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

slide8
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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