞。点突变MI在凋亡过程中对蛋白水解降解具有抗性，并且可以保护细胞免受肿瘤坏死因子（TNF）诱导的死亡。我们已经在小鼠Rb基因中创建了胚系MI突变，并发现这些Rb-MI小鼠对败血性休克具有抗性。我们拟建立一个Rb-N757 F小鼠模型，以进一步研究RB在终末分化中的作用。我们将使用蛋白质组学方法来鉴定RB-N757 F的蛋白质结合缺陷。我们将使用基因组学方法来鉴定在表达RB或RB-N757 F的肌细胞中差异表达的基因。我们还将鉴定在TNF刺激条件下RB与RB-MI细胞中差异表达的基因。通过比较这两个独立衍生的基因集，我们将能够确定RB是否调节共同的转录组以使细胞对促有丝分裂和凋亡信号产生抗性。RB调控的遗传程序的鉴定是理解RB在分化和凋亡中的功能的基础。这些遗传学程序可能会在肿瘤发展过程中受到影响。因此，拟议的研究将导致确定癌症治疗干预的重要靶点。

The long-range goal of our research is to understand the biological function of the retinoblastoma tumor suppressor protein (RB). During the current funding period, we have obtained results to support the idea that RB functions as an inhibitor of cell proliferation and cell death. This dual role of RB allows for terminal differentiation that is coupled to long-term survival, e.g., of muscles and neurons. Inactivation of RB by genetic mutation, by viral oncoproteins, or by phosphorylation, is associated with tumor development. The inactivation of RB, we reason, must be complemented by defects in apoptosis to cause tumor development. The proposed research is designed to identify genetic programs that are regulated by RB to establish terminal growth arrest and the resistance to apoptosis. We are interested in the hypothesis that RB may regulate a single "transcriptome" to block mitogenic and apoptotic stimulation of differentiated cells. The proposed research is based on two interesting RB mutants we have created. The point- mutant N757F can inhibit S-phase entry, but cannot establish mitogen- resistant growth arrest during muscle differentiation. The point-mutant MI is resistant to proteolytic degradation during apoptosis and can protect cells from tumor necrosis factor (TNF)-induced death. We have created a germline MI mutation in the mouse Rb gene and found these Rb-MI mice to be resistant to septic shock. We propose to create a Rb- N757F mice as a model to further study the function of RB in terminal differentiation. We will use proteomics methods to identify the protein- binding defects of RB-N757F. We will use genomics methods to identify genes that are differentially expressed in myocytes that express RB or RB-N757F. We will also identify genes that are differentially expressed in RB versus RB-MI cells under conditions of TNF stimulation. By comparing these two independently derived gene sets, we will be able to determine if RB regulates a common transcriptome to render cells resistant to mitogenic and apoptotic signals. Identification of RB-regulated genetic programs is fundamental to the understanding of RB function in differentiation and apoptosis. These genetics programs are likely to be affected during tumor development. Therefore, the proposed research will result in the identification of important targets for therapeutic intervention in cancer treatment.