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参与癌症进展的细胞反应，如细胞周期调控和耐药性。我们的长期目标是研究癌症进展中的早期事件，以及它们如何从转录因子（TF）活性的角度影响临床结果，这与信号通路的活性有关。我们假设，基于细胞的检测报告的各种信号通路的活性的癌细胞将基因型与表型。本提案的目的是开发一种转染细胞阵列，以乳腺癌为模型，用于癌细胞中TF活性的高通量分析。我们的假设和提出的研究基于以下观察：a）cDNA或蛋白质微阵列不能预测细胞活性，因为活性基于特定的蛋白质构象或状态和细胞定位，B）基于细胞的测定提供了检查细胞活性的生理背景，c）TF活性被广泛用于使用双质粒系统测量细胞活性：第一个测量特定TF的活性，第二个标准化转染效率：基于这些观察结果，该提议的实验目标是开发一种高通量系统来定量许多TF的活性。该提案的具体目标是：具体目标1：创建具有100个点的转染细胞阵列，能够局部有效转染乳腺癌细胞系。我们将a）开发在斑点中存款脂质复合物同时保留其生物活性的程序，和B）定量斑点中转染细胞和转基因表达的百分比。具体目标二：通过a）确定两种质粒的比例和阵列点之间的位置一致性和B）使用能够随时间跟踪TF活性的成像技术非侵入性地定量TF活性，来检测转染的细胞阵列以定量TF活性。具体目标3：比较因一种或多种基因型突变而不同的细胞的TF活性。我们将表征a）通过TERT（76 NTERT）或通过突变型p53（76 Ndel239）永生化的乳腺上皮细胞和B）原发性肿瘤和恶性肿瘤细胞的TF活性，所述原发性肿瘤和恶性肿瘤细胞从同一患者建立。 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: