Minority Predoctoral Fellowship Program

描述(由申请人提供)：该项目的长期目标是确定癌细胞变得不依赖生长因子的机制。我们希望通过调节营养转运蛋白的内化和胞内转运来识别作为肿瘤抑制因子或癌蛋白的新蛋白。调节这一过程的蛋白质可能是癌症化疗的潜在靶点。具体目标1：确定PKCd诱导影响营养转运蛋白内化和运输的哪些阶段。假设：生长因子停用后PKCd的诱导增加了营养转运体的内吞作用，减少了循环，并增加了泛素化。在存在或不存在生长因子的情况下，营养转运体内化和再循环的速率将使用针对营养转运体的抗体通过流式细胞术进行测量。PKCd的作用将通过表达shRNA的细胞来评估，shRNA是PKCd的显性阴性，也是PKCd的化学抑制物(Rotlerin)。泛素化将通过转运蛋白的免疫沉淀和泛素的Western印迹进行评估。具体目标2：确定与生长因子诱导的营养转运蛋白下调有关的PKCd底物。假设：生长因子停用后PKCd的诱导导致一种新的PKCd底物的磷酸化，该底物负责营养转运蛋白的下调。被Scansite确定为潜在的PKCd底物的候选底物将通过以下几种方法进行分析：1)正磷酸标记，2)体外激酶测定和3)二维电泳法。目标1的结果将有助于确定我们将专注于哪些底物。我们还将使用生化方法来鉴定新的PKCd底物、支架蛋白和结合伙伴。这些研究试图确定新的化疗靶点，这些靶点将对治疗多种类型的癌症有用。我们控制癌症的方法是新颖的：通过了解癌细胞是如何生长的，而正常细胞却因为营养物质而失去了生命，我们可能会学习如何通过饿死癌细胞来杀死癌细胞。阻断单独的途径可能会杀死癌细胞，同时影响有营养吸收后备途径的正常细胞。许多类型的癌症应该对这种治疗敏感，因为所有的癌细胞都需要营养才能存活。

DESCRIPTION (provided by applicant): The long term goals of this project are to identify the mechanisms by which cancer cells become growth factor-independent. We hope to identify novel proteins that act as tumor suppressors or oncoproteins by regulating nutrient transporter internalization and endocytic trafficking. Proteins that regulate this process may represent potential targets for cancer chemotherapy. Specific Aim 1: Determine which stages of nutrient transporter internalization and trafficking are affected by PKCd induction. Hypothesis: PKCd induction following growth factor withdrawal increases nutrient transporter endocytosis, decreases recycling, and increases ubiquitination. Rates of nutrient transporter internalization and recycling will be measured in the presence or absence of growth factor using antibodies specific for nutrient transporters by flow cytometry. The role of PKCd will be assessed using cells expressing shRNA, a dominant negative, and a chemical inhibitor of PKCd (rottlerin). Ubiquitination will be assessed by immunoprecipitation of the transporters followed by Western blot for ubiquitin. Specific Aim 2: Identify the PKCd substrates responsible for growth factor induced nutrient transporter down-regulation. Hypothesis: PKCd induction following growth factor withdrawal leads to phosphorylation of a novel PKCd substrate responsible for nutrient transporter down-regulation. Candidate substrates identified by Scansite as potential PKCd substrates will be analyzed for PKCd phosphorylation by: 1) orthophosphate labeling, 2) in vitro kinase assays and 3) 2-D electrophoresis. Results from Aim 1 will help to determine which substrates we will focus on. We will also use a biochemical approach to identify novel PKCd substrates, scaffolding proteins and binding partners. These studies seek to identify new chemotherapeutic targets that would be useful in treating numerous types of cancer. Our approach to cancer control is novel: by understanding how cancer cells grow while normal cells lose asses to nutrients we may learn how to kill cancer cell by starving them to death. Blocking individual pathways may kill cancer cells while affecting normal cells that have back-up pathways for nutrient uptake. Many types of cancer should be sensitive to this type of treatment because all cancer cells need nutrients to survive.