Transfected cell arrays for cancer research

描述(申请人提供)：癌症生物学的一个主要挑战是了解基因异常或突变如何改变细胞反应，如细胞周期调节和耐药性，参与癌症进展。我们的长期目标是从转录因子(TF)活性的角度研究癌症进展的早期事件及其对临床结果的影响，转录因子(TF)活性与信号通路的活性有关。我们假设，报告癌细胞内各种信号通路活性的基于细胞的分析将把基因型与表型联系起来。该方案的目的是建立一种高通量分析肿瘤细胞中转铁蛋白活性的转基因细胞阵列，以乳腺癌为模型。我们的假设和拟议的研究是基于以下观察结果：a)cDNA微阵列或蛋白质微阵列不能预测细胞的活性，因为活性是基于特定的蛋白质构象或状态和细胞定位的，b)基于细胞的分析提供了检测细胞活性的生理环境，c)转铁蛋白活性被广泛用于使用双质粒系统来测量细胞活性：一个测量特定转运蛋白的活性，第二个标准化以提高转染率：基于这些观察，本方案的实验目标是开发一个高通量的系统来定量多个转铁蛋白的活性。该提案的具体目标是：具体目标1：创建一个具有100个点的转基因细胞阵列，能够对乳腺癌细胞系进行定位、高效的转染。我们将a)开发在保留其生物活性的同时在斑点沉积脂复合物的程序，以及b)在斑点中量化转基因细胞和转基因表达的百分比。具体目标2：通过a)确定两个质粒的比例和阵列点之间的位置一致性，以及b)使用能够随时间跟踪TF活性的成像技术来非侵入性地定量TF活性，从而验证转基因细胞阵列用于定量TF活性。具体目标3：比较因一个或多个基因突变而不同的细胞的转铁蛋白活性。我们将对a)经TERT(76NTERT)或突变型p53(76Ndel239)永生化的乳腺上皮细胞和b)来自同一患者的原发肿瘤和恶性肿瘤细胞的TF活性进行表征。 评估：

DESCRIPTION (provided by applicant): A major challenge in cancer biology is to understand how genotypic abnormalities or mutations will alter cellular responses, such as cell cycle regulation and drug resistance, involved in cancer progression. Our long-term goal is to investigate the early events in cancer progression and how they impact clinical outcome from the perspective of transcription factor (TF) activity, which relates to the activity of the signaling pathways. We hypothesize that cell-based assays that report on the activity of various signaling pathways within the cancer cells will link genotype to phenotype. The objective of this proposal is to develop a transfected cell array for high throughput analysis of TF activity in cancer cells, with breast cancer serving as a model. Our hypothesis and proposed research are based on the following observations: a) cDNA or protein microarrays cannot predict cellular activity, as activity is based on a specific protein conformation or state and cellular localization, b) cell-based assays provide the physiological context in which to examine cellular activity, c) TF activity is widely used to measure cellular activity using a two-plasmid system: one measures the activity of a specific TF and the second normalizes for the transfection efficiency: Based on these observations, the experimental objectives of the proposal are to develop a high throughput system to quantify activity of many TFs. The specific aims of the proposal are: Specific Aim 1: Create a transfected cell array with 100 spots capable of localized, efficient transfection of breast cancer cell lines. We will a) develop the procedures to deposit lipoplexes in spots while retaining their bioactivity and b) quantify the percentage of transfected cells and transgene expression in the spot. Specific Aim 2: Validate the transfected cell array for quantifying TF activity by a) determining ratio of the two plasmids and positional consistency between array spots and b) quantifying TF activity non-invasively using an imaging technique that enables following the TF activity over time. Specific Aim 3: Compare the TF activity for cells differing by one or more genotypic mutations. We will characterize TF activity for a) mammary epithelial cells immortalized by TERT (76NTERT) or by mutant p53 (76Ndel239) and b) primary tumor and malignant tumor cells, which were established from the same patient. ASSESSMENT: