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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-13R1 and IL-13R2 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.