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A.TITLE OF PROPOSED PROJECT:
Transcriptomic profiling of the Human Jurkat Cell Line
Genomics and Transcriptomics
To understand the various cellular and molecular processes going on in living organisms, we need model systems that can mimic the physiological conditions of the organism under study. However, studies have shown that no model system can represent the natural environment of living organisms to the full extent. Variations exist and the need is to determine the extent and significance of these variations so that appropriate models can be selected while designing experiments and the results can be interpreted and generalized accordingly. We propose to evaluate one such model system, the Jurkat Cell line, which is derived from White blood cells, and then compare the levels of expressed genes to the normal tissue. Such a study will determine the significance of the results of studies conducted on these model systems.
1. PROJECT SUMMARY
Describe the proposed research. (max. 100 words)
The Jurkat cell line was derived from the blood of a patient with T-cell leukemia. Years of research on Jurkat cells have shed a great deal of light on the T-cell receptor signaling. Various studies, however, have shown that the expression profiles of cell lines differ significantly from the tissues of their origin, raising a question on the practicality of using these models for mimicking the physiological conditions and the significance of the results. We propose to profile transcriptomes of the Jurkat cell line and the tissue of its origin using to evaluate the differences in expression patterns.
HYPOTHESIS/BASIS OF RESEARCH
Based on previous studies we hypothesize that significant differences exist in the transcriptomes of cell lines and the tissues from which they are derived and the need is to determine the significance of these variations in context of interpreting the results.
Human Jurkat cell line, derived from blood of a Leukemic patient, was first discovered in 1970s. Over the years, it has turned into an important model system for elucidating the T-cell signaling pathways. Expression of Interleukin-2 has also been established in it (Gillis and Watson 1980). Despite the fact that cell lines are extensively used as model systems for studying biological processes, the variation observed between them and their origin are quite significant (Shaw, Morse et al. 2002; Boess, Kamber et al. 2003; Landry, Pyl et al. 2013).
We are proposing a genomics and transcriptomics study involving creating a transcriptomic resource for the human Jurkat cell line using deep RNA sequencing. Jurkat transcriptome will be profiled along with the normal tissue of origin (white blood cells) and compared to the RNA-Seq data available from Illumina Human BodyMap 2.0.
We aim to identify the differences in pattern and level of expression between the in vitro and in vivo system so that while designing experiments and interpreting their results, these differences can be considered.
4A.BACKGROUND OF THE RESEARCH PROBLEMS TO BE ADDRESSED (Not to exceed two pages)
The Jurkat cell line was first established in 1970s. It was derived from the blood of a patient with T-cell leukemia (Schneider, Schwenk et al. 1977). Since then it has gained importance for elucidating the signalling pathways involving T-cells, T-cell Leukemia, cancers and their response to radiation therapy, etc.
Soon after this cell line was derived, expression of interleukin 2 was established in it (Gillis and Watson 1980), aiding the characterization of this immunoregulatory molecule. Years of research on Jurkat cells have shed a great deal of light on the T-cell receptor signalling (Spinozzi, Pagliacci et al. 1994; Meisner, Conway et al. 1995; Abraham and Weiss 2004). In another study cholecystokininbinding sites were identified in the Jurkat cells (Lignon, Bernad et al. 1991). cholecystokininis a peptide involved in the digestion of fats and proteins (Gibbs, Young et al. 1973), and this discovery helped in further elucidating the regulation of CCK receptors. Studies were carried out, attempting to explain mechanisms of T-cells activation and apoptosis, under stress conditions (Katika, Hendriksen et al. 2012). In other studies, components of the apoptotic pathway including several Heat Shock proteins and caspases were identified in Jurkat cells (Samali, Cai et al. 1999). Jurkat cells have been extensively used to study immune responses by inducing apoptosis of T-cells (Dhein, Walczak et al. 1995; Martinez-Lorenzo, Alava et al. 1998).
These studies although have led to a number of important discoveries, it needs to be kept it mind that they were conducted on immortalized cell lines and it needs to be seen how much divergence have they gone through in terms of gene expression to achieve this state so that the results can be generalized to the in vivo conditions. Transcriptomic profiling of the Jurkat cell line and the tissue of its origin can reveal the potential differences in expression levels.
A similar study was conducted on HeLa cell line, one of the most commonly used model for studying cellular and molecular biology. Results showed that the cell line showed markedly variable patterns of expression when compared to normal tissues of same origin (Landry, Pyl et al. 2013). In another study the transcriptome of HEK293 cells was profiled to identify the kidney cell type from which the cell line originated (Shaw, Morse et al. 2002).
Hepatic cell lines were used to carry out a similar study, revealing major differences between the expression levels of the liver cells and the cell lines (Boess, Kamber et al. 2003)
As mentioned above, the importance of Jurkat cells in elucidating important signaling pathways such as apoptosis and T-cell signaling which are an essential component of the immune regulation process calls for analyzing its transcriptome in order to ascertain that the results obtained do indeed correspond to the normal physiological conditions. These findings highlight the practicality of using of such in vitro systems for mimicking in vivo conditions, and extrapolation of the results.
4B.RESEARCH PLAN/ METHODOLOGY: SCHEDULE/PHASING
Abraham, R. T. and A. Weiss (2004). "Jurkat T cells and development of the T-cell receptor signalling paradigm." Nat Rev Immunol 4(4): 301-308.
Boess, F., M. Kamber, et al. (2003). "Gene expression in two hepatic cell lines, cultured primary hepatocytes, and liver slices compared to the in vivo liver gene expression in rats: possible implications for toxicogenomics use of in vitro systems." Toxicological Sciences 73(2): 386-402.
Dhein, J., H. Walczak, et al. (1995). "Autocrine T-cell suicide mediated by APO-1/(Fas/CD95)." Nature 373(6513): 438-441.
Gibbs, J., R. C. Young, et al. (1973). "Cholecystokinin decreases food intake in rats." Journal of comparative and physiological psychology 84(3): 488.
Gillis, S. and J. Watson (1980). "Biochemical and biological characterization of lymphocyte regulatory molecules. V. Identification of an interleukin 2-producing human leukemia T cell line." J Exp Med 152(6): 1709-1719.
Katika, M. R., P. J. Hendriksen, et al. (2012). "Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights." Toxicol Appl Pharmacol 264(1): 51-64.
Landry, J. J., P. T. Pyl, et al. (2013). "The genomic and transcriptomic landscape of a HeLa cell line." G3 (Bethesda) 3(8): 1213-1224.
Lignon, M. F., N. Bernad, et al. (1991). "Pharmacological characterization of type B cholecystokinin binding sites on the human JURKAT T lymphocyte cell line." Mol Pharmacol 39(5): 615-620.
Martinez-Lorenzo, M. J., M. A. Alava, et al. (1998). "Involvement of APO2 ligand/TRAIL in activation-induced death of Jurkat and human peripheral blood T cells." Eur J Immunol 28(9): 2714-2725.
Meisner, H., B. R. Conway, et al. (1995). "Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells." Mol Cell Biol 15(7): 3571-3578.
Samali, A., J. Cai, et al. (1999). "Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of jurkat cells." EMBO J 18(8): 2040-2048.
Schneider, U., H. U. Schwenk, et al. (1977). "Characterization of EBVâ€genome negative “null” and “T” cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed nonâ€Hodgkin lymphoma." International journal of cancer 19(5): 621-626.
Shaw, G., S. Morse, et al. (2002). "Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells." FASEB J 16(8): 869-871.
Spinozzi, F., M. C. Pagliacci, et al. (1994). "The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in Jurkat T-leukemia cells." Leuk Res 18(6): 431-439.
5. IMPACT (of proposed research on teaching/training of manpower, institutional capability building and on local industry)
This study, along with previous researches will allow researchers to design and interpret experiments in the light of the fact that the model systems we use do not mimic the normal physiological conditions 100% so we need to keep these differences in mind when we extrapolate the findings to in vivo systems.