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Research Mentor and Department
Dr. David Spencer, Division of Oncology
Mutations in isocitrate dehydrogenase (IDH) 1 or 2 are found in about 23% of acute myeloid leukemia (AML) samples and about 90% of gliomas. Mutations result in neomorphic function of the IDH enzyme that yields the novel molecule 2-hydroxyglutarate (2HG) instead of alpha-ketoglutarate (aKG). 2HG is known to be associated with hypermethylation of DNA and histones, a phenotype seen in AML. Our lab intends to study the mechanism by which hypermethylation is achieved and how this mechanism relates to the onset of cancer. In this experiment, we utilized a line of H9 stem cells which we had developed. These cells have been transfected with the IDH2 gene with R140Q mutation. Before the gene is a doxycycline (DOX) promoter, and after is the green fluorescent protein (GFP) gene. Treating these cells with DOX induces production of mutant IDH2. In this set of experiments, we wanted to characterize what happened once mutant IDH2 was turned on in cells. Cancer phenotype is marked by two major symptoms: increased proliferation and inhibited differentiation. We analyzed rate of cell growth over timepoints in cells treated with and without DOX. We discovered that the rate of cell growth was significantly less, almost halted, in cells treated with DOX. To verify that these cells were also producing mutant IDH2, we utilized flow cytometry to measure the amount of GFP present. Cells were confirmed to express GFP and therefore mutant IDH2.
Available for download on Thursday, May 17, 2018