Characterization of Methods for Preservation of Phosphoproteins in fixed tissues

描述（由申请人提供）：蛋白质磷酸化是调节蛋白质功能和活性的重要机制，依赖于激酶和磷酸酶的竞争系统。这是一个动态过程，在许多疾病状态下发生改变。例如，激活的酪氨酸激酶是慢性髓性白血病（BCR-ABL1）和胃肠道间质瘤（KIT）发病机制的核心。通过原位免疫组织学方法检测固定组织中的磷蛋白（PPs）可能对癌症患者具有诊断、预后和治疗意义。初步研究表明，PPs相当不稳定。关于保持组织中磷酸化状态的方法知之甚少。本应用程序的目的是开发最佳的组织处理方法，该方法将适用于检测固定组织中的PPs，同时在临床环境中保持中等实际限制。为此，我们打算1)开发一种定量免疫荧光（IF）方法，使用量子点来定量固定细胞块中的PP状态；2)描述人类细胞系小鼠异种移植物的最佳固定条件（时间、固定剂、磷酸酶抑制剂的要求），作为可用对照材料的对照模型，通过定量Western blot和IF进行检测；4)在含有BCR-ABL1的细胞系异种移植物的小鼠模型中，甲甲酸伊马替尼（imatinib mesylate， IM）和疑似携带JAK2 V617F突变的慢性骨髓增生性疾病患者的骨髓活检中证明了其原理。已知在这两种系统中磷酸化- stats都增加。在经过最佳处理的组织中，预计在经im处理的异种移植物中，磷酸化stat5免疫染色表达降低，而在JAC2 V617F+骨髓巨核细胞中表达增加。这一应用在恶性肿瘤和其他疾病的诊断、预后和治疗中具有相关性，这些疾病改变了PP水平作为其致病途径的一部分。它将定义组织处理条件，以充分保留体内PP状态，用于随后的诊断和预后测试。此外，该应用程序将产生具有确定的许多PPs相对表达水平的对照材料，并允许实验室评估其单个分析的性能。

DESCRIPTION (provided by applicant): Protein phosphorylation is an important mechanism of regulating protein function and activity that depends on a competing system of kinases and phosphatases. It is a dynamic processes that is altered in many disease states. For example, activated tyrosine kinases are central to the pathogenesis of chronic myelogenous leukemia (BCR-ABL1) and gastrointestinal stromal tumors(KIT). Detection of phosphoproteins (PPs) in fixed tissues by in situ immunohistologic methods may have diagnostic, prognostic, and therapeutic implications for cancer patients. Initial studies have shown that PPs are quite labile. Little is known regarding methods to preserve phosphorylation status in tissues. The purpose of this application is to develop optimal tissue handling methods that will be suitable for detection of PPs in fixed tissues, keeping in mid practical limitations in the clinical setting. To this end we intend to 1) develop a quantitative immunofluorescence (IF) method using quantum dots to quantitate PP status in fixed cell blocks; 2) characterize optimal fixation conditions (time, fixative, requirement of phosphatase inhibitors) in murine xenografts of human cell lines as a controlled model of available control material that is assayed both by quantitative Western blot and IF; and 4) show proof of principle of in a murine model of BCR-ABL1 containing cell line xenograft treated with imatinib mesylate (IM) and bone marrow biopsies from patients suspected of chronic myeloproliferative disorder harboring the JAK2 V617F mutation. Phospho-STATS is known to be increased in both these systems. Decreased expression by phospho-STAT5 immunostaining in IM-treated xenografts and increased expression in JAC2 V617F+ bone marrow megakarycotyes is expected in optimally handled tissues. This application has relevance in the diagnosis, prognosis, and therapy of malignancies and other diseases that have altered PP levels as part of their pathogenic pathways. It will define tissue handling conditions that adequately preserve in vivo PP status for subsequent diagnostic and prognostic testing. Furthermore, control material with defined relative expression levels of many PPs will result from this application and allow laboratories to assess performance of their individual assays.