UBC Theses and Dissertations
A high-throughput screen to identify compounds that modulate quiescence in mesenchymal progenitor cells Baker, Samuel F.
Hypermethylated in Cancer 1 (Hic1) is a gene that marks a population of cells known as mesenchymal progenitors (MPs) in humans as well as mice. MPs can be found in almost all adult tissues, where they support repair and regeneration processes after injury. Hic1 regulates MP quiescence, a state where cells are growth arrested, but are poised to enter the cell cycle following activation with various stimuli (i.e., injury). When Hic1 is deleted, MPs exit quiescence and exhibit a mildly activated state, which is associated with an accumulation of MPs and their progeny. These conditions also provide an environment conducive for the development of various cancers and fibrosis. Both diseases are significant contributors to global mortality and morbidity, and treatment options are limited. This project investigated mechanisms for modifying MP activity, in part, through inducing quiescence. Using Hic1 expression as a surrogate for quiescence, an assay was developed to measure Hic1 transcript abundance. The assay relies on the use of cells from a genetically engineered mouse model, in which a nuclear LacZ gene was knocked-in to the Hic1 locus. In this manner, Hic1 expression and by extension quiescence, can be followed by measuring LacZ activity. CPRG is a chromogenic substrate of LacZ and was used to measure the abundance of LacZ after treating Hic1ⁿᴸªᶜᶻ/⁺ mouse embryonic fibroblasts (within the MP umbrella) with a library of drugs that passed at least Phase I of FDA clinical trials. High-throughput techniques such as robotic cell seeding and 96-well parallel drug administration by pinning tool were used to efficiently work through the over ~ 4,000 drugs. The screen identified various retinoids as hits, which was expected due to the direct targeting of Hic1 by retinoic acid (RA) receptors (Rs), and no non-retinoid compounds resulted in significant increases in Hic1 expression. Screen results were further investigated through a high content X-gal and nucleic acid stain screen, as well as through RNA sequencing. Secondary screening indicated that hit compounds were acting through modulating RAR signaling. Collectively, these findings reinforce the concept that MP quiescence is predominantly regulated by RAR-mediated signaling.
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