Probing the expression patterns of system x c in human glioma cells
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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

Probing the Expression Patterns of System xc- in Human Glioma Cells

Mazi Condelee

Chase Lab

Summer 2007

REACH Program


Background research
Background Research

  • System xc- is a neurotransmitter transport system important in glutathione homeostasis

    • Exchanges Cystine for Glutamate

      • Increases amount of glutathione


Research question
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


Protocol
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-


Immunocytochemistry
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


Results t 0
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


Results t 2
Results T=2

Expression is more diffuse

Some remaining staining in

ER, but fewer vesicles are

apparent


T=4

Less expression in ER

And vesicles

More expression on the

Plasma membrane


Results t 6
Results, T=6

Similar expression as T=0

with expression primarily

in ER


Conclusions and future work
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


Acknowledgements
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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