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Quantification and DNA Sequencing of IL-13Rα1 and IL-13Rα2 On Various Cancer Cell Lines

Erin Dolac, Department of Biology, York College

Introduction•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.

•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.

•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.

•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.

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 RT-PCR 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 two-way 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.

Results

Literature CitedBernard, J., Treton, D., Vermot-Desroches, C., Boden, C., Horellou, P., Angevin, E., Galanaud, P., Wijdenes, J., & Richard Y. 2001. Expression of interleukin 13 receptor in glioma and renal cell carcinoma: IL13Rα2 as a decoy receptor for IL13. Laboratory Investigation 81: 1223-1231.Debinski, W., Gibo, D. M., Hulet, S. W., Connor, J. R., and Gillespie, G. Y. 1999. Receptor for Interleukin 13 is a marker and therapeutic target for human high-grade gliomas. Clinical Cancer Research [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 the STAT6 pathway. Blood 97: 2673-2679.

AcknowledgementsThanks to Dr. Thompson for helping me with all the lab techniques and to Dr. Kleiner for helping with the statistical analysis.

Objectives1)Quantify IL-13Rα1 and IL-13Rα2 for each cancer cell line.2)Determine which receptor is more abundant for each cancer cell line.3)Extract the DNA to determine any differences between the DNA sequences of IL-13Rα1 and IL-13Rα2.

Hypotheses1)There will be no difference between the quantity of IL13R-α1 and IL13R-α2 between the cancer cell lines: U87MG, U251, DU145, Jurkat, MCF-7, clone 5, MDA435, and HT29.2) 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.

MethodsCell 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

Growing cellsGrown and maintained in tissue culture flasks

(Corning) containing appropriate growth media

RNA isolation Protein isolation

RT-PCR BSA standard curve and protein

concentration

DNA extraction

DNA sequencing

Western Blot (Figure 1)

ELISA (Figure 1)

Protein quantification

Two-way ANOVA

IL-13Rα1 or α2

1) Primary antibody

2) Secondary antibody

HRP

3) Super Signal

Figure 1. Sequence of substances for Western Blot and ELISA. The total protein for each cell line was added into different wells of the Western Blot gel and the maleic anhydride filled wells for the ELISA. Then the following sequence was added for quantification of IL-13Rα1 or IL-13Rα2: 1) Primary antibody specific for the protein, 2) Secondary antibody specific for the primary antibody with Horseradish Peroxidase attached, and 3) Super Signal.

Figure 3. Western Blot specific for IL-13Rα2. A chemiluminescent substrate revealed that Jurkat cells have IL-13Rα2 on their membranes running at 50kD.

α2Jurkat

50kD

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.

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 revealed that none of the cell lines expressed IL-13Rα1.• Western Blot revealed that Jurkat and clone 5 cells expressed IL-13Rα2.•ELISA revealed that all cell lines expressed IL-13Rα1 and IL-13Rα2.•Not enough DNA was extracted for DNA sequencing.

Conclusions•Overall, cell lines expresses more IL-13Rα2 than IL-13Rα1 (p<0.0001).

•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.

http://www.novusbio.com/data_sheet/index/NB600-1384

Figure 4. DNA bands for IL-13Rα2 from various cancer cells’ total protein. Specific IL-13Rα2 primers were used for the RT-PCR. IL-13α2 runs at 1100bp. The ladder is a 500bp ladder.

Clone 5

50kD

α2

Ladder

U87MGU251

DU145Jurkat

MCF-7

1100bp700bp400bp

HT29MDA435

controlLadder

Alpha-1 Alpha -20

25

50

75

100

125

150

175

U87MGU251

DU145

Jurkat

MCF-7

clone 5

MDA435

HT29

Quantification of IL-13R1 and IL-13R2 pertotal protein on various cancer cell lines

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