Transfected cell arrays for cancer research

描述（由申请人提供）：癌症生物学的一个主要挑战是了解基因型异常或突变如何改变细胞反应，如细胞周期调节和耐药性，涉及癌症进展。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如何影响临床结果，转录因子（TF）活性与信号通路的活性有关。我们假设基于细胞的检测报告了癌细胞内各种信号通路的活性，将基因型与表型联系起来。本提案的目的是开发一种转染细胞阵列，用于高通量分析肿瘤细胞中的TF活性，以乳腺癌为模型。我们的假设和提出的研究是基于以下观察：a) cDNA或蛋白质微阵列不能预测细胞活性，因为活性是基于特定的蛋白质构象或状态和细胞定位，b)基于细胞的检测提供了检查细胞活性的生理背景，c) TF活性广泛用于使用双质粒系统测量细胞活性。一种是测量特定TF的活性，另一种是对转染效率进行归一化：基于这些观察结果，本提案的实验目标是开发一种高通量系统来量化许多TF的活性。该提案的具体目标是：具体目标1：创建具有100个位点的转染细胞阵列，能够定位，有效地转染乳腺癌细胞系。我们将a)开发在保留其生物活性的情况下将脂质体沉积在斑点上的程序，b)量化转染细胞的百分比和在斑点上的转基因表达。具体目标2：验证转染的细胞阵列，通过a)确定两个质粒的比例和阵列点之间的位置一致性，b)使用成像技术非侵入性地定量TF活性，从而跟踪TF活性随时间的变化。特异性目的3：比较一个或多个基因型突变不同的细胞的TF活性。我们将对来自同一患者的原发肿瘤细胞和恶性肿瘤细胞的TF活性进行表征：a) TERT （76NTERT）或突变型p53 （76Ndel239）永活的乳腺上皮细胞；b)来自同一患者的原发肿瘤细胞和恶性肿瘤细胞。

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: