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Molecular & Cellular Analysis Technologies
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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: