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内吞转运来鉴定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个阶段。假设：生长因子撤除后PKCd的诱导增加了营养转运蛋白的内吞作用，减少了再循环，并增加了泛素化。在存在或不存在生长因子的情况下，使用营养转运蛋白特异性抗体通过流式细胞术测量营养转运蛋白内化和再循环的速率。PKCd的作用将使用表达shRNA（显性阴性）和PKCd的化学抑制剂（rottlerin）的细胞来评估。将通过转运蛋白的免疫沉淀，然后通过蛋白质印迹法评估泛素化。具体目标2：确定负责生长因子诱导的营养转运蛋白下调的PKCd底物。假设：生长因子撤除后PKCd诱导导致负责营养转运蛋白下调的新型PKCd底物磷酸化。将通过以下方法分析Scansite鉴定为潜在PKCd底物的候选底物的PKCd磷酸化：1）正磷酸盐标记，2）体外激酶试验和3）2-D电泳。目标1的结果将有助于确定我们将关注哪些底物。我们还将使用生物化学方法来鉴定新的PKCd底物、支架蛋白和结合伴侣。这些研究旨在确定新的化疗靶点，这些靶点可用于治疗多种类型的癌症。我们控制癌症的方法是新颖的：通过了解癌细胞如何生长，而正常细胞如何失去营养，我们可以学习如何通过饥饿杀死癌细胞。阻断个别途径可能会杀死癌细胞，同时影响具有营养摄取备用途径的正常细胞。许多类型的癌症应该对这种类型的治疗敏感，因为所有癌细胞都需要营养才能生存。

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.