In Vivo Bioimaging Model to Study Inducible Drug Resistance in Cancer

描述（由申请人提供）：转录调节代表了控制基因表达的主要机制，然而我们对基因在整个身体中如何调节几乎没有直接的了解，这主要是由于在真实的生理条件下和在真实的时间内不能观察和测量基因表达的变化。由于类似的原因，如果在转录水平介导，则不可能直接研究给定癌症疗法对其靶点的影响。然而，最近的革命分子成像已经产生了新的工具进行非侵入性的，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道基因靶向内源基因的基因组基因座，使得可以非侵入性地、真实的时间地并且响应于内部和外部信号来监测该基因的受调节的表达。我们已经建立了这样一个系统，使用小鼠mdr 1a基因座作为原理的证明和作为生物学上重要的基因。多药耐药（MDR）仍然是癌症患者治愈性化疗的严重障碍。MDR的机制之一是MDR 1基因表达增强。MDR 1过表达与许多人类癌症的耐药性有关，但其对临床结果的贡献仍未得到解决。确定MDR 1在耐药性中作用的系统性纵向研究很难（如果不是不可能的话）在人类中进行，并且以前没有足够的动物模型来研究这些问题。MDR 1在药物药代动力学中的作用已得到充分证实，但对MDR 1在参与药物摄取和清除的正常器官中的调节知之甚少。在我们的初步研究中：1）我们已经改造了mdr 1a +/fLUC小鼠，这些小鼠具有靶向mdr 1a基因的基因组位点的萤火虫荧光素酶（fLUC），使得fLUC的框内表达以Cre介导的重组为条件。2)我们已经证明，在内源性mdr 1a基因位点的控制下，fLUC的表达是mdr 1a在基础稳态表达的可靠体内报告基因。3)我们还证明了fLUC可用于监测外源性刺激对mdr 1a基因表达的诱导。我们现在建议使用这种非侵入性的模型系统，在体内环境中研究mdr 1a基因的表达。我们还将扩展该模型，以便能够研究在转录、转录后和翻译水平控制mdr 1a基因表达的顺式和反式作用因子。目的1是确定mdr 1a是否以组织特异性方式在正常器官中被诱导。目的2是确定mdr 1a是否在乳腺癌进展和/或治疗过程中被诱导。目的3是确定在特定组织中mdr 1a的诱导是否需要特定的反式和顺式作用因子。

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.