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Role of Bcl11b, Bim, and E4BP4 in Regulation Of Apoptosis in the RS4;11 Human Leukemic Cell Line
Apoptosis is a mode of programmed cell death where characteristic morphological changes occur in a controlled manner, thereby avoiding damage to surrounding cells or tissues. This method of cell death plays an important role in development and homeostasis but requires a delicate balance. Without this balance, inhibition of apoptosis may result in the formation of neoplasms or autoimmune diseases while excessive apoptosis has been linked to neurological diseases. Three genes of interest, Bcl11b, E4BP4, and Bim are believed to be involved in apoptosis. Bcl11b is thought to be a transcriptional repressor of apoptosis and has a major role in T cell development. Previous research has shown that glucocorticoid-evoked upregulation of E4BP4 consequently results in upregulation of the pro-apoptotic Bim during apoptosis. The RS4;11 cell line is a human acute lymphoblastic leukemia cell line which expresses both myeloid and lymphoid markers and is associated with a poor prognosis. Daunorubicin is an anthracycline commonly used to treat leukemia while 5-fluorouracil is an antimetabolite used to treat a wide range of solid tumors in addition to sometimes being used in the treatment of leukemia. In observing gene regulation, we can better understand the role of these genes in apoptosis as well as gain knowledge in potential targets of chemotherapeutic agents. The hypothesis of this project is that Bcl11b suppression correlates with upregulation of E4BP4 and Bim in response to the anti-leukemic agents above. Cell viability assays confirmed the susceptibility of RS4;11 cells to treatment by daunorubicin and 5-fluorouracil. Fluorescent microscopy was utilized to further confirm that apoptosis was occurring as opposed to necrosis. RNA extractions of this cell line 24 hours after treatment and subsequent reverse transcription reactions yielded intact cDNA which was utilized in qRT-PCR reactions. The expression levels of the aforementioned genes were analyzed in response to treatment by the anti-leukemic agents daunorubicin and 5-fluorouracil. However, a relationship between the drug treatments and gene expressions was shown not to occur. A possible reason for this outcome is that the drugs used in this study induce apoptosis by upregulation or downregulation of genes other than those analyzed in this project.