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Probing the Expression Patterns of System x c - in Human Glioma Cells

Probing the Expression Patterns of System x c - in Human Glioma Cells. Mazi Condelee Chase Lab Summer 2007 REACH Program. Background Research. System x c - is a neurotransmitter transport system important in glutathione homeostasis Exchanges Cystine for Glutamate

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Probing the Expression Patterns of System x c - in Human Glioma Cells

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  1. Probing the Expression Patterns of System xc- in Human Glioma Cells Mazi Condelee Chase Lab Summer 2007 REACH Program

  2. Background Research • System xc- is a neurotransmitter transport system important in glutathione homeostasis • Exchanges Cystine for Glutamate • Increases amount of glutathione

  3. Research Question • As a cell proceeds through the cell cycle, different amounts of reactive oxygen species are produced • Previous work in the lab suggest that System xc- expression patterns change in response to the level of reactive oxygen species • We hypothesize that the expression patterns of System xc- will change as the cell progresses through the cell cycle

  4. Protocol • Utilize U138MG (human glioma) cells to study the trafficking of System xc- through the cell cycle • Maintain cell line in MEM media supplemented with Fetal Bovine Serum • To synchronize cell division, we serum starve cells for 24 hours • Addition of MEM media + FBS at T=0 to initiate cell cycle progression • Fix cells at T=0, 2, 4, 6 hours to visualize System xc-

  5. Immunocytochemistry • Use immunocytochemistry to examine expressions and cellular localization of xCT and 4F2HC (components of System xc-) during cell cycle progression All signals that are yellow, indicate colocalization of 4F2HC and xCT

  6. Results T=0 xCT and 4F2HC appear primarily in endoplasmic reticulum and in vesicles outside of the nucleus Very little transporter is observed on the membrane

  7. Results T=2 Expression is more diffuse Some remaining staining in ER, but fewer vesicles are apparent

  8. T=4 Less expression in ER And vesicles More expression on the Plasma membrane

  9. Results, T=6 Similar expression as T=0 with expression primarily in ER

  10. Conclusions and Future Work • Expression of System xc- is initially more concentrated in the ER • Expression becomes more diffuse • Next step: use flow cytometry to better examine expression through the cell cycle. • We will also use organelle markers to confirm our hypotheses about transporter location

  11. Acknowledgements • Dr. Leah Chase • Lab Members • A. Goltz • T. Henderson • A. Hilbrand • L. Jones • S. Sherburn • M. Wixson • Hope College Departments of Biology and Chemistry • REACH Program • The Campbell Foundation • NSF-MRI

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