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Leszek Wojnowski

Publications and source records attributed to Leszek Wojnowski.

At least 19 recordsLinked to original sources
PubMed2006

Topoisomerase II beta expression level correlates with doxorubicin-induced apoptosis in peripheral blood cells.

Anthracyclines are widely used in oncology. Both the response and side-effects of anthracyclines are individually variable, but determinants or predictive markers of this variability are not available. We investigated the variability in the expression of the anthracycline targets topoisomerases II (topo II) alpha and beta and its significance for the apoptotic response following exposure to the anthracycline doxorubicin. Only topo II beta protein expression was detected in peripheral blood cells. Usually considered a constitutively expressed protein, topo II beta varied 3-, 18-, and 16-fold on the mRNA, protein and activity levels, respectively, among the volunteers tested. In addition, the expression of topo II beta was modified by several mitogens, suggesting a role in the regulation of cell cycle. Strikingly, topo II beta activity correlated statistically significantly with the apoptotic response in peripheral blood leukocytes exposed to 1 microM doxorubicin. A longitudinal study in a subset of study subjects demonstrated that 30% of the topo II expression variability may be inherited. However, resequencing of the TOP2B gene in 48 unrelated individuals revealed only 8 gene variants, none of them with obvious effects on the expression or protein sequence of topo II beta. Taken together, the apoptotic response to doxorubicin in peripheral blood cells may be mediated by topo II beta. The expression level of topo II beta is intra- and inter-individually variable, and may in part determine the apoptotic response to doxorubicin and other anthracyclines.

Antibiotics, Antineoplastic↗
PubMed2006

Dominant contribution of P450 3A4 to the hepatic carcinogenic activation of aflatoxin B1.

The hepatic carcinogen aflatoxin B1 (AFB1) is metabolized in the liver by at least four different P450s, all of which exhibit large interindividual differences in the expression levels. These differences could affect the individual risk of hepatocellular carcinoma (HCC). We investigated the metabolism of AFB1 in a panel of 13 human liver microsomal preparations using a hepatic abundance model, which takes into account the specific kinetic parameters and the expression levels of these P450s. We found a 12-fold variability in the production rate of the carcinogenic metabolite AFB1-8,9-epoxide (AFBO) and a 22-fold variability in the production of the detoxification product AFQ1. The ratio between the AFBO and the AFQ1 production rates varied between 1:19 and 1:1.7. P450 3A4 contributed a majority of AFBO and AFQ1, and its expression level was the most important determinant of the AFB1 disposition toward these primary metabolites. P450 3A5, which exclusively produced AFBO, was the second-most important enzyme activating AFB1 to AFBO, followed by P450 3A7 and P450 1A2. The relative contribution of AFBO by P450 3A5 strongly depended on the concomitant expression of P450 3A4, and it was as high as 15% in a P450 3A5 high expressor with the lowest P450 3A4 expression of all livers. The P450 1A2-specific AFB1 detoxification product AFM1 was not detected. In conclusion, the variable expression of P450s has a major effect on the carcinogenic activation of AFB1, which may affect the individual predisposition to HCC. P450 3A4 expression is the most important determinant of AFB1 activation to AFBO. The contribution of P450 1A2 to AFB1 metabolism appears to be negligible and may have been overestimated. Targeted chemoprevention of AFB1-associated HCC should consider P450 3A4 inhibitors and avoidance of P450 3A4 inducers.

Aflatoxin B1↗
PubMed2006

Association of genetic polymorphism in ABCC2 with hepatic multidrug resistance-associated protein 2 expression and pravastatin pharmacokinetics.

OBJECTIVE: Our aim was to investigate possible effects of sequence variations in ABCC2, encoding the multidrug resistance-associated protein 2 (MRP2), on the pharmacokinetics of the MRP2 substrate pravastatin. METHODS: Deoxyribonucleic acid samples of 41 healthy volunteers, in whom SLCO1B1 single nucleotide polymorphisms (SNPs) and haplotypes had previously been found to be associated with increased plasma pravastatin concentrations, were investigated. Each study participant had ingested a single 40-mg dose of pravastatin followed by blood sampling for pharmacokinetic characterization in standardized conditions. The exons, exon-intron boundaries, promoter region and 3'-untranslated region of the ABCC2 gene of six individuals with the highest and six individuals with the lowest pravastatin area under the plasma concentration-time curve (AUC) values were sequenced. RESULTS: Of the 26 sequence variations found, the synonymous c.1446C>G SNP was observed heterozygously in three (50%) of the six individuals with a low pravastatin AUC and in none (0%) of the six individuals with a high AUC (P=0.06 for allele frequency). The remaining 29 participants were then also genotyped for c.1446C>G, but none of them carried the SNP. In addition, the effect of c.1446C>G on MRP2 mRNA expression was investigated in 93 human liver samples. A multiple linear regression analysis in the 41 participants with pravastatin pharmacokinetic data indicated that the ABCC2 c.1446C>G SNP and the previously identified SLCO1B1 haplotype *17 were independent predictors of the AUC0-12 and Cmax of pravastatin (r=32 and 29%, respectively) (P<0.01). In the participants heterozygous for the ABCC2 c.1446C>G SNP (n=3), who were not carriers of the SLCO1B1*17 haplotype, the AUC0-12 and Cmax of pravastatin were 67 and 68% lower than in those carrying neither the SLCO1B1*17 haplotype nor the ABCC2 c.1446C>G SNP (n=35) (P<0.05). MRP2 mRNA expression was 95% higher in livers with the c.1446CG genotype (n=7) than in those with the c.1446CC genotype (n=86) (P<0.05). CONCLUSIONS: These results support the idea that the ABCC2 c.1446C>G SNP is associated with reduced systemic exposure to pravastatin as a consequence of increased MRP2 expression. The underlying mechanism may involve either a modulating effect of the SNP on mRNA stability or linkage to other polymorphism(s) acting at the transcriptional level.

Cells, Cultured↗
PubMed2006

Clinical implications of CYP3A polymorphisms.

Due to their enormous substrate spectrum CYP3A4, -3A5 and -3A7 constitute the most important drug-metabolising enzyme subfamily in humans. CYP3As are expressed predominantly, but not exclusively, in the liver and intestine, where they participate in the metabolism of 45 - 60% of currently used drugs and many other compounds such as steroids and carcinogens. CYP3A expression and activity vary interindividually due to a combination of genetic and nongenetic factors such as hormone and health status, and the impact of environmental stimuli. Over the past several years, genetic determinants have been identified for much of the variable expression of CYP3A5 and -3A7, but not for CYP3A4. Using these markers, an effect of CYP3A5 expression status has been demonstrated beyond doubt for therapies with the immunosuppressive drug tacrolimus. Further associations are likely to emerge for drugs metabolised predominantly by CYP3A5 or -3A7, especially for individuals or tissues with concomitant low expression of CYP3A4. However, as exemplified by the controversial association between CYP3A4*1B and prostate cancer, the detection of clinical effects of CYP3A gene variants will be difficult. The most important underlying problems are the continuing absence of activity markers specific for CYP3A4 and the strong contribution of nongenetic factors to CYP3A variability.

Cyclosporine↗
PubMed2005

NAD(P)H oxidase and multidrug resistance protein genetic polymorphisms are associated with doxorubicin-induced cardiotoxicity.

BACKGROUND: A significant number of patients treated with anthracyclines develop cardiotoxicity (anthracycline-induced cardiotoxicity [ACT]), mainly presenting as arrhythmias (acute ACT) or congestive heart failure (chronic ACT). There are no data on pharmacogenomic predictors of ACT. METHODS AND RESULTS: We genotyped participants of the German non-Hodgkin lymphoma study (NHL-B) who were followed up for the development of heart failure for a median of >3 years. Single-nucleotide polymorphisms (SNPs) were selected from 82 genes with conceivable relevance to ACT. Of 1697 patients, 55 developed acute and 54 developed chronic ACT (cumulative incidence of either form, 3.2%). We detected 5 significant associations with polymorphisms of the NAD(P)H oxidase and doxorubicin efflux transporters. Chronic ACT was associated with a variant of the NAD(P)H oxidase subunit NCF4 (rs1883112, -212A-->G; symbols with right-pointing arrows, as edited?' odds ratio [OR], 2.5; 95% CI, 1.3 to 5.0). Acute ACT was associated with the His72Tyr polymorphism in the p22phox subunit (rs4673; OR, 2.0; 95% CI, 1.0 to 3.9) and with the variant 7508T-->A (rs13058338; OR, 2.6; 95% CI, 1.3 to 5.1) of the RAC2 subunit of the same enzyme. In agreement with these results, mice deficient in NAD(P)H oxidase activity, unlike wild-type mice, were resistant to chronic doxorubicin treatment. In addition, acute ACT was associated with the Gly671Val variant of the doxorubicin efflux transporter multidrug resistance protein 1 (MRP1) (OR, 3.6; 95% CI, 1.6 to 8.4) and with the Val1188Glu-Cys1515Tyr (rs8187694-rs8187710) haplotype of the functionally similar MRP2 (OR, 2.3; 95% CI, 1.0 to 5.4). Polymorphisms in adrenergic receptors previously demonstrated to be predictive of heart failure were not associated with ACT. CONCLUSIONS: Genetic variants in doxorubicin transport and free radical metabolism may modulate the individual risk to develop ACT.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Contribution of CYP3A5 to the in vitro hepatic clearance of tacrolimus.

BACKGROUND: Tacrolimus is metabolized predominantly to 13-O-demethyltacrolimus in the liver and intestine by cytochrome P450 3A (CYP3A). Patients with high concentrations of CYP3A5, a CYP3A isoenzyme polymorphically produced in these organs, require higher doses of tacrolimus, but the exact mechanism of this association is unknown. METHODS: cDNA-expressed CYP3A enzymes and a bank of human liver microsomes with known CYP3A4 and CYP3A5 content were used to investigate the contribution of CYP3A5 to the metabolism of tacrolimus to 13-O-demethyltacrolimus as quantified by liquid chromatography-tandem mass spectrometry. RESULTS: Demethylation of tacrolimus to 13-O-demethyltacrolimus was the predominant clearance reaction. Calculated K(m) and V(max) values for CYP3A4, CYP3A5, and CYP3A7 cDNA-expressed microsomes were 1.5 micromol/L and 0.72 pmol x (pmol P450)(-1) x min(-1), 1.4 micromol/L and 1.1 pmol x (pmol P450)(-1) x min(-1), and 6 micromol/L and 0.084 pmol x (pmol P450)(-1) x min(-1), respectively. Recombinant CYP3A5 metabolized tacrolimus with a catalytic efficiency (V(max)/K(m)) that was 64% higher than that of CYP3A4. The contribution of CYP3A5 to 13-O-demethylation of tacrolimus in human liver microsomes varied from 1.5% to 40% (median, 18.8%). There was an inverse association between the contribution of CYP3A5 to 13-O-demethylation and the amount of 3A4 protein (r = 0.90; P <0.0001). Mean 13-O-demethylation clearances in CYP3A5 high and low expressers, estimated by the parallel-tube liver model, were 8.6 and 3.57 mL x min(-1) x (kg of body weight)(-1), respectively (P = 0.0088). CONCLUSIONS: CYP3A5 affects metabolism of tacrolimus, thus explaining the association between CYP3A5 genotype and tacrolimus dosage. The importance of CYP3A5 status for tacrolimus clearance is also dependent on the concomitant CYP3A4 activity.

Baculoviridae↗
PubMed2005

A critical function for B-Raf at multiple stages of myelopoiesis.

Raf kinases play an integral role in the classic mitogen-activated protein (MAP) kinase (Raf/MEK/extracellular signal-related kinase [ERK]) intracellular signaling cascade, but their role in specific developmental processes is largely unknown. Using a genetic approach, we have identified a role for B-Raf during hematopoietic progenitor cell development and during megakaryocytopoiesis. Fetal liver and in vitro embryonic stem (ES) cell-derived myeloid progenitor development is quantitatively impaired in the absence of B-Raf. Biochemical data suggest that this phenotype is due to the loss of a normally occurring rise in B-Raf expression and associated ERK1/2 activation during hematopoietic progenitor cell formation. However, the presence of B-raf-/- ES cell-derived myeloid progenitors in the bone marrow of adult chimeric mice indicates the lack of an obligate cell-autonomous requirement for B-Raf in myeloid progenitor development. The lack of B-Raf also impairs megakaryocytopoiesis. Thrombopoietin (Tpo)-induced in vitro expansion of ES cell-derived megakaryocyte-lineage cells fails to occur in the absence of B-Raf. Moreover, this quantitative in vitro defect in megakaryocyte-lineage expansion is mirrored by chimeric mice data that show reduced B-raf-/- genotype contribution in megakaryocytes relative to its contribution in myeloid progenitors. Together, these data suggest that B-Raf plays a cell-autonomous role in megakaryocytopoiesis and a permissive role in myeloid progenitor development.

Animals↗
PubMed2005

Genome-wide single-nucleotide polymorphism arrays demonstrate high fidelity of multiple displacement-based whole-genome amplification.

Whole-genome DNA amplification by multiple displacement (MD-WGA) is a promising tool to obtain sufficient DNA amounts from samples of limited quantity. Using Affymetrix' GeneChip Human Mapping 10K Arrays, we investigated the accuracy and allele amplification bias in DNA samples subjected to MD-WGA. We observed an excellent concordance (99.95%) between single-nucleotide polymorphisms (SNPs) called both in the nonamplified and the corresponding amplified DNA. This concordance was only 0.01% lower than the intra-assay reproducibility of the genotyping technique used. However, MD-WGA failed to amplify an estimated 7% of polymorphic loci. Due to the algorithm used to call genotypes, this was detected only for heterozygous loci. We achieved a 4.3-fold reduction of noncalled SNPs by combining the results from two independent MD-WGA reactions. This indicated that inter-reaction variations rather than specific chromosomal loci reduced the efficiency of MD-WGA. Consistently, we detected no regions of reduced amplification, with the exception of several SNPs located near chromosomal ends. Altogether, despite a substantial loss of polymorphic sites, MD-WGA appears to be the current method of choice to amplify genomic DNA for array-based SNP analyses. The number of nonamplified loci can be substantially reduced by amplifying each DNA sample in duplicate.

Genome, Human↗
PubMed2005

Application of genomewide SNP arrays for detection of simulated susceptibility loci.

The prospect of SNP-based genomewide association analysis has been extensively discussed, but practical experiences remain limited. We performed an association study using a recently developed array of 11,555 SNPs distributed throughout the human genome. A total of 104 DNA samples were hybridized to these chips with an average call rate of 97% (range 85.3-98.6%). The resulting genomewide scans were applied to distinguish between carriers and noncarriers of 37 test variants, used as surrogates for monogenic disease traits. The test variants were not contained in the chip and had been determined by other methods. Without adjustment for multiple testing, the procedure detected 24% of the test variants, but the positive predictive value was low (2%). Adjustment for multiple testing eliminated most false-positive associations, but the share of true positive associations decreased to 10-12%. We also simulated fine-mapping of susceptibility loci by restricting testing to the immediate neighborhood of test variants (+/-5 Mb). This increased the proportion of correctly identified test variants to 22-27%. Simulation of a bigenic inheritance reduced the sensitivity to 1%. Similarly adverse effect had reduction of allelic penetrance. In summary, we demonstrate the feasibility and considerable specificity of SNP array-based association studies to detect variants underlying monogenic, highly penetrant traits. The outcome is affected by allelic frequencies of chip SNPs, by the ratio between simulated "cases" and "controls," and by the degree of linkage disequilibrium. A major improvement is expected from raising the density of the SNP array.

Alleles↗
PubMed2005

Three haplotypes associated with CYP2A6 phenotypes in Caucasians.

The human cytochrome P450 2A6 (CYP2A6) enzyme metabolizes several xenobiotic compounds of clinical or toxicological importance. We aimed to identify genetic variants and major CYP2A6 haplotypes associated with CYP2A6 phenotypic variation. CYP2A6 mRNA level, protein level, activity and haplotypes were determined in Caucasian liver samples via real-time polymerase chain reaction, Western blot, coumarin 7-hydroxylation, DNA sequencing and genotyping, respectively. Phenotypes were then analyzed for associations with haplotypes. CYP2A6 transcript, protein and activity levels were correlated among each other. In 45 African-American, 156 Caucasian, 47 Chinese, 50 Japanese and 47 Korean DNA samples, we detected 95 different polymorphisms in the CYP2A6 gene, 49 of which had not been described previously. Caucasian variants formed 33 haplotypes which built four clades. Allele *9B and the CYP2A7/2A6 partial deletion allele CYP2A6*12B were both associated with decreased expression. The latter haplotype extends at least over 147 kb up into the CYP2B6 gene. A haplotype almost identical to allele *1A was associated with decreased expression and activity of CYP2A6 compared to all other haplotypes. In summary A CYP2A6*1A-like allele, *9B and *12B are major genetic determinants of CYP2A6 phenotype variation in Caucasians.

Alleles↗
PubMed2005

A model for PTCH1/Ptch1-associated tumors comprising mutational inactivation and gene silencing.

Mutations of the Sonic hedgehog (SHH) receptor, Patched1 (PTCH1), have been identified in a variety of tumors. PTCH1 is usually considered to be a tumor suppressor gene. However, one normal allele is retained in many tumors. We investigated the mechanism of tumorigenesis in murine heterozygous Ptch1 knock-out mice. Here we show that Ptch1 transcripts, which are consistently overexpressed in tumors in these mice, are derived predominantly from the mutated allele. These transcripts give rise to a mutant protein incapable of pathway inhibition. In contrast, the expression of wild-type transcripts in the tumor is reduced. The transcriptional activity of a Ptch1 promoter is sensitive to methylation. Based on these results, we propose a model, in which tumorigenesis begins with the transcriptional silencing of one PTCH1/Ptch1 allele. This alone has no functional consequences. Upon mutational inactivation of the other allele, the resulting loss of PTCH1/Ptch1 function activates PTCH1/Ptch1 transcription from the non-silenced, i.e. the mutant, allele. These events can occur in an opposite order. This model is consistent with the expression of PTCH1/Ptch1-derived transcripts and proteins found in tumors, with the sensitivity of the murine Ptch1 promoter to methylation, and with the recently reported effect of demethylating agents on Ptch1 expression. These latter agents could be effective in treatment of, at least, some tumors associated with loss of PTCH1 function.

Alleles↗
PubMed2004

The induction of cytochrome P450 3A5 (CYP3A5) in the human liver and intestine is mediated by the xenobiotic sensors pregnane X receptor (PXR) and constitutively activated receptor (CAR).

Induction of cytochrome P450 3A (CYP3A) by xenobiotics may lead to clinically relevant drug interactions. In contrast with other CYP3A family members, studies on the inducibility of CYP3A5 indicate conflicting results. We report the induction of CYP3A5 mRNA in 13 of 16 hepatocyte preparations exposed to rifampin. Furthermore, induction of CYP3A5 mRNA was observed in intestinal biopsies in three of eight probands following exposure to the antibiotic. The highest absolute levels of CYP3A5 transcripts were found following rifampin treatment in hepatocytes and intestines from carriers of CYP3A5*1 alleles. Elucidation of the mechanism involved in CYP3A5 induction revealed that constitutively activated receptor (CAR) and pregnane X receptor (PXR) transactivated the CYP3A5 promoter (-688 to +49) and that the transactivation was dependent on an everted repeat separated by 6 bp (ER6-dependent). Treatment with the prototypical PXR ligand rifampin led to a 2-fold induction of the CYP3A5 promoter activity. In agreement with these observations, PXR and CAR bound specifically to the ER6 motif. Hepatic expression of PXR correlated with that of CYP3A5 mRNA levels in a bank of liver samples. Taken together, studies here revealed the presence of a functional ER6 motif in the CYP3A5 promoter located -100 bp upstream from the transcription start site, suggesting that CYP3A5 is inducible by mechanisms similar to those involved in CYP3A4 induction. Enhanced expression of CYP3A5 caused by exposure to inducers may phenocopy the effects of the high expression allele CYP3A5*1. In this manner, induction of CYP3A5 may contribute to the overall importance of this P450 in drug metabolism and drug interactions.

Alleles↗
PubMed2004

Limited contribution of CYP3A5 to the hepatic 6beta-hydroxylation of testosterone.

The polymorphic expression of CYP3A5 in human livers is well established, but its significance for the entire hepatic CYP3A activity is disputed. We investigated the contribution of CYP3A5 to the CYP3A activity assessed as 6beta-hydroxylation of testosterone using baculovirus-expressed CYP3A4 and CYP3A5 and microsomes isolated from 47 Caucasian human livers. Under comparable conditions, the specific activities of baculovirus-expressed CYP3A4 and CYP3A5 were nearly identical. Among human livers tested, the Vmax of 6beta-testosterone hydroxylation varied 28-fold. Of the enzymes that are capable of catalyzing 6beta-hydroxylation of testosterone (CYP3A and CYP1A1), only CYP3A4 mRNA and protein expression correlated significantly with the Vmax values (r=0.51, p<0.001 and r=0.66, p<0.001, respectively). Neither consideration of the CYP3A5 polymorphism nor combining CYP3A4 mRNA expression with the expression of other CYP3A mRNA species increased the correlation. The five livers heterozygous for the CYP3A5*1 allele had a mean 6beta-testosterone hydroxylation Vmax value of 2,976 pmol/mg/min, compared with 3,798 pmol/mg/min in the homozygous CYP3A5*3 livers. Together, these data suggest that the specific activities of CYP3A4 and CYP3A5 towards testosterone are comparable. However, the contribution of CYP3A5 to 6beta-hydroxylation of testosterone in Caucasian livers is limited, due to the much lower expression levels of CYP3A5.

Baculoviridae↗
PubMed2004

Genetics of the variable expression of CYP3A in humans.

CYP3A isozymes participate in the metabolism of 45-60% of currently used drugs and of a variety of other compounds such as steroid hormones, toxins, and carcinogens. The CYP3A expression status is a major determinant of drug efficacy and safety, and it may also affect an individual's predisposition to certain cancers. The inter- and intraindividual expression of CYP3A is variable because of a complex interplay between genetic and environmental factors. Markers predictive of the individual CYP3A activity could improve therapies with CYP3A substrates by personalised dose adjustments, but their development has been slower than for other drug-metabolizing enzymes. Here we summarize the recent progress in genomics and regulation of CYP3A. The recently described markers of the CYP3A5 and CYP3A7 polymorphisms should facilitate the development of isozyme-specific activity markers for the individual CYP3A isozymes, including CYP3A4.

Aryl Hydrocarbon Hydroxylases↗
PubMed2004

Single nucleotide polymorphism characterization by mRNA expression imbalance assessment.

The functional characterization of single nucleotide polymorphism (SNPs) represents a major challenge for pharmacogenetics and related research areas. Here, we propose a procedure, termed mRNA expression imbalance assessment, that can be applied to detect cis-acting SNPs with an effect on mRNA expression. The procedure is based on the observation that the relative transcript levels derived from the two alleles of an autosomal gene are reflected in the sequence of the amplified cDNA. The key element of the procedure is the detection of a discrepancy between the specific nucleotide signal intensity of a marker SNP in the genomic DNA and in the cDNA. We used the CYP3A5*1/*3 polymorphism as a proof principle for this approach. In this case, we could even demonstrate that the procedure works equally well with the appropriate tissue samples and in silico, using the existing databanks of sequences. In conclusion, the procedure provides a fast and easy tool, which may facilitate identification of functional SNPs.

Base Sequence↗
PubMed2004

Increased levels of aflatoxin-albumin adducts are associated with CYP3A5 polymorphisms in The Gambia, West Africa.

OBJECTIVES: Major risk factors for hepatocellular carcinoma (HCC) are hepatitis viruses and exposure to aflatoxins, including aflatoxin B1 (AFB1). The mutagenic effect of AFB1 results from hepatic bioactivation to AFB1-exo-8,9-epoxide. This is in part catalysed by CYP3A5, an enzyme expressed polymorphically. We investigated the role of CYP3A5 polymorphisms in the formation of AFB1-exo-8,9-epoxide in The Gambia, a population exposed to high aflatoxin levels. METHODS: Common CYP3A5 polymorphisms were identified in an African-American population. Subsequently, 288 Gambian subjects were genotyped and CYP3A5 activity predicted using haplotypes of the three variant loci (CYP3A5*3, *6 and *7) associated with decreases in protein expression. CYP3A5 expression was then compared to aflatoxin-albumin (AF-alb) adduct, a biomarker of AFB1 bioactivation; data were also analysed in relation to expression of other aflatoxin-metabolizing enzymes. RESULTS: CYP3A5 haplotypes reflecting high CYP3A5 protein expression were associated with increased AF-alb. Compared to individuals with predicted low expression those predicted to express CYP3A5 from one allele displayed 16.1% higher AF-alb (95% CI: -2.5, 38.2, P = 0.093) and homozygous expressers displayed 23.2% higher AF-alb levels (95% CI: -0.01, 52.0, P = 0.051). The effect of the CYP3A5 polymorphism was strongest in individuals with low CYP3A4 activity with a 70.1% increase in AF-alb (95% CI: 11.8, 158.7, P < 0.05) in high compared to low expressers. A similar effect was observed for individuals with null alleles of GSTM1, which conjugates the AFB1-exo-8,9-epoxide to reduced glutathione. CONCLUSIONS: The CYP3A5 polymorphism is associated with increased levels of the mutagenic AFB1-exo-8,9-epoxide, particularly in individuals with low CYP3A4, and this may modulate individual risk of HCC.

Adolescent↗
PubMed2004

B-Raf acts via the ROCKII/LIMK/cofilin pathway to maintain actin stress fibers in fibroblasts.

Recent data have shown that the BRAF gene is mutated at a high frequency in human malignancies. We have analyzed the migratory characteristics of B-raf(-/-) mouse embryonic fibroblasts (MEFs) and compared these with the organization of the actin cytoskeleton and the activity of signaling pathways that are known to influence this organization. Disruption of B-raf significantly reduced the levels of phospho-ERK1/2 and, surprisingly, induced an approximately 1.5-fold increase in cell migration. Consistent with these findings, the high level of actin stress fibers normally present in MEFs was considerably reduced following disruption of B-raf, and the F-actin content of B-raf(-/-) cells was less than half that of B-raf(+/+) cells. Phosphorylation of the myosin light chain on Thr18/Ser19 residues was not reduced in B-raf(-/-) cells. Rather, reduced ROCKII expression and attenuated phosphorylation of ADF/cofilin on serine 3 occurred. Normal stress fiber and phosphocofilin levels were restored by the expression of human B-Raf and catalytically active MEK and by the overexpression of LIM kinase (LIMK). These results have important implications for the role of the B-Raf/ERK signaling pathway in regulating cell motility in normal and malignant cells. They suggest that B-Raf is involved in invasiveness by regulating the proper assembly of actin stress fibers and contractility through a ROCKII/LIMK/cofilin signaling pathway.

Actin Depolymerizing Factors↗