Imaging Rho-C Oncogene Function in Human Mammary Cells

人乳腺细胞中 Rho-C 癌基因功能的成像

• 批准号: 6860900
• 负责人: Mary-Ann Mycek
• 金额: $ 15.27万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6860900
• 关键词: bioimaging /biomedical imaging; biomarker; breast neoplasm /cancer diagnosis; breast neoplasms; diagnosis design /evaluation; diagnostic tests; enzyme activity; enzyme inhibitors; fluorescence resonance energy transfer; genetic markers; guanine nucleotide binding protein; guanosinetriphosphatases; imaging /visualization /scanning; mammary epithelium; molecular /cellular imaging; molecular oncology; neoplastic cell; oncogenes; protein localization; tissue /cell culture

DESCRIPTION (provided by applicant): The ability to rapidly characterize molecular function and/or activation of oncogenes in living cells would be a fundamental advance in the diagnosis of cancer, the planning of molecular-based therapies, and in cancer prevention. Inflammatory breast cancer (IBC) is an aggressive form of locally advanced breast cancer that is highly angiogenic, invasive, and metastatic. RhoC-GTPase has been identified in the Merajver lab as a specific marker of aggressive breast cancer phenotypes and of IBC in particular. RhoC behaves as a transforming oncogene of human mammary epithelial cells whose overexpression can lead to a highly invasive, angiogenic, and metastatic phenotype, extremely akin to IBC. The biophysical mechanisms for activation and inhibition of this oncogene (including detailed molecular associations and cellular localization) are not completely understood; however, these features impact on function and biological activity in tumors. We propose in this R21 application to investigate RhoC-GTPase localization, activation, and inhibition in living human cellular models of IBC using intensity and lifetime fluorescence resonance energy transfer (FRET) technology and methods developed in the Mycek lab for cellular and molecular imaging. FRET measurements detect molecular localization and binding with nanoscale spatial sensitivity. The use of fluorescence lifetime sensing offers an additional source of contrast for performing quantitative FRET measurements in living cells, while being generally independent of artifacts influencing fluorescence intensity (including fluorophore concentration, photobleaching, and sources of optical loss (absorption and scattering) in biological systems) that may complicate the interpretation of intensity measurements. Specific Aim 1: Characterize RhoC-GTPase oncogene localization in living normal mammary epithelial cells and in RhoC-driven malignant cells. Specific Aim 2: Determine RhoC activation states with and without binding to the key effector, rhotekin. Specific Aim 3: Define the changes in FRET and RhoC localization caused by known inhibitors of RhoC-GTPase activation: C3 exotransferase and a farnesyl transferase inhibitor (FTI 832). The molecular imaging methods developed in this R21 proposal are cross-cutting in nature in that they are broadly applicable to basic biological studies of cellular processes and structures in living cells obtained from patients, and would find rapid translation to the clinic.

描述（由申请人提供）：在活细胞中迅速表征分子功能和/或激活癌基因的能力将是癌症诊断，基于分子疗法的计划和预防癌症的基本进步。炎症性乳腺癌（IBC）是一种高度血管生成，侵入性和转移性的局部晚期乳腺癌的侵略性形式。 RHOC-GTPase已在Merajver Lab中被鉴定为侵略性乳腺癌表型的特定标记，尤其是IBC的特定标记。 RHOC表现为人类乳腺上皮细胞的转化癌基因，其过表达可以导致高度侵入性，血管生成和转移性表型，非常类似于IBC。尚不完全了解这种致癌基因（包括详细的分子关联和细胞定位）的生物物理机制。但是，这些特征会影响肿瘤的功能和生物活性。我们在此R21应用中建议使用强度和寿命荧光共振能量转移（FRET）技术以及在MyCEK Lab中开发的用于细胞和分子成像的MyCEK LAB中开发的RHOC-GTPase定位，激活和抑制IBC的人类细胞模型。 FRET测量检测分子定位并与纳米级空间敏感性结合。荧光寿命传感的使用提供了对活细胞进行定量特征测量的附加对比来源，同时通常独立于影响荧光强度（包括荧光团浓度，光漂白和光泽和散射的生物学系统）的伪像，这可能会使强度测量的解释复杂化。具体目标1：表征RHOC-GTPase癌基因在活的正常乳腺上皮细胞和RHOC驱动的恶性细胞中的定位。特定目标2：确定有或没有与关键效应子Rhotekin结合的RHOC激活状态。特定目标3：定义由已知的RHOC-GTPase激活抑制剂引起的FRET和RHOC定位的变化：C3外促转移酶和Farnesyl转移酶抑制剂（FTI 832）。在本R21提案中开发的分子成像方法本质上是横切的，因为它们广泛适用于从患者获得的活细胞中细胞过程和结构的基本生物学研究，并且会发现快速转化为诊所。