ABSTRACT
We compared the expression of the ABCB1 gene in healthy male and female Thai subjects; this gene encodes the P-glycoprotein transporter in peripheral blood mononuclear cells (PBMCs). We also identified the most suitable housekeeping genes for normalization of ABCB1 expression levels in PBMCs. PBMCs from 30 females and 26 males were isolated. Total RNA was extracted, followed by reverse transcription (100 ng total RNA per sample). The internal normalization controls were actin-beta, beta-2M and GAPDH. Real-time quantitative PCR was then performed to determine the expression levels of the ABCB1 gene. The expression levels were found to be 1.5- to 2.5-fold higher in males, depending on the endogenous control used for normalization. Actin-beta was the most stable control gene and could be used as a single endogenous control for normalization of ABCB1 expression levels in PBMCs. However, more than one endogenous control genes are recommended for normalization of gene expression. We conclude that the expression levels of ABCB1 in PBMCs is influenced by gender; this helps, in part, explain the gender difference in pharmacokinetics and pharmacodynamics of drugs that are P-glycoprotein substrates. ABCB1 gene expression profiles need to be carefully interpreted with regards to the endogenous control genes that are involved.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Gene Expression Profiling , Leukocytes, Mononuclear/metabolism , ATP Binding Cassette Transporter, Subfamily B , Actins/metabolism , Adult , DNA Primers/genetics , DNA, Complementary/metabolism , Female , Gene Expression Regulation , Humans , Male , Polymerase Chain Reaction , Sex Factors , Thailand , beta 2-Microglobulin/metabolismABSTRACT
1. Methods for the co-expression in Escherichia coli of human cytochrome P450 (CYP) 2C8 and CYP2C9 with NADPH-cytochrome P450 reductase (OxR) to produce a catalytically active system were compared. 2. Approaches assessed were expression of a CYP:OxR fusion construct, bicistronic plasmids, simultaneous transformation with CYP and OxR plasmids, and separate expression of CYP and OxR with reconstitution of activity by mixing the bacterial membranes. Two N-terminal modifications (Delta3-20 and 17alpha-leader) of the individual P450s were additionally investigated. 3. Each approach gave efficient expression of CYP2C8 and CYP2C9, but the bicistronic constructs under the expression conditions used gave low OxR expression and low catalytic activity. CYP expression was higher with the Delta3-20 construct for CYP2C9 and with the 17alpha-presequence construct for CYP2C8. 4. Using torsemide as substrate, all methods gave catalytically active systems with K(m) values similar to human liver microsomes. Mixing bacterial membranes containing separately expressed CYP and OxR reconstituted a catalytically active system with the Delta3-20 construct for CYP2C9 but not for CYP2C8, and with neither of the 17alpha- presequence constructs. OxR co-expressed with CYP in the same membrane interacted with CYP to reconstitute activity more effectively than addition of exogenous OxR membranes. 5. Expression construct and OxR co-expression strategy should be individualized for CYP isoforms.
