Al-Dosari, Mohammed S
(2005)
FUNCTIONAL ANALYSIS OF 5'-FLANKING REGION OF CYTOCHROME P450 GENES THROUGH MOLECULAR CLONING AND TRANSFECTION IN VITRO AND IN VIVO.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Cytochrome P450 (CYP) enzymes are an important class of heme-containing proteins that catalyze oxidation reactions leading toward the removal of a wide variety of endogenous and exogenous substrates including prescription drugs. The activities of CYP enzymes are regulated primarily at the transcription level involving the regulatory sequences at the 5'-flanking region of the CYP genes. The objective of this dissertation study was to characterize the function of the 5'-flanking sequences of selected CYP genes primarily responsible for drug metabolism. Various sequences from the 5'-flanking regions of different CYP genes (CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP2E1, and CYP3A4) were cloned in expression vectors and tested for their activity in driving reporter gene expression in mouse livers and in transfected HepG2, 293, and BL-6 cells under optimized conditions. It was demonstrated that among the tested 5'-flanking regions of CYP genes, the CYP2D6 promoter showed the highest activity both in vivo and in vitro. The activities of various 5'-flanking regions of CYP genes in sustaining transgene expression were then tested in mouse liver and compared to those of other promoter sequences. As a result, the CYP2D6 promoter showed the highest activity and its activity was comparable to that of many established promoters. The mechanism underlying CYP promoter activities in vivo and in vitro were then studied using the CYP2C9 promoter as a model. Activities of various 5'-flanking sequences of CYP2C9 were evaluated by using deletion mutations of plasmid constructs in combination with transfection in mouse livers and in HepG2 cells. Finally, the role of PXR and CAR nuclear receptors in regulating CYP2C9 activation was investigated. The results show that both CAR and PXR are essential for CYP2C9 activation and that the regulatory elements reside in the proximal 1-2 kb region upstream of the CYP2C9 gene.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
24 May 2005 |
| Date Type: |
Completion |
| Defense Date: |
5 April 2005 |
| Approval Date: |
24 May 2005 |
| Submission Date: |
7 April 2005 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Pharmacy > Pharmaceutical Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Cytochrome; Metabolism; P450; Promoter; Regulation |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-04072005-160230/, etd-04072005-160230 |
| Date Deposited: |
10 Nov 2011 19:34 |
| Last Modified: |
19 Dec 2016 14:35 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/6844 |
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