Date of Award
5-12-2023
Document Type
Thesis
Degree Name
Bachelor of Arts
Department
Biology
First Advisor
Wade Powell
Abstract
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates toxicity of dioxin-like compounds. In the absence of xenobiotics, AHR also plays roles in development and physiology, including promoting cell cycle progression through the G1/S checkpoint. Unlike humans and rodents, the frog Xenopus laevis expresses two paralogous AHR proteins, AHR1α and AHR1β, the result of a genome duplication approximately 17-18 million years ago. We recently generated mutant derivatives of the XLK-WG cell line lacking a functional version of either AHR1α or AHR1β. Growth of each strain was measured using colony formation assays and the water-soluble tetrazolium (WST-8) method, each demonstrating that AHR1α-/- cells proliferate approximately 30% more slowly than wild-type or the AHR1β-/- line. To investigate a potential mechanism underlying the slow growth phenotype, cell cycle distribution of each line was determined by propidium iodide staining and flow cytometry. AHR1β-/- cells and wild-type lines exhibited similar distributions of cells in the various stages. In contrast, AHR1α-/- cells exhibited a unique sub-G1 population. This indicates breakdown of genomic DNA and suggests that this mutant line has a heightened propensity for apoptosis. We tested this hypothesis directly using two approaches: Annexin V staining with flow cytometry for fixed cells and a caspase-3/7 assay in live cells. Neither approach detected increased apoptosis in AHR1α-/- cells. We next tested the hypothesis that AHR1α-/- cells in the sub-G1 population undergo pyroptosis, a distinct pathway of programmed cell death that resembles apoptosis in the breakdown of genomic DNA. A luminescent caspase-1 inflammasome assay revealed no increased pyroptosis, even in wild-type cells treated with talabostat, resiquimod, or PAM3CSK4, inducers of the pyroptosis pathway. We tentatively conclude that the slow growth and apparent degradation of genomic DNA in AHR1α-/- mutants occurs by a unique mechanism, as yet undetermined.
Recommended Citation
Milhon, Paige, "Mechanisms Underlying Emergence of a Sub-G1 Population of Cells in a Mutant Xenopus laevis Line Lacking AHR1α" (2023). Honors Theses. 638.
https://digital.kenyon.edu/honorstheses/638
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