Discovery Tools for Chemotherapy Resistance to Cell Death.

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DESCRIPTION (provided by applicant): Apoptosis is an evolutionarily conserved cell death process that involves over 100 gene products. In response to cellular stress or to maintain tissue homeostasis, the apoptotic machinery initiates and carries out a series of biochemical events leading to cell death in the absence of inflammation characteristic of necrosis. Apoptosis is essential to remove damaged or dangerous cells, and defects in apoptosis contribute both to tumorigenesis and resistance to anti-cancer chemotherapeutic regimens. The complexity of the apoptotic response to chemotherapy coupled with functional crosstalk between apoptosis and the cell survival process of autophagy presents a significant challenge in our understanding of the cellular resistance to chemotherapy. To help characterize the cellular response to different classes of chemotherapeutic agents, particularly in tumor cells with defects in apoptosis, we propose to develop a set of isogenic human cell lines as discovery tools for characterizing the apoptosis genes involved in chemotherapy resistance. In this Phase I feasibility project, we will prepare and characterize shRNA expressing lentiviruses specific for six human proteins that are key nodes in either the extrinsic or intrinsic apoptotic pathways (DR4, Caspase-8, PUMA, BAX, Caspase-9 and Caspase-3). These lentiviruses will be used for the development of stable cell lines with specific gene knockdown in both the glioma cell line LN428 and the colon cancer cell line HCT-116, followed by mRNA expression (qRT-PCR) characterization of each of the knockdown cells and single-cell clones. This will be coupled with analysis to validate apoptosis deficiency via protein expression loss, functional analysis of multiple apoptotic and autophagy endpoints and selective response to apoptosis inducing agents (Temozolomide, Camptothecin, staurosporine and Sulindac). The optimum shRNA for each will then inform for the development of cell lines with the specific gene knockdown together with (i) a far-red fluorescent reporter (FP635) for selection, (ii) a luciferase reporter amenable to real-time imaging of apoptosis and (iii) expression of LC3-EGFP for a direct analysis of autophagy induction, linked via T2A sequences in a single gene cassette. These cells will function as valuable tools for the identification of key apoptotic targets in chemoresistance and the discovery of agents designed to overcome gene-specific defects in apoptosis. In addition, these novel cell lines are designed to be amenable to high-throughput drug testing or analysis using cell-based and xenograft models. The development of such isogenic human cells specific for an additional 100 genes coding for apoptosis proteins will be the topic of the second phase of this proposal. PUBLIC HEALTH RELEVANCE: We describe the creation of isogenic human cell lines as discovery tools for the identification of key apoptotic targets in chemoresistance and the discovery of agents designed to overcome gene-specific defects in apoptosis. In this Phase I project, we will demonstrate the feasibility of this approach by developing isogenic LN428 and HCT-116 cell lines functionally deficient in one of six human proteins that are key nodes in either the extrinsic or intrinsic apoptotic pathways. Finally, these cell lines will be modified by co-expression of fluorescent markers for utility as valuable tools for discovery of agents designed to be amenable to high-throughput drug testing or analysis using cell-based and xenograft models.