How do Tumor Cells Gain Anchorage Independency?

摘要 DNA肿瘤病毒的转化蛋白与内源性相互作用 在细胞生长调节中发挥广泛作用的细胞因子。这个 内源性细胞周期调节蛋白的最佳表征示例 与DNA病毒相互作用的转化蛋白是pRb。然而，还有其他的 举个例子。一种非常高分子量(600 KDa)的细胞蛋白(以下简称 P600)形成紧密的蛋白质：与E7转化的蛋白质复合体 人和牛乳头瘤病毒(HPV)的因子。在初步研究中，我的 合作者和我已经证明了P600在锚定中起着普遍的作用 多种肿瘤细胞类型的独立生长和恶变。这个 这里提出的工作的总体目标是了解锚地依赖 和通过纯化和酶特性鉴定的恶性转化 在E7可置换的P600结合蛋白中。我有两个明确的目标： 目标1是确定E7依赖的P600结合的细胞因子 时尚。P600蛋白太大，不能进行基因操作或异位 使用常规方法进行表达。在初步研究中，我使用了 “敲入”策略创造表达串联亲和力表位标记的ES细胞 P600。在这些细胞的抗体下拉实验中，我有几个P600- 结合被HPV E7取代的细胞蛋白。目标1的学习计划 描述我将如何使用以下工具提纯和识别这些E7可替换因素 生化方案和质谱学。 目标2是了解E7和E7可置换的细胞蛋白如何 调节P600的转化功能。在初步研究中，我已经证明 P600具有泛素连接酶活性。我推测Ubuquitin连接酶的活性 Of P600受E7和E7-置换细胞因子相互作用的调节。 为了验证这一假设，我将描述P600和P600的泛素连接酶结构域。 鉴定E7特异性泛素连接酶底物。 在针对这两个具体目标的概念验证研究中，我已经 鉴定出一种与P600结合、E7可置换的蛋白为PKM2-a蛋白激酶 最近已经证明，这在肿瘤的合成代谢生长中发挥着广泛的作用。我相信，这些实验将为我们提供对 恶性表型的分子机制包括失巢失巢和 依靠锚地的增长。到最后，这项工作可能会导致 抗癌药物开发的新靶点。

Abstract The transforming proteins of DNA tumor viruses interact with endogenous cellular factors that can play broad roles in the regulation of cell growth. The best-characterized example of an endogenous cell cycle regulator protein that interacts with a DNA virus transforming protein is pRB. However, there are other examples. A very high molecular weight (600 kDa) cellular protein (hereafter termed "p600") forms a tight protein:protein complex with the E7 transforming factor of human and bovine papillomaviruses (HPV). In preliminary studies, my collaborators and I have shown that p600 plays a generalized role in anchorage independent growth and malignant transformation of many tumor cell types. The broad objective of work proposed here is to understand anchorage dependency and malignant transformation through purification and enzymatic characterization of E7-displacible, p600 binding proteins. I have two specific aims: Aim 1 is to identify cellular factors that bind to p600 in an E7-dependent fashion. The p600 protein is too large for genetic manipulation or ectopic expression using conventional approaches. In preliminary studies, I have used a "knock in" strategy to create ES cells that express tandem affinity epitope-tagged p600. In antibody pull down experiments with these cells, I have several p600- binding cellular proteins that are displaced by HPV E7. The study plan for aim 1 describes how I will purify and identify these E7-displacible factors using biochemical protocols and mass spectroscopy. Aim 2 is to understand how E7 and E7-displacible cellular proteins might regulate p600 transforming functions. In preliminary studies, I have shown that p600 has ubiquitin ligase activity. I hypothesize that the ubuquitin ligase activity of p600 is regulated by interactions with E7 and E7-displacibile cellular factors. To test this hypothesis, I will characterize the ubiquitin ligase domain of p600 and identify E7-specific ubiquitin ligase substrates. In proof-of-concept studies for these two specific aims, I have already identified one p600-binding, E7-displacible protein as PKM2 - a protein kinase that has recently been shown to play a broad role in anabolic growth of tumors. I believe that these experiments will provide new insights into the molecular mechanisms of the malignant phenotype including anoikis and anchorage-dependent growth. In the fullness of time, the work could lead to novel new targets for anti cancer drug development.