In Vivo Bioimaging Model to Study Inducible Drug Resistance in Cancer

描述(申请人提供)：转录调控是控制基因表达的主要机制，但我们对基因在全身是如何调控的直接了解很少，主要是因为无法观察和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，如果在转录水平上调节，不可能直接研究给定癌症疗法对其靶点(S)的影响。然而，最近的分子成像革命已经产生了新的工具来进行非侵入性的、活体的基因表达成像。成像技术在生物学上的相关应用是通过同源重组将报告基因定位到内源基因的基因组位置，从而可以非侵入性地、实时地并响应于内部和外部信号来监测该基因的调节表达。我们已经建立了这样一个系统，使用小鼠的mdr1a基因作为原理的证明，并将其作为生物学上重要的基因。多药耐药(MDR)仍然是癌症患者根治性化疗的严重障碍。MDR的一个机制是MDR1基因表达增强。在许多人类癌症中，MDR1的过度表达与耐药有关，但其对临床结果的贡献仍未得到解决。要确定多药耐药基因S在耐药性中的作用，即使不是不可能，也很难在人类身上进行系统的纵向研究，而且以前还没有足够的动物模型来研究这类问题。多药耐药基因在药物药代动力学中的作用已得到证实，但对参与药物摄取和清除的正常器官中多药耐药基因的调节知之甚少。在我们的初步研究中：1)我们已经培育了mdr1a+/flc小鼠，它们具有针对mdr1a基因基因组位置的萤火虫荧光素酶(flrefly Luciferase，FLUC)，使其在框内表达的条件是Cre介导的重组。2)我们已经证明，内源性mdr1a基因座控制的flc的表达是mdr1a在基础、稳定状态下表达的可靠的体内报告基因。3)我们还证明了Flc可以用来监测mdr1a基因对异种刺激的诱导表达。我们现在建议使用这个非侵入性的模型系统来研究体内环境中mdr1a基因的表达。我们还将扩展该模型，以能够研究在转录、转录后和翻译水平上控制mdr1a基因表达的顺式作用和反式作用因素。第一个目标是确定mdr1a是否在正常器官中以组织特异性的方式被诱导。目的2是确定mdr1a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是否需要特定的反式作用因子和顺式作用因子来诱导mdr1a。公共卫生相关性：多药耐药性仍然是癌症患者根治性化疗的严重障碍。多药耐药的一个机制是mdr1基因表达增强。在许多人类癌症中，MDR1的过度表达与耐药有关，但其在临床耐药中的作用仍未解决。我们的工作使我们有可能在原位、实时地研究mdr1对发育、生理或环境信号的调节。项目成果将提供对癌症进展和药物治疗过程中mdr1被激活的机制的更大洞察力，从而可能导致在人类中避免此类事件的新方法。

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.