Regulatory Proteins Bind at the Polymorphic NFSE Element of CYP3A4.

CYP3A4 is the most abundant cytochrome P450 enzyme in the liver and is involved in the metabolism of most clinically used drugs. An A to G substitution in the nifedipine responsive element (NFSE) in the promoter of this gene has been found to be associated with a lower incidence of pediatric therapy-related leukemia (Felix, Proc Natl Acad Sci USA95:13176, 1998) and adult therapy-related leukemia (Rund et al, Leukemia, accepted for publication). To study the effect of this polymorphism on gene expression in hematopoietic cells, we constructed reporter plasmids with the luciferase gene (in pGL3E) under control of the CYP3A4 promoter, using both the polymorphic and normal sequences. These plasmids were transfected into several cell lines of hematopoietic origin and luciferase was quantitated. We used KG1a (myeloid leukemia), K562 (CML blast crisis), and as controls, MelA1, a melanoma line and HepG2, a hepatoma line. Experiments were repeated at least three times for each cell line. The results consistently demonstrated 20–30% lower luciferase activity (in KG1a and K562 respectively) using the polymorphic sequence as compared to the normal sequence while the MelA1 and HepG2 lines showed the opposite effect, a 25% higher luciferase expression with the variant sequence. The results for HepG2 were in agreement with those reported by Rebbeck (Environmental and Molecular Mutagenesis49:299, 2003). To identify the factors binding at NFSE which may influence expression, electrophoretic mobility shift assays were performed using nuclear extracts of both cell lines (K562, KG1a, and HL60) and patient leukemia cells with a DNA probe representing the normal and polymorphic sequences. A gel shift was demonstrated, indicating binding of nuclear extracts to the region of the polymorphism. The database of transacting factors states complete homology of the polymorphic sequence of the NFSE region with the consensus binding site of HSF-1. We therefore performed a series of experiments to determine if HSF-1 is the protein binding at that site. HSF-1 is a multimeric transcription factor which binds to heat shock elements in many promoters which are rapidly transcribed following stress by increases in temperature. We found that recombinant HSF-1 did not bind to the DNA probe alone. However, nuclear extracts of cells which underwent stress by heating to 43°C for one hour (which is known to increase HSF-1 production) demonstrated increased binding to the probe representing the region of the polymorphism and Western blotting demonstrated more HSF-1 in these extracts. Using a Streptavidin-biotin system with a DNA fragment representing the NFSE region, we demonstrated that DNA binding activity to the probe was present in the elution fractions which contained HSF-1, as detected by ECL (enhanced chemoluminescence). Elution fractions which did not show DNA binding activity did not contain detectable HSF-1. We conclude that HSF-1 may be the protein which binds at the NFSE element of the CYP3A promoter but that it binds either as a multimer or as part of a complex of several proteins, which complicates its detection as a DNA binding protein.