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成为生长因子独立的机制。我们希望通过调节营养转运蛋白的内化和内吞运输来鉴定作为肿瘤抑制蛋白或癌蛋白的新蛋白。调节这一过程的蛋白质可能代表癌症化疗的潜在靶点。具体目标1：确定PKCd诱导影响营养转运体内化和运输的哪个阶段。假设：生长因子停用后PKCd诱导增加营养转运体内吞作用，减少循环，增加泛素化。在存在或不存在生长因子的情况下，营养转运蛋白内化和再循环的速率将通过流式细胞术使用针对营养转运蛋白的抗体来测量。PKCd的作用将通过表达shRNA（显性阴性）和PKCd的化学抑制剂（rotlerin）的细胞来评估。泛素化将通过转运蛋白的免疫沉淀和泛素蛋白的免疫印迹来评估。特异性目标2：确定PKCd底物负责生长因子诱导的营养转运蛋白下调。假设：生长因子退出后PKCd诱导导致一种新的PKCd底物磷酸化，负责营养转运蛋白下调。Scansite确定的候选底物作为潜在的PKCd底物，将通过以下方法分析PKCd磷酸化：1)正磷酸盐标记，2)体外激酶测定和3)二维电泳。Aim 1的结果将有助于确定我们将重点关注哪些底物。我们还将使用生化方法鉴定新的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，这将有助于治疗多种类型的癌症。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，而正常细胞失去了大量的营养，我们可以学习如何通过饿死癌细胞来杀死它们。阻断单个通路可能会杀死癌细胞，同时影响具有营养摄取备用通路的正常细胞。许多类型的癌症对这种治疗应该是敏感的，因为所有的癌细胞都需要营养来生存。

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.