Ablation of Succinate Dehydrogenase Subunits A and B and Their Impacts on Cancer Phenotypes
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Date
2023-05
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The Ohio State University
Abstract
Succinate dehydrogenase (SDH), also known as succinate-coenzyme Q reductase or complex II, is a crucial metabolic enzyme that plays a dual role in cellular energy production. It participates in both the citric acid cycle, catalyzing the conversion of succinate to fumarate, and the electron transport chain, facilitating the reduction of ubiquinone to ubiquinol. The enzyme complex comprises four core subunits (SDHA, SDHB, SDHC, and SDHD) and four associated assembly factors (SDHAF1-SDHAF4). Loss of function in any of these components results in reduced SDH activity, which has been increasingly linked to a wide range of pathological conditions. To gain insight into the effects of SDH mutations on thyroid cancer phenotypes, CRISPR-mediated knockout FTC133 cell lines were created for SDHA and SDHB, and their phenotypic characteristics were documented in comparison to parental lines. For each SDHA and SDHB, two clones with knockout (KO) activity were selected for further functional analysis. SDHA and SDHB KO clones were subjected to in vitro functional assays (proliferation, colony survival, and soft agar growth). One of the SDHA knockout clones exhibited a reduction in proliferation, colony formation, and anchorage-independent growth, while one showed no significant change. Of the SDHB clones, one showed a reduction in colony formation and proliferation, and the other showed a reduction in anchorage-independent growth. These results suggest that further investigation is required for each knockdown clone to establish the cause of this variable phenotypic expression between the lines, and to paint a clearer picture of the impacts of SDH subunit ablation on enzyme function.
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Keywords
Cancer, Metabolic Proteins, Succinate Dehydrogenase, CRISPR/cas9, Cell culture