Oliver Burk

The genetic determinants of the CYP3A5 polymorphism.

CYP3A proteins comprise a significant portion of the hepatic cytochrome P450 (CYP) protein and they metabolize around 50% of drugs currently in use. The dissection of the individual contributions of the four CYP3A genes identified in humans to overall hepatic CYP3A activity has been hampered by sequence and functional similarities. We have investigated the expression of CYP3A5 and its genetic determinants in a panel of 183 Caucasian liver samples. CYP3A5 expression is increased in 10% of livers in this ethnic group. Using a high density map of CYP3A5 variants, we searched for genetic markers of the increased CYP3A5 expression. In agreement with an independent, recent study, we report that a SNP within intron 3 (g.6986G>A) is the primary cause of the CYP3A5 protein polymorphism. The frequencies of the g.6986A variant which allow for normal splicing of CYP3A5 transcripts are 5% in Caucasians, 29% in Japanese, 27% in Chinese, 30% in Koreans and 73% in African-Americans. In the last ethnic group, the expression of CYP3A5 in some individuals who carry the g.6986A variant is affected adversely by a frame shift mutation (CYP3A5*7, D348., q = 0.10). In summary, these results should add to efforts to identify clinically relevant, CYP3A5-specific reactions and to further elucidate traits responsible for variable expression of the entire CYP3A family.

Association of the P-Glycoprotein Transporter MDR1C3435T Polymorphism with the Susceptibility to Renal Epithelial Tumors

Except for hereditary disease, genetic factors that contribute to the development of renal epithelial tumors are unknown. There is a possibility that the MDR1 encoded plasma membrane transporter P-glycoprotein (PGP) influences the risk of development of renal neoplasms. PGP is known to be involved in uptake, binding, transport, and distribution of xenobiotics. There is evidence that the MDR1(C3435T) polymorphism drives expression and modulates disease risk. In an explorational case-control study, constitutional genotype frequencies were established at MDR1(C3435T) of 537 healthy control subjects and compared with those of 212 patients with renal epithelial tumors. There were 179 clear cell renal cell carcinoma (CCRCC) and 33 tumors collectively assigned as non-CCRCC. In a second study, genotypes of another 150 healthy control subjects and 50 patients with three non-CCRCC types (26 papillary RCC, 11 chromophobe RCC, and 13 renal oncocytic adenoma) were compared. PCR-restriction fragment length polymorphism-based analysis of constitutional DNA, and statistical analysis were applied. PGP expression was analyzed by quantitative immunohistochemistry. The explorational study showed a significant association between T allele frequency and the occurrence of tumors (P = 0.007). When tumors were histopathologically distinguished into frequent CCRCC and less frequent non-CCRCC, both patient groups contributed to this effect with a seemingly strong influence by the latter (P = 0.0419). The second study established the T allele as a risk factor especially for non-CCRCC (P = 0.0005) with the highest risk for homozygote TT allele carriers (P < 0.0001). Independently, MDR1(C3435T) genotype associated variations in PGP expression were shown in normal renal parenchyma with a 1.5-fold difference of median values (TT, 1.9; CC, 2.8; P = 0.0065). The data provide evidence for PGP to influence the susceptibility to develop renal epithelial tumors by virtue of its MDR1(C3435T) polymorphism and changes in expression. Especially T and TT carriers are at risk for developing non-CCRCC, i.e., papillary and chromophobe RCC as well as oncocytic adenomas.

Genomic organization of the human CYP3A locus: identification of a new, inducible CYP3A gene.

Proteins encoded by the human CYP3A genes metabolize every second drug currently in use. The activity of CYP3A gene products in the general population is highly variable and may affect the efficacy and safety of drugs metabolized by these enzymes. The mechanisms underlying this variability are poorly understood, but they include gene induction, protein inhibition and unknown genetic polymorphisms. To better understand the regulation of CYP3A expression and to provide a basis for a screen of genetic polymorphisms, we determined and analysed the sequence of the human CYP3A locus. The 231 kb locus sequence contains the three CYP3A genes described previously (CYP3A4, CYP3A5 and CYP3A7), three pseudogenes as well as a novel CYP3A gene termed CYP3A43. The gene encodes a putative protein with between 71.5% and 75.8% identity to the other CYP3A proteins. The highest expression level of CYP3A43 mRNA is observed in the prostate, an organ with extensive steroid metabolism. CYP3A43 is also expressed in several other tissues including liver, where it can be induced by rifampicin. CYP3A43 transcripts undergo extensive splicing. The identification of a new member of the CYP3A family and the characterization of the full CYP3A locus will aid efforts to identify the genetic variants underlying its variable expression. This, in turn, will lead to a better optimization of therapies involving the numerous substrates of CYP3A proteins.

Synergistic activation of the chicken mim-1 gene by v-myb and C/EBP transcription factors.

The retroviral oncogene v‐myb encodes a transcriptional activator which is responsible for the activation of the mim‐1 gene in myelomonocytic cells transformed by v‐myb. The mim‐1 promoter contains several myb consensus binding sites and has previously been shown to be regulated directly by v‐myb. Here we report that the mim‐1 gene is activated synergistically by v‐myb and different C/EBP transcription factors. We have cloned a chicken C/EBP‐related gene that is highly expressed in myeloid cells and identified it as the chicken homolog of C/EBP beta. A dominant‐negative variant of chicken C/EBP beta interferes with the v‐myb induced activation of the mim‐1 gene in these cells, suggesting that C/EBP beta or another C/EBP transcription factor is required for the activation of mim‐1 by v‐myb. We found that C/EBP beta and other C/EBP transcription factors confer to fibroblasts the ability to induce the mim‐1 gene in the presence of v‐myb. Finally we show that, in contrast to v‐myb, c‐myb synergizes with C/EBP transcription factors only at low concentrations of c‐myb protein. Our results suggest a role for C/EBP beta, and possibly for other C/EBP transcription factors, in v‐myb function and in myeloid‐specific gene activation.

Cytochrome P450 3A and their regulation

CYP3A isozymes collectively comprise the largest portion of the liver and small intestinal CYP protein and they are involved in the metabolism of 45–60% of all currently used drugs. In addition to drugs, CYP3A isozymes metabolise a variety of other compounds including steroid hormones, toxins and carcinogens. It is also well known that the hepatic expression and activity of CYP3A isozymes varies from individual to individual. The involvement of this variability in harmful interactions frequently encountered in development and application of drugs that are CYP3A substrates is well documented. It has also been postulated that variable CYP3A expression could affect an individual’s predisposition to cancers caused by environmental carcinogens that are metabolised by CYP3A. The elucidation of factors controlling an individual’s CYP3A activity could permit personalised dose adjustments in therapies with its substrates and may also possibly lead to the identification of sub-populations at increased risk for several common cancers. However, until recently, the development of markers predictive for the individual CYP3A expression has been slower than for other drug metabolising enzymes. Here we summarise the current status of our understanding of the genetics and regulation of the expression of CYP3A, including the recently described markers of the CYP3A5 and CYP3A7 polymorphisms. These latter markers are expected to speed up the development of activity probes for the individual CYP3A isozymes and to aid in our understanding of their individual functions.

A role for constitutive androstane receptor in the regulation of human intestinal MDR1 expression.

Abstract MDR1/P-glycoprotein is an efflux transporter determining the absorption and presystemic elimination of many xenobiotics in the gut. Thus, interindividual differences in MDR1 expression may affect the efficacy of drug treatment. The expression of MDR1 is partially controlled by the pregnane X receptor (PXR), which mediates induction by many xenobiotics. Since it has been described that the nuclear receptors PXR and constitutive androstane receptor (CAR) can bind to the same binding sites, we investigated the role of CAR in the regulation of MDR1 gene expression. We demonstrate here by gel shift and transfection experiments that CAR binds to distinct nuclear receptor response elements in the -7.8 kbp enhancer of MDR1 and transactivates MDR1 expression through DR4 motifs to which the receptor binds as a heterodimer with RXR or as a monomer, respectively. Expression of the endogenous MDR1 gene is elevated in cells stably expressing CAR, thus arguing for the functional relevance of CAR-dependent activation of MDR1. The physiological relevance of the regulation of MDR1 by CAR is further suggested by correlation of the expression of CAR and MDR1 in the human small intestine. In summary, our data suggest that CAR plays a role in the regulation of intestinal MDR1 expression.

CYP3A genetics in drug metabolism

Most drugs are metabolized by CYP3A enzymes, and variations in expression levels of these enzymes are believed to determine whether patients will have a positive or adverse drug response. Little is known about the mechanisms that underlie inter-individual differences in CYP3A expression, but the mapping of human genome sequence variations will facilitate the search for answers.

Estrogen-dependent alterations in differentiation state of myeloid cells caused by a v-myb/estrogen receptor fusion protein.

The oncogene v‐myb and its cellular progenitor c‐myb encode nuclear, DNA binding phosphoproteins that are thought to regulate the expression of myb‐responsive genes during myeloid differentiation. To identify such myb‐regulated genes, and to explore the mechanisms by which v‐myb affects their expression, we have established a conditional expression system for v‐myb. We have converted the v‐myb protein to an estrogen‐inducible transactivator by fusing the protein to the hormone binding domain of the human estrogen receptor. Expression of the chimeric protein in a chicken macrophage cell‐line causes estrogen‐dependent, reversible changes in the differentiation state as well as alterations in the gene expression program of the cells. We have used this estrogen‐dependent v‐myb expression system to identify a novel v‐myb regulated gene.

tom‐1, a novel v‐Myb target gene expressed in AMV‐ and E26‐transformed myelomonocytic cells

The retroviral oncogene v‐myb is a mutated and truncated version of the c‐myb proto‐oncogene and encodes a transcription factor (v‐Myb) that specifically transforms myelomonocytic cells. Two different variants of v‐myb, transduced independently by the oncogenic chicken retroviruses AMV and E26, have been characterized. It is believed that both variants of v‐Myb transform myelomonocytic cells by affecting the expression of specific genes; however, no target genes common to both oncogenic viruses have been identified. Here, we describe the identification of a novel v‐Myb target gene, designated as tom‐1 (target of myb 1). The tom‐1 gene has two promoters, one of which is Myb‐inducible. tom‐1 is expressed at elevated levels in AMV‐transformed as well as in E26‐transformed myeloid cells. We show that tom‐1 activation by v‐Myb does not require de novo protein synthesis and that the Myb‐inducible tom‐1 promoter contains a functional Myb binding site. Thus, tom‐1 is the first example of a direct target gene for both oncogenic forms of the v‐myb gene. Further analysis of the Myb‐inducible tom‐1 promoter shows that a C/EBP binding site is juxtaposed to the Myb binding site and that C/EBP is required for the Myb‐dependent activation of the promoter. Together with previous work our results suggest that C/EBP may be a general cooperation partner for v‐Myb in myelomonocytic cells.

Direct Transcriptional Regulation of Human Hepatic Cytochrome P450 3A4 (CYP3A4) by Peroxisome Proliferator–Activated Receptor Alpha (PPARα)

The nuclear receptor peroxisome proliferator–activated receptor (PPAR)α is known primarily as a regulator of fatty acid metabolism, energy balance, and inflammation, but evidence suggests a wider role in regulating the biotransformation of drugs and other lipophilic chemicals. We investigated whether PPARα directly regulates the transcription of cytochrome P450 3A4, the major human drug-metabolizing enzyme. Using chromatin immunoprecipitation in human primary hepatocytes as well as electrophoretic mobility shift and luciferase reporter-gene assays, we identified three functional PPARα-binding regions (PBR-I, -II, and -III) within ∼12 kb of the CYP3A4 upstream sequence. Furthermore, a humanized CYP3A4/3A7 mouse model showed in vivo induction of CYP3A4 mRNA and protein by [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (WY14,643) in liver but not in intestine, whereas hepatic occupancy of PBRs by PPARα was ligand independent. Using lentiviral gene knock-down and treatment with WY14,643 in primary human hepatocytes, PPARα was further shown to affect the expression of a distinct set of CYPs, including 1A1, 1A2, 2B6, 2C8, 3A4, and 7A1, but not 2C9, 2C19, 2D6, or 2E1. Interestingly, the common phospholipid 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (16:0/18:1-PC), previously proposed to reflect nutritional status and shown to be a specific endogenous ligand of PPARα, induced CYP3A4 (up to 4-fold) and other biotransformation genes in hepatocytes with similar selectivity and potency as WY14,643. These data establish PPARα as a direct transcriptional regulator of hepatic CYP3A4. This finding warrants investigation of both known and newly developed PPARα-targeted drugs for their drug-drug interaction potential. Furthermore, our data suggest that nutritional status can influence drug biotransformation capacity via endogenous phospholipid signaling.