Transcript Quantification and DNA Sequencing of IL-13Rα1 and IL

Erin Dolac, Department of Biology, York College
Methods
Abstract
IL-13Rα1 is found on all normal cells while IL-13Rα2 is found to
be overexpressed only on certain lines of cancer cells. IL-13Rα2 acts
as a decoy receptor, stopping the normal STAT6 pathway that
normally initiates an immune response when the IL-13 ligand binds to
the IL-13α1/IL-4α receptor complex. Eight different cancer cell lines
were obtained. RNA was isolated from each line and subjected to RTPCR for DNA sequencing. Total protein was isolated from each cell
line and then a Western Blot and an ELISA were ran to quantify the
amount of IL-13Rα1 and IL-13Rα2 for each cancer cell line. A twoway ANOVA shows that there is more IL-13Rα2 than IL-13Rα1 per
total protein for DU145 (p<0.001), Jurkat (p<0.001), MCF-7
(p<0.001), and clone 5 (p<0.01). These four cell lines would be
useful in cancer research and treatment because they contain a large
amount of IL-13Rα2 which is not found on normal cell lines, making
them ideal for specific targeting.
Introduction
Cell lines
•U87MG-glioblastoma
•U251-glioblastoma
•DU145-prostate carcinoma
•Jurkat-T-lymphocyte
•MCF-7-Breast adenocarcinoma
•Clone 5-glioblastoma
•MDA435-Breast ductal carcinoma
•HT29-Colon carcinoma
•Western Blot revealed that none
of the cell lines expressed IL13Rα1.
• Western Blot revealed that Jurkat
and clone 5 cells expressed IL13Rα2.
•ELISA revealed that all cell lines
expressed IL-13Rα1 and IL-13Rα2.
•Not enough DNA was extracted
for DNA sequencing.
Growing cells
Grown and maintained in tissue culture flasks (Corning)
containing appropriate growth media
•IL-13α2 receptor (IL-13Rα2) is found to be on some cancer cell lines
such as glioblastoma cells (brain tumor cells) and is known to be
overexpressed.
•IL-13Rα2 is thought to be a decoy receptor because when the IL-13
cytokine is released from the T cells, the ligand attaches to the IL-13Rα2
instead of the IL-13α1 receptor (IL-13Rα1) which is found on both
normal and cancer cells.
α2
Clone 5
50kD
RNA isolation
•Since IL-13Rα2 is found only on cancer cells it would be a good target
for cancer treatment, but only for those cancer lines that have a high
amount of the receptor.
Objectives
Quantification of IL-13R1 and IL-13R2 per
total protein on various cancer cell lines
175
DU145
150
Jurkat
125
100
MCF-7
75
clone 5
50
HT29
25
MDA435
U87MG
U251
0
RT-PCR
BSA standard curve and
protein concentration
DNA extraction
Figure 2. Western Blot specific for
IL-13Rα2. A chemiluminescent
substrate revealed that clone 5
cells have IL-13Rα2 on their
membranes running at 50kD.
Western Blot (Figure 1)
Jurkat
DNA sequencing
ELISA (Figure 1)
α2
50kD
•The immune response pathway is never initiated if the ligand attaches
to the IL-13Rα2 so the body does not fight against the invading cancer
cells.
•Chemotherapy is the popular method of treating cancer at this time,
but it is not specific to just targeting cancer cells, making the patient
very sick.
http://www.novusbio.com/data_sheet/index/NB600-1384
Protein isolation
•A signaling ligand called Interleulin 13 (IL-13) is produced and released
by activated T cells which normally attach to the intramembranous
protein receptor complex IL-13α1/IL-4α.
•This triggers an immune response pathway to fight against foreign
invaders.
Results
ng of protein
http://www.targepeutics.com/il13.htm
Quantification and DNA Sequencing of IL-13Rα1 and IL-13Rα2
On Various Cancer Cell Lines
Alpha-1
Alpha -2
Receptor Type (IL-13)
Figure 5. Quantification of IL-13Rα1 and IL-13Rα2 per
total protein for various cancer cell lines through an
ELISA. A two-way ANOVA shows that overall, the
cancer cell lines studied do express significantly more
IL-13Rα2 than IL-13Rα1 per total protein (p<0.0001).
Four cell lines express more IL-13Rα2 than IL-13Rα1
per total protein: DU145 (p<0.0001), Jurkat
(p<0.0001), MCF-7 (p<0.0001), and clone 5 (p<0.01).
HT-29, MDA435, U87MG, and U251 all do not have a
significant difference between the amount of IL-13Rα1
and IL-13Rα2 expressed.
Conclusions
3) Super Signal
•Overall, cell lines expresses more IL-13Rα2
than IL-13Rα1 (p<0.0001).
Protein quantification
HRP
2) Secondary antibody
Two-way ANOVA
1) Primary antibody
1)Quantify IL-13Rα1 and IL-13Rα2 for each cancer cell
IL-13Rα1 or α2
line.
2)Determine which receptor is more abundant for each
Figure 1. Sequence of substances for Western
cancer cell line.
3)Extract the DNA to determine any differences between Blot and ELISA. The total protein for each cell
line was added into different wells of the
the DNA sequences of IL-13Rα1 and IL-13Rα2.
Western Blot gel and the maleic anhydride filled
wells for the ELISA. Then the following
Hypotheses
sequence was added for quantification of IL1)There will be no difference between the quantity of
13Rα1 or IL-13Rα2: 1) Primary antibody specific
IL13R-α1 and IL13R-α2 between the cancer cell lines:
for the protein, 2) Secondary antibody specific
U87MG, U251, DU145, Jurkat, MCF-7, clone 5, MDA435,
for the primary antibody with Horseradish
and HT29.
Peroxidase attached, and 3) Super Signal.
2) There will be no difference in the DNA sequence for
IL13R-α2 in the cancer cell lines U87MG, U251, DU145,
Jurkat, MCF-7, clone 5, MDA435, and HT29.
Figure 3. Western Blot specific for IL13Rα2. A chemiluminescent substrate
revealed that Jurkat cells have IL-13Rα2
on their membranes running at 50kD.
•DU145 (p<0.0001), Jurkat (p<0.0001). MCF-7
(p<0.0001), and clone 5 (p<0.01) express
more IL-13Rα2 than IL-13Rα1.
Literature Cited
Bernard, J., Treton, D., Vermot-Desroches, C., Boden,
C., Horellou, P., Angevin, E., Galanaud, P.,
Wijdenes, J., & Richard Y. 2001. Expression of
Ladder
Ladder
control
interleukin 13 receptor in glioma and renal cell
MDA435
carcinoma: IL13Rα2 as a decoy receptor for IL13.
U251 Jurkat
HT29
Laboratory Investigation 81: 1223-1231.
U87MG DU145 MCF-7
Debinski, W., Gibo, D. M., Hulet, S. W., Connor, J. R.,
and Gillespie, G. Y. 1999. Receptor for Interleukin
1100bp
13 is a marker and therapeutic target for human
700bp
high-grade gliomas. Clinical Cancer Research
400bp
[serial online] 5:985-990. Available from: PubMed.
Kawakami, K., Taguchi, J., Murata, T., Puri, R. K. 2001.
The interleukin-13 receptor α2 chain: an essential
component for binding and internalization by not for
interleukin-13-induced signal transduction through
Figure 4. DNA bands for IL-13Rα2 from various
the STAT6 pathway. Blood 97: 2673-2679.
cancer cells’ total protein. Specific IL-13Rα2
Acknowledgements
primers were used for the RT-PCR. IL-13α2 runs Thanks to Dr. Thompson for helping me with all the lab techniques and to Dr.
Kleiner for helping with the statistical analysis.
at 1100bp. The ladder is a 500bp ladder.