Characterization of Methods for Preservation of Phosphoproteins in fixed tissues

固定组织中磷蛋白保存方法的表征

• 批准号: 7282643
• 负责人: Eric Hsi
• 金额: $ 16.88万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7282643
• 关键词: Accounting; Affect; Antibodies; Area; Autolysis; BCR/ABL1; Biological Assay; Biological Preservation; Biological Process; Biopsy Specimen; Bone Marrow; Bone marrow biopsy; Calibration; Cancer Patient; Categories; Cell Line; Cells; Chronic; Chronic Myeloid Leukemia; Chronic Myeloproliferative Disorder; Clinical; Clinical Trials; Condition; Confocal Microscopy; Data; Detection; Diagnosis; Diagnostic; Disease; ERBB2 gene; Event; Excision; Fixatives; Flow Cytometry; Fluorescent Dyes; Formalin; Gastrointestinal Stromal Tumors; Gene Expression Profiling; Gold; HT29 Cells; Heating; Hematologic Neoplasms; Hen Egg Lysozyme; Human Cell Line; Imatinib; Imatinib mesylate; Immunofluorescence Immunologic; Immunohistochemistry; In Situ; Individual; Investigation; JAK2 gene; K-562; K562 Cells; LY294002; Laboratories; Malignant Neoplasms; Maps; Measurement; Medical; Megakaryocytes; Metals; Methods; Modeling; Molecular; Molecular Abnormality; Monitor; Mus; Mutation; Myeloproliferative disease; Necrosis; Operative Surgical Procedures; Pathogenesis; Pathway interactions; Patients; Performance; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Process; Protein Overexpression; Protein Tyrosine Kinase; Proteins; Protocols documentation; Purpose; Quantum Dots; Relative (related person); Research Personnel; SCID Mice; Serine; Signal Transduction; Solid Neoplasm; Staining method; Stains; Standards of Weights and Measures; System; Techniques; Technology; Testing; Therapeutic; Threonine; Time; Tissue Microarray; Tissues; Trastuzumab; Trephine Biopsy; Tumor Cell Line; Tyrosine; Tyrosine Kinase Inhibitor; Western Blotting; Xenograft Model; Xenograft procedure; Zinc; base; cell fixing; crosslink; day; fusion gene; human tissue; immunoreactivity; in vivo; kinase inhibitor; leukemia/lymphoma; malignant breast neoplasm; method development; neoplastic cell; outcome forecast; phosphatase inhibitor; prognostic; programs; protein function; sample fixation; sarcoma; tissue fixing; tissue processing; tumor

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.

描述(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，它依赖于一种相互竞争的激酶和磷酸酶系统。这是一个动态的过程，在许多疾病状态下都会发生变化。例如，激活的酪氨酸激酶是慢性粒细胞白血病(BCR-ABL1)和胃肠道间质瘤(KIT)发病机制的核心。用原位免疫组织学方法检测固定组织中的磷蛋白(PPS)可能对癌症患者的诊断、预后和治疗有意义。初步研究表明，PPS相当不稳定。关于在组织中保持磷酸化状态的方法知之甚少。本应用的目的是开发适合于检测固定组织中PPS的最佳组织处理方法，以保持在临床环境中的实用限制。为此，我们打算1)建立一种使用量子点的定量免疫荧光(IF)方法来定量固定细胞块中的PP状态；2)表征小鼠人类细胞系异种移植中的最佳固定条件(时间、固定剂、磷酸酶抑制剂的需求)，以此作为可用对照材料的对照模型，并通过定量Western印迹和IF进行检测；以及4)在含BCR-ABL1的小鼠模型中提供甲磺酸伊马替尼(IM)治疗的BCR-ABL1细胞株的原理证据，并从疑似携带JAK2 V617F突变的慢性骨髓增殖性疾病患者的骨髓活检组织中进行检测。已知在这两个系统中，磷酸化STATS都增加了。在IM处理的异种移植中，通过磷酸化STAT5免疫染色减少表达，而在JAC2 V617F+骨髓巨核细胞中增加表达，在处理最好的组织中是可望的。这种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，以充分保存体内PP状态，以便进行后续诊断和预后测试。此外，这一应用将产生具有定义的许多PPS的相对表达水平的对照材料，并允许实验室评估其单独检测的性能。