Regulation of UGT1A1 and HNF1 transcription factor gene expression by DNA methylation in colon cancer cells.

BACKGROUND: UDP-glucuronosyltransferase 1A1 (UGT1A1) is a pivotal enzyme involved in metabolism of SN-38, the active metabolite of irinotecan commonly used to treat metastatic colorectal cancer. We previously demonstrated aberrant methylation of specific CpG dinucleotides in UGT1A1-negative cells, and revealed that methylation state of the UGT1A1 5'-flanking sequence is negatively correlated with gene transcription. Interestingly, one of these CpG dinucleotides (CpG -4) is found close to a HNF1 response element (HRE), known to be involved in activation of UGT1A1 gene expression, and within an upstream stimulating factor (USF) binding site. RESULTS: Gel retardation assays revealed that methylation of CpG-4 directly affect the interaction of USF1/2 with its cognate sequence without altering the binding for HNF1-alpha. Luciferase assays sustained a role for USF1/2 and HNF1-alpha in UGT1A1 regulation in colon cancer cells. Based on the differential expression profiles of HNF1A gene in colon cell lines, we also assessed whether methylation affects its expression. In agreement with the presence of CpG islands in the HNF1A promoter, treatments of UGT1A1-negative HCT116 colon cancer cells with a DNA methyltransferase inhibitor restore HNF1A gene expression, as observed for UGT1A1. CONCLUSIONS: This study reveals that basal UGT1A1 expression in colon cells is positively regulated by HNF1-alpha and USF, and negatively regulated by DNA methylation. Besides, DNA methylation of HNF1A could also play an important role in regulating additional cellular drug metabolism and transporter pathways. This process may contribute to determine local inactivation of drugs such as the anticancer agent SN-38 by glucuronidation and define tumoral response.

In vitro glucuronidation of fenofibric acid by human UDP-glucuronosyltransferases and liver microsomes.

Fenofibric acid (FA), the active moiety of fenofibrate, is an agonist of the peroxisome proliferator-activated nuclear receptor alpha that modulates triglyceride and cholesterol profiles. Lipid response to fenofibrate and FA serum concentrations is highly variable. Although FA is reported to be almost exclusively inactivated by UDP-glucuronosyltransferases (UGTs) into FA-glucuronide (FA-G), the contribution of UGT isoenzymes has never been systematically assessed. Heterologously expressed human UGT1A and UGT2B and their coding variants were tested for FA glucuronidation using liquid chromatography/mass spectrometry. Recombinant UGT2B7 presented the highest V(max)/K(m) value (2.10 microl/min/mg), 16-fold higher than the activity of other reactive UGTs, namely, UGT1A3, UGT1A6, and UGT1A9 (0.13, 0.09, and 0.02 microl/min/mg, respectively). UGT2B7.1 (His(268)) and UGT2B7.2 (Tyr(268)) enzyme activity was similar, whereas UGT1A3.2 (R(11)A(47)), UGT1A3.3 (Trp(11)), and UGT1A9.3 (Thr(33)) showed 61 to 96% reduced V(max)/K(m) values compared with the respective (1) reference proteins. FA-G formation by a human liver bank (n = 48) varied by 10-fold, but the rate of formation was not associated with common genetic variations in UGT1A3, UGT1A6, UGT1A9, and UGT2B7. Correlation with activities for the probe substrates zidovudine (UGT2B7; r(2) = 0.75), mycophenolic acid (UGT1A9; r(2) = 0.42), fulvestrant (UGT1A3; r(2) = 0.36), but not serotonin (UGT1A6; r(2) = 0.06) indicated a primary role for UGT2B7 and lesser roles of UGT1A9 and UGT1A3 in hepatic FA glucuronidation. This was confirmed by a strong correlation of FA-G formation with UGT2B7 protein content and inhibition by fluconazole, a known UGT2B7 selective inhibitor. Additional studies are required to identify genetic factors contributing to the observed FA glucuronidation variability.

Characterization of common UGT1A8, UGT1A9, and UGT2B7 variants with different capacities to inactivate mutagenic 4-hydroxylated metabolites of estradiol and estrone.

The oxidative metabolism of estrone (E1) and estradiol (E2) to form carcinogenic 4-hydroxy-catecholestrogens (4-OHCE) is associated with uterine and breast carcinogenesis. In this study, we conducted functional analyses of genetic variants in the UDP-glucuronosyltransferase UGT1A8, UGT1A9, and UGT2B7 enzymes primarily involved in the inactivation of 4-OHCEs. Compared with UGT2B7*2 (H268Y), UGT2B7*1 exhibited a 2-fold lower efficiency (intrinsic clearance) at conjugating 4-hydroxyestrone and 4-hydroxyestradiol at positions 3 and 4 caused by altered capacities (Vmax) and affinities (Km). The -79 G>A promoter variation, characterizing the UGT2B7*2g haplotype, leads to a 50% reduction of transcription (P < 0.001) in human endometrial carcinoma-1B cells. Furthermore, a >12-fold decreased intrinsic clearance of the *1 proteins was induced by selected amino acid substitutions in UGT1A8 (*3 C277Y) and UGT1A9 (*3 M33T). Frequencies of the low-activity alleles in Caucasians were 45% for UGT2B7*1, 5% for the -79A promoter variant, 1.2% for UGT1A8*3, and 2.2% for UGT1A9*3. Supporting a protective role in two organs sensitive to 4-OHCE-induced damages, the expression of UGT enzymes was shown by immunohistochemistry in normal breast and endometrial tissues and confirmed by Western blotting in a subset of samples. Altogether, findings suggest that specific polymorphisms in UGT genes may modulate the exposure to carcinogenic metabolites of E2 and potentially lead to an altered risk of breast and endometrial cancers in women carrying the variant alleles.

A pharmacogenomics study of the human estrogen glucuronosyltransferase UGT1A3.

UGT1A3 is one of the most efficient at conjugating estrone, a precursor for biosynthesis of estradiol in peripheral tissues. We established the genetic mechanisms that might contribute to individual variation in UGT1A3 expression and activity. UGT1A3 first exon and 5'-flanking regions were sequenced in 249 Caucasians. We identified 17 polymorphisms, among them seven regulatory and 10 exonic polymorphisms with six leading to amino-acid changes. Luciferase reporter assays, site-directed mutagenesis and electrophoretic mobility shift assays using hepatoma HepG2 cells were carried out to show functionality of variant promoters. Reduced transcriptional activity was associated with all six variant promoters (two-fold; P<0.001). One of the potential mechanisms would involve the -148 T>C and -581 C>T variations that modulate gene function by affecting hepatocyte nuclear factor-1alpha and hepatocyte nuclear factor-4alpha binding, respectively. Then, estrone-conjugating activity was assessed with 11 heterologously expressed allozymes. Three phenotypes were observed; UGT1A3*1, *2 (WR, VA) and *3 (WR) with high intrinsic clearance values; UGT1A3*5 (QR, WR), *7 (FI), *9 (WR, ML), *10 (VA) and *11 (WR, VA and MI) had intermediate CLint (2X-10X lower vs. *1), whereas UGT1A3*4 (RW), *6 (WR, VA, MV) and *8 (AV) had low CLint (>10X lower vs. *1). Diplotype analyses indicate that 20.1% of individuals carry two alleles affecting UGT1A3 expression and/or activity. This study did not investigate genotype-phenotype association, but raise the possibility that genetically determined variation might contribute to variability in the inactivation of estrone by UGT1A3 and subsequent changes in lifetime exposure to estrogens potentially modifying risk of cancer.

Influence of nonsynonymous polymorphisms of UGT1A8 and UGT2B7 metabolizing enzymes on the formation of phenolic and acyl glucuronides of mycophenolic acid.

Mycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil (MMF), a standard immunosuppressive drug approved for clinical use in the prevention of acute allograft rejection after organ transplantation. This study examines the role of the genetic variants of UDP-glucuronosyltransferase (UGT) 1A8 and 2B7 enzymes involved in the formation of the primary metabolite of MPA, the inactive phenolic glucuronide (MPAG), and the reactive acyl glucuronide (AcMPAG). The first exon of UGT1A8 was first resequenced in the region encoding for the substrate binding domain in 254 Caucasians and 41 African Americans. Eight nonsynonymous changes were observed and led to the following amino acid substitutions: S43L, H53N, S126G, A144V, A173G, A231T, T240A, and C277Y. Thirteen haplotypes were inferred, comprising only two previously described alleles, namely, UGT1A8*2 (A173G) and UGT1A8*3 (C277Y). Upon stable expression in human embryonic kidney 293 cells, the UGT1A8*3 (C277Y), *5 (G173A240), *7 (A231T), *8 (S43L), and *9 (N53G) proteins were associated with the most profound decreases in the formation of MPAG and AcMPAG, indicating that these amino acids are critical for substrate binding and enzyme function. Altogether, the low-activity UGT1A8 enzymes are carried by 2.8 to 4.8% of the population. The variant of the UGT2B7 protein (UGT2B7*2 Y268), the main enzyme involved in the formation of AcMPAG, demonstrated a catalytic efficiency comparable with that of UGT2B7*1 (H268). In conclusion, although the common UGT2B7*2 variant is predicted to have limited impact, several UGT1A8 variants identified may potentially account for the large interindividual variance in MMF pharmacokinetics and deserve further clinical investigations.