In Vivo Bioimaging Model to Study Inducible Drug Resistance in Cancer

描述（由申请人提供）：转录调控是调控基因表达的主要机制，但我们对基因在整个机体中的调控机制知之甚少，这主要是由于我们无法实时观察和测量真实生理条件下基因表达的变化。由于类似的原因，如果在转录水平上介导，则不可能直接研究给定的癌症治疗对其靶点的影响。然而，最近的分子成像革命已经产生了新的工具来执行无创的，在体内的基因表达成像。成像技术的生物学相关应用是通过同源重组将报告基因定位到内源性基因的基因组位点，从而可以对该基因的调节表达进行非侵入性的实时监测，并对内部和外部信号作出反应。我们已经建立了这样一个系统，使用小鼠mdr1a位点作为原理的证明，并作为一个重要的生物学基因。多药耐药（MDR）仍然是癌症患者治疗性化疗的一个严重障碍。MDR的机制之一是MDR1基因的表达增强。在许多人类癌症中，MDR1过表达与耐药有关，但其对临床结果的影响仍未得到解决。确定MDR1在耐药性中的作用的系统纵向研究即使不是不可能，也很难在人类中进行，而且以前还没有适当的动物模型来研究这些问题。MDR1在药物代谢动力学中的作用已被证实，但MDR1在参与药物摄取和清除的正常器官中的调节作用尚不清楚。在我们的初步研究中：1)我们设计了mdr1a+/fLUC小鼠，使萤火虫荧光素酶（fLUC）靶向mdr1a基因的基因组位点，使fLUC在帧内的表达以cre介导的重组为条件。2)我们已经证明，内源性mdr1a基因位点控制下的fLUC表达是mdr1a基本稳定状态下忠实的体内报告基因。3)我们也证明了fLUC可以用来监测mdr1a基因在外源刺激下的表达诱导。我们现在建议使用这种非侵入性模型系统来研究mdr1a基因在体内的表达。我们还将扩展该模型，以便能够在转录、转录后和翻译水平上研究控制mdr1a基因表达的顺式作用和反式作用因子。目的1是确定mdr1a是否在正常器官中以组织特异性方式诱导。目的2是确定mdr1a是否在乳腺癌进展和/或治疗期间被诱导。目的3是确定在特定组织中诱导mdr1a是否需要特定的反式作用和顺式作用因子。公共卫生相关性：多药耐药仍然是癌症患者治疗性化疗的严重障碍。多药耐药的机制之一是mdr1基因的表达增强。在许多人类癌症中，Mdr1过表达与耐药有关，但其对临床耐药的贡献仍未得到解决。我们的工作使得研究mdr1的原位、实时调控以及对发育、生理或环境信号的响应成为可能。项目结果将为mdr1在癌症进展和药物治疗过程中被激活的机制提供更深入的见解，从而可能导致避免人类发生此类事件的新方法。

DESCRIPTION (provided by applicant): Transcriptional regulation represents the major mechanism of controlling gene expression, yet we have little direct knowledge of how genes are regulated in the whole body, due largely to an inability to observe and measure changes in gene expression under real physiological conditions and in real time. For similar reasons, it has not been possible to study directly the effects of given cancer therapies on their target(s), if mediated at the transcriptional level. However, the recent revolution in molecular imaging has yielded novel tools for performing noninvasive, in vivo imaging of gene expression. A biologically relevant application of imaging technology is to target, by homologous recombination, a reporter gene into the genomic locus of an endogenous gene so that the regulated expression of that gene can be monitored non-invasively, in real time, and in response to internal and external signals. We have established such a system using the mouse mdr1a locus as a proof of principle and as a biologically important gene. Multidrug resistance (MDR) remains a serious impediment to curative chemotherapy in cancer patients. One mechanism of MDR is the enhanced expression of the MDR1 gene. MDR1 overexpression has been associated with drug resistance in many human cancers, but its contribution to clinical outcomes remains unresolved. Systematic longitudinal studies to determine MDR1's role in resistance are difficult, if not impossible, to perform in humans and an adequate animal model to study such questions has not previously been available. The role of MDR1 in drug pharmacokinetics is well established, but the regulation of MDR1 in normal organs involved in drug uptake and clearance is poorly understood. In our preliminary studies: 1) We have engineered mdr1a+/fLUC mice that have firefly luciferase (fLUC) targeted to the mdr1a gene's genomic locus in a way that makes in-frame expression of fLUC conditional on Cre-mediated recombination. 2) We have shown that expression of fLUC under the control of the endogenous mdr1a gene locus is a faithful in vivo reporter for mdr1a expression in the basal, steady state. 3) We have also demonstrated that fLUC can be used to monitor induction of mdr1a gene expression in response to xenobiotic stimuli. We now propose to use this non-invasive model system to study mdr1a gene expression in the in vivo setting. We will also extend the model to be able to study both the cis-acting and trans-acting factors that control mdr1a gene expression at the transcriptional, post-transcriptional and translational level. Aim 1 is to determine if mdr1a is induced in normal organs in a tissue-specific fashion. Aim 2 is to determine if mdr1a is induced during breast cancer progression and/or treatment. Aim 3 is to determine if specifi trans-acting and cis-acting factors are required for mdr1a induction in specific tissues. PUBLIC HEALTH RELEVANCE: Multidrug resistance remains a serious impediment to curative chemotherapy in cancer patients. One mechanism of multidrug resistance is the enhanced expression of the mdr1 gene. Mdr1 overexpression has been associated with drug resistance in many human cancers, but its contribution to clinical drug resistance remains unresolved. Our work makes it possible to study the regulation of mdr1 in situ, in real time, and in response to developmental, physiological, or environmental signals. Project results will provide greater insights into mechanisms by which mdr1 is turned on during cancer progression and drug treatment, thus potentially leading to novel approaches to avoiding such events in humans.