PhD Thesis Defence
Marcia Taylor
Department of Biochemistry and Molecular Biology
Degrees
- BSc (Applied Biochemistry), University of Guelph
Title of Thesis:
- Phosphatidylcholine Metabolism and Its Role in the Execution of Farnesol-Induced Apoptosis
Time/Date:
- 9:30 am, Thursday, October 28, 2004
Place:
- Lord Dalhousie Room, 1st Floor, Henry Hicks Academic Administration Building
Examining Committee:
- Dr. Roy Baker, Department of Biochemistry, University of Toronto (External Examiner)
- Dr Kirill Rosen, Department of Biochemistry & Molecular Biology and Pediatrics (Reader)
- Dr. David Hoskin, Department of Microbiology and Immunology (Reader)
- Dr. Christopher Sinal, Department of Pharmacology (Reader)
- Dr Christopher McMaster, Department of Biochemistry & Molecular Biology and Pediatrics (Supervisor)
- Dr. Michael Gray, Department of Biochemistry & Molecular Biology (Departmental Representative)
Chair:
- Dr. Thomas MacRae, PhD Defence Panel, Faculty of Graduate Studies
Abstract
Phosphatidylcholine (PC) is the most abundant phospholipid present in eukaryotic cellular membranes, comprising approximately 50% (w/w). The enzyme catalyzing the terminal reaction in the biosynthesis of PC is cholinephosphotransferase (CPT). This work has confirmed the localization of the CEPT1 isoform of mammalian CPT activity to the endoplasmic reticulum (ER) and the nuclear membrane. A determining factor between the Golgi localization of CPT1 and the ER / nuclear membrane localization of CEPT1 was determined to lie within the amino terminal portion of the proteins. The kinetic parameters of CEPT1 were established and a variety of potential inhibitor compounds were tested. Farnesol is a catabolite of the cholesterol biosynthetic pathway that has been demonstrated to inhibit PC biosynthesis and to preferentially cause apoptosis in transformed cells versus normal cells. Farnesol addition to cells was proposed to cause apoptosis by inhibition of PC synthesis through direct competition with cholinephosphotransferase for its substrate diacylglycerol (DAG). Cell lines capable of inducibly over-expressing CPT1 and CEPT1 proteins 10 to 20 fold were established. It was determined that increased expression of CEPT1 or CPT1 in Chinese hamster ovary cells (CHO-K1) restored rates of PC synthesis to normal levels in the presence of farnesol but did not alter the dose of farnesol required to cause apoptosis. Second, it was observed that the addition of DAG to cells prevented farnesol induced apoptosis but did not increase the rate of PC synthesis. Third, farnesol did not inhibit cholinephosphotransferase activity in vitro but instead resulted in an assay dependent inactivation of the cholinephosphotransferase protein. Thus, the hypothesis that farnesol caused apoptosis by inhibition of PC synthesis through direct inhibition of cholinephosphotransferase enzyme activity was disproved. This work has shown that inhibition of PLD signalling augmented, while addition of its product phosphatidic acid attenuated farnesol-induced apoptosis. In addition, over-expression of a phosphatidic acid phosphohydrolase prevented farnesol-induced apoptosis. The results of these studies are consistent with a model whereby farnesol inhibits the production of a PLD generated DAG pool required for activation of a proliferative signal, likely a protein kinase C, necessary for life.
