Transcript Probing the Expression Patterns of System xc

Probing the Expression
Patterns of System xc in
Human Glioma Cells
Mazi Condelee
Chase Lab
Summer 2007
REACH Program
Background Research

System xc- is a
neurotransmitter
transport system
important in
glutathione
homeostasis

Exchanges Cystine
for Glutamate

Increases amount of
glutathione
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 xcexpression patterns change in response to the
level of reactive oxygen species
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We hypothesize that the expression
patterns of System xc- will change as the
cell progresses through the cell cycle
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
x c
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
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
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
Similar expression as T=0
with expression primarily
in ER
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


Dr. Leah Chase
Lab Members
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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