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THE MEASUREMENT OF ENDOGENOUS mRNA EXPRESSION OF PLD ISOFORMS IN HL-60 CELLS USING QRT-PCR AND THE IMPACT OF THESE ISOFORMS ON GENE EXPRESSION OF mTOR AND S6K
| Title: | THE MEASUREMENT OF ENDOGENOUS mRNA EXPRESSION OF PLD ISOFORMS IN HL-60 CELLS USING QRT-PCR AND THE IMPACT OF THESE ISOFORMS ON GENE EXPRESSION OF mTOR AND S6K |
| Author: | Tabatabaian, Farnaz |
| Description: | Phospholipase D (PLD) is an enzyme that hydrolyzes the phosphodiester bond in phosphatidylcholine, yielding choline and phosphatidic acid (PA). There are two main isoforms: PLD1 and PLD2, which share 50 % genetic homology. Both PLD isoforms are highly regulated by fatty acids, small GTPase proteins, protein kinase C, Ca2+, and phosphorylation. PLDs are involved in physiological and cellular signaling pathways, primarily through the production of the lipid second messengers PA and diacylglycedrol (DAG). While PA is involved in a variety of intracellular mechanisms, the role of PLD, independent of PA, in regulation of signaling protein kinases is not fully understood. This is particularly the case for the protein kinases mammalian target of rapamycin (mTOR) and ribosomal p70S6K. mTOR is a kinase, activated by growth factors and inhibited by rapamycin, that contributes to cell growth and proliferation. p70S6K, also a kinase, is a substrate of mTOR and catalyzes the phosphorylation of the S6 protein, a component of the eukaryotic ribosomal 40S subunit. P70S6K also plays a role in the regulation of cell growth. The objectives of the present study were to analyze: (a) if the expression of the PLD mRNA isoforms were subjected to regulation by the expression of members in other cell signaling pathways, namely mTOR and S6K, and (b) if such regulation should exist, how it might be modulated by granulocyte agonists, specifically chemoattractant agents Epithelial-neutrophil activating peptide (ENA-78), N-formyl-methionyl-leucyl-phenylalanine (fMLP) and Interleukin-8 (IL-8). Using a variety of molecular methodologies, we were able to establish a working method for high-yield transfection of the human promyleocyitic leukemic (HL-60), a hematopoietic cell line that grows in cell culture in suspension. Real Time PCR (qRT-PCR) was optimized for the four genes of interest in this study: PLD1, PLD2, mTOR, S6K. Using qRT-PCR, we found that DMSO differentiated HL-60 cells (dHL-60) that express the neutrophilic phenotype, were able to respond to ENA-78, fMLP, and IL-8. Gene expression of PLD1, PLD2, mTOR and S6K increased in presence of ENA-78 and IL-8. On the other hand, fMLP increased gene expression of PLD2, mTOR and S6K, but not PLD1. Endogenous gene expression after silencing with specific, double strand, small interfering RNAs (si RNA) was also investigated. PLD1 RNA interference effectively silenced PLD1, but showed a small degree of non-specific silencing towards PLD2 as well. PLD2, mTOR and S6K were effectively silenced by RNA interference. Interestingly, the silencing of the latter genes was not rescued (brought back to basal levels) by fMLP or IL-8 induction. Further, a relationship between the PLD2 and the mTOR/S6K kinases was found, namely, that when either mTOR or S6K gene expression is silenced, PLD2 expression is dramatically potentiated. We propose that mTOR and S6K expression negatively regulate PLD2 gene expression, and this cannot be rescued by cell stimulation with chemoattractants. The mechanism by which RNA from a set of cell signaling molecules influence the expression of other genes is not addressed in this thesis, but several exploring possibilities are discussed. |
| Permanent Link: |
http://rave.ohiolink.edu/etdc/view?acc_num=wright1165950834
http://hdl.handle.net/2374.OX/19638 |
| Date: | 2006 |
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