as follows: For the PXR expression vector, a full-length PXR open
reading frame was amplified by RT–PCR from human liver RNA using
the gene-specific primers 5^0 -AACCTGGAGGTGAGACCCAAAGA-3^0 and
50 -ATCTCGAGGATCCTCAGCTACCTGTGATGCCGA-3^0 (Bertilsson et al.,
1998; Lehmann et al., 1998). Theresulting amplicon was digested withEcoRI
andXhoI and subcloned into the polylinker site of pcDNA3 for expression,
hereafter designated pcDNA3–hPXR. The correct PXR sequence and insert
orientation were confirmed by DNA sequence analysis. A chimeric CYP3A4
luciferase reporter vector was prepared following the described method
(Goodwin et al., 1999). The resulting construct contains two fragments of the
CYP3A45 flanking region (836 to7208 and 62 to +53) linked to the
luciferase reporter sequence of the pGL3-basic vector (Promega, Madison,
WI) and hereafter designated pGL3–3A4. Correct insert orientation was
confirmed by DNA sequence analysis.
The PXR reporter gene assay itself is performed in our laboratory as
follows: HepG2/C3A cells (American Type Culture Collection, Manassas,
VA) are plated in 96-well plates (Packard Bioscience, Meriden, CT) (2 104
cells/well) in 100mL Plating Medium (phenol-red-free Dulbecco Modified
Eagle Medium (DMEM) supplemented with 10% fetal bovine serum
(charcoal stripped), 2 mML-glutamine and 1 mM nonessential amino acids),
and preincubated overnight. The cells are then transfected by lipofection using
Fugene-6 (Roche Molecular Biochemicals, Indianapolis, IN) in the presence
of serum per the manufacturer’s instructions. Transfection mixes applied to
each well contain 2.5 ng of hPXR expression vector pcDNA3–hPXR, 50 ng of
luciferase reporter plasmid pGL3–3A4, and 20 ng of alkaline phosphatase
expression vector pSEAP2-Control (Clontech, Plao Alto, CA). The media are
replaced with 100mL of fresh Dosing Medium (Plating Medium + test
compounds in 0.25% DMSO) in 6 h following transfection, and incubated for
an additional 42 h. All test compounds except phenobarbital were tested at
final concentrations of 2, 10, 20, and 50mM. Phenobarbital was tested at 20,
50, 150, and 250mM. The activities of firefly luciferase and alkaline
phosphatase were determined by Steady-GloTMLuciferase Assay System
(Promega, Madison, WI) and GreatEscAPeTMchemiluminescence detection
kits (Clontech, Palo Alto, CA), respectively, according to the supplier’s
specifications.
Although the overall principles of the assay and its basic design are
essentially the same in all laboratories, specific details may vary from one
laboratory to another. Table 17.2 shows details of how the three key
components of the assay (cell line, PXR expression plasmid, and reporter
gene construct) may vary among individual laboratories. There are basic
requirements for each of the three key components. Cell lines should be
immortalized, relatively easy to propagate and maintain in a consistent
manner, readily amenable to plasmid transfection, and have little or no
background or constitutive expression of PXR. Caco-2 cells, CV-1 (Africa
green monkey kidney fibroblast) cells, and HepG2 cells all meet these criteria.
560 TESTING DRUG CANDIDATES FOR CYP3A4 INDUCTION