Predictive Modeling of Tacrolimus Dose Requirement Based on High-Throughput Genetic Screening.

Any biochemical reaction underlying drug metabolism depends on individual gene-drug interactions and on groups of genes interacting together. Based on a high-throughput genetic approach, we sought to identify a set of covariant single-nucleotide polymorphisms predictive of interindividual tacrolimus (Tac) dose requirement variability. Tac blood concentrations (Tac C0 ) of 229 kidney transplant recipients were repeatedly monitored after transplantation over 3 mo. Given the high dimension of the genomic data in comparison to the low number of observations and the high multicolinearity among the variables (gene variants), we developed an original predictive approach that integrates an ensemble variable-selection strategy to reinforce the stability of the variable-selection process and multivariate modeling. Our predictive models explained up to 70% of total variability in Tac C0 per dose with a maximum of 44 gene variants (p-value <0.001 with a permutation test). These models included molecular networks of drug metabolism with oxidoreductase activities and the multidrug-resistant ABCC8 transporter, which was found in the most stringent model. Finally, we identified an intronic variant of the gene encoding SLC28A3, a drug transporter, as a key gene involved in Tac metabolism, and we confirmed it in an independent validation cohort.

Long-Term Clinical Impact of Adaptation of Initial Tacrolimus Dosing to CYP3A5 Genotype.

Pretransplantation adaptation of the daily dose of tacrolimus to CYP3A5 genotype is associated with improved achievement of target trough concentration (C0 ), but whether this improvement affects clinical outcomes is unknown. In the present study, we have evaluated the long-term clinical impact of the adaptation of initial tacrolimus dosing according to CYP3A5 genotype: The transplantation outcomes of the 236 kidney transplant recipients included in the Tactique study were retrospectively investigated over a period of more than 5 years. In the Tactique study, patients were randomly assigned to receive tacrolimus at either a fixed dosage or a dosage determined by their genotype, and the primary efficacy end point was the proportion of patients for whom tacrolimus C0 was within target range (10-15 ng/mL) at day 10. Our results indicate that the incidence of biopsy-proven acute rejection and graft survival were similar between the control and the adapted tacrolimus dose groups, as well as between the patients who achieve the tacrolimus C0 target ranges earlier. Patients' death, cancer, cardiovascular events, and infections were also similar, and renal function did not change. We conclude that optimization of initial tacrolimus dose using pharmacogenetic testing does not improve clinical outcomes.

Kidney transplant recipients carrying the CYP3A4*22 allelic variant have reduced tacrolimus clearance and often reach supratherapeutic tacrolimus concentrations.

CYP3A4*22 is an allelic variant of the cytochrome P450 3A4 associated with a decreased activity. Carriers of this polymorphism may require reduced tacrolimus (Tac) doses to reach the target residual concentrations (Co). We tested this hypothesis in a population of kidney transplant recipients extracted from a multicenter, prospective and randomized study. Among the 186 kidney transplant recipients included, 9.3% (18 patients) were heterozygous for the CYP3A4*22 genotype and none were homozygous (allele frequency of 4.8%). Ten days after transplantation (3 days after starting treatment with Tac), 11% of the CYP3A4*22 carriers were within the target range of Tac Co (10-15 ng/mL), whereas among the CYP3A4*1/*1 carriers, 40% were within the target range (p = 0.02, OR = 0.19 [0.03; 0.69]). The mean Tac Co at day 10 in the CYP3A4*1/*22 group was 23.5 ng/mL (16.6-30.9) compared with 15.1 ng/mL (14-16.3) in the CYP3A4*1/*1 group, p < 0.001. The Tac Co/dose significantly depended on the CYP3A4 genotype during the follow-up (random effects model, p < 0.001) with the corresponding equivalent dose for patients heterozygous for CYP3A4*22 being 0.67 [0.54; 0.84] times the dose for CYP3A4*1/*1 carriers. In conclusion, the CYP3A4*22 allelic variant is associated with a significantly altered Tac metabolism and carriers of this polymorphism often reach supratherapeutic concentrations.

Influence of genetics and non-genetic factors on acenocoumarol maintenance dose requirement in Moroccan patients.

WHAT IS KNOWN AND OBJECTIVE: Coumarin derivatives such as acenocoumarol represent the therapy of choice for the long-term treatment and prevention of thromboembolic diseases. Many genetic, clinical and demographic factors have been shown to influence the anticoagulant dosage. Our aim was to investigate the contribution of genetic and non-genetic factors to variability in response to acenocoumarol in Moroccan patients. METHODS: Our study included 114 adult Moroccan patients, receiving long-term acenocoumarol therapy for various indications. Tests for VKORC1 -1639G>A promoter polymorphism (rs9923231), CYP2C9*2 rs1799853, CYP2C9*3 rs1057910, and CYP4F2 rs2108622 alleles were undertaken using Taq Man(®) Pre-Developed Assay Reagents for allelic discrimination. The statistical analysis was performed using the SAS V9 statistical package. RESULTS AND DISCUSSION: Genotyping showed that the allele frequencies for the SNPs studied were no different to those found in Caucasians population. A significant association was observed between the weekly maintenance dose and the VKORC1 (P = 0·0027) and CYP2C9 variant genotypes (P = 0·0082). A final multivariate regression model that included the target International Normalized Ratio, VKORC1 and CYP2C9 genotypes explained 36·2% of the overall interindividual variability in acenocoumarol dose requirement. WHAT IS NEW AND CONCLUSION: Our study shows large interindividual variability in acenocoumarol maintenance dose requirement in our population. VKORC1 and CYP2C9 variants significantly affected acenocoumarol dose, in-line with results in other populations. For the Moroccan population, the SNPs that have the largest effect on acecoumarol dose are CYP2C9 rs1799853, CYP2C9 rs1057910 and VKORC1 rs9923231.

Cyclosporine-induced endoplasmic reticulum stress triggers tubular phenotypic changes and death.

The molecular mechanisms by which cyclosporine induces chronic nephrotoxicity remain poorly understood. A previous transcriptomic study suggested that cyclosporine might induce endoplasmic reticulum (ER) stress in human tubular cells. The aim of the present study was to characterize the features of tubular ER stress induced by cyclosporine and to investigate its effects on cell differentiation and viability. Using primary cultures of human tubular cells, we confirmed that cyclosporine is responsible for ER stress in vitro. This was also confirmed in vivo in the rat. In vitro, cyclosporine and other ER stress inducers were responsible for epithelial phenotypic changes leading to the generation of protomyofibroblasts, independent of transforming growth factor-beta signaling. RNA interference directed against cyclophilin A supported the role of its inhibition in triggering ER stress as well as epithelial phenotypic changes induced by cyclosporine. Salubrinal, which is known to protect cells from ER stress, significantly reduced epithelial phenotypic changes and cytotoxicity induced by cyclosporine in vitro. Salubrinal also reduced cyclosporine nephrotoxicity in rat kidneys. Thus, we describe a novel mechanism that initiates dedifferentiation and tubular cell death upon cyclosporine treatment. These results provide an interesting framework for further nephroprotective therapies by targeting ER stress.

Anti-liver endoplasmic reticulum autoantibodies are directed against human cytochrome P-450IA2. A specific marker of dihydralazine-induced hepatitis.

Sera from patients with dihydralazine-induced hepatitis were shown to contain anti-liver microsomal autoantibodies (anti-LM) by indirect immunofluorescence. These anti-LM antibodies were different from anti-liver/kidney microsomes (anti-LKM) 1 or 2 autoantibodies which have been previously described. Sera recognized a single 53,000 = Mr polypeptide in human liver microsomes as judged by immunoblotting, and the target antigen was identified as cytochrome P-450IA2 (P-450IA2) by (a) comparison of immunoblotting patterns with anti-human P-450IA2 and anti-rat P-450IA2 and with five anti-LM sera, and (b) specific immunoinhibition of microsomal ethoxyresorufin and phenacetin O-deethylation activities (both P-450IA2 supported reactions) by anti-LM antibodies. Finally, purified human P-450IA2 was recognized by these anti-LM sera. The anti-LM antibodies are specific for the disease because none of the other antisera tested behaved in the same manner as anti-LM, even those from patients treated with dihydralazine and without hepatic disease. A possible role of P-450IA2 in the metabolism of dihydralazine was suggested by competitive inhibition of ethoxyresorufin-O-deethylase observed in microsomal incubations. Thus, a new example is presented in which a cytochrome P-450 may be a target for autoantibodies in drug-induced hepatitis.

UDP-glucuronosyltransferase UGT1A7 genetic polymorphisms in hepatocellular carcinoma: a differential impact according to seropositivity of HBV or HCV markers?

BACKGROUND: We conducted a case-control study to evaluate the role of UDP-glucuronosyltransferase 1A7 (UGT1A7) polymorphisms in the onset of hepatocellular carcinoma (HCC). METHODS: The study included 165 patients with HCC, 134 with cirrhosis and 142 controls without liver disease, matched for age and hospital. All were men younger than 75 years. HCC and cirrhosis patients were stratified according to time since cirrhosis diagnosis. RESULTS: We found a positive association between the UGT1A7*3/*3 genotype and HCC when the comparison was restricted to patients whose disease was of viral origin [OR = 3.4 (0.3-45)] but a negative association when it included only alcoholic patients [OR = 0.1 (0.02-0.6), p = 0.01]. CONCLUSION: Our study shows that UGT1A7 may play a role in hepatocellular carcinogenesis and that this role may differ according to the primary cause of the cirrhosis.

[Pharmacogenetics: from basic research to clinical applications]. / La pharmacogénétique: de la recherche fondamentale aux applications cliniques.

Pharmacogenetics is the study of the genetic factors implicated in the pharmacological or toxicological response to drugs. This response is very variable and depends on three steps: metabolism/transport, target and organism reaction. Each step is very variable as a function of endogenous (patho-physiological, genetics...) or exogenous (environment: drugs, diet, smoking, alcohol...) factors. The scientific bases of pharmacogenetics have been strongly established: genetic polymorphisms have an impact on pharmacological activity or on the toxicity of numerous drugs and examples are given in this review. By contrast, present clinical applications are more limited although, in some instances, medical and economic interest of the pre-treatment determination of genotype or phenotype of the patients has been clearly demonstrated. Certain hospital laboratories now include pharmacogenetic activity. The predictive or explicative value of pharmacogenetics is scientifically demonstrated but prospective studies for validation are now necessary to identify situations where a clinical application will be actually beneficial for patients.

[Molecular pharmacogenetics in hospital laboratories in France: current data and future prospects]. / La pharmacogénétique moléculaire hospitalière en France: données actuelles et perspectives.

Molecular pharmacogenetic units have recently been established in several hospital laboratories in France. The clinical impact of these units is still limited and numerous problems of organizational, ethical, legal, technical, social and economical nature remain to be resolved. However, an increasing number of these units, a rise in their activities and an enlargement of their scope of application are foreseeable in the future. Ultimately, these units would significantly contribute to limit the public health problem caused by interindividual variabilities in drug effects. In view of these prospects, it seems essential that such hospital activity should be quickly recognised by the authorities and the various health sectors in France. It is also essential that the problems that arise from such pharmacogenetic activities should be considered by the authorities and would profit from the organization of a national network and from financial guarantees.

Interindividual variability in dabigatran and rivaroxaban exposure: contribution of ABCB1 genetic polymorphisms and interaction with clarithromycin.

Essentials Rivaroxaban and dabigatran are substrates of the P-glycoprotein (P-gp) encoded by the ABCB1 gene. We tested the effect of ABCB1 polymorphisms and of a P-gp inhibitor on both drugs' pharmacokinetics. The ABCB1 genotype was not a clinically relevant determinant of both drugs' pharmacokinetics. Administration of P-gp inhibitors with dabigatran or rivaroxaban should be exercised with caution. SUMMARY: Background The direct oral anticoagulants (DOACs) dabigatran and rivaroxaban are both substrates of the P-glycoprotein (P-gp) transporter, encoded by the ABCB1 gene. Rivaroxaban is metabolized by cytochrome P450 A4 (CYP3A4). Interindividual variability in DOAC exposure and frequent P-gp-associated drug-drug interactions have been described in patients. Objective To assess the influence of ABCB1 polymorphisms on the pharmacokinetics of dabigatran and rivaroxaban, associated or not with clarithromycin, a P-gp and CYP3A4 inhibitor. Methods Sixty healthy male volunteers, selected according to ABCB1 genotype (20 homozygous mutated, 20 heterozygous mutated, and 20 wild-type for haplotype 2677-3435), were included in this randomized, two-center, crossover study. All received sequentially a single dose of dabigatran etexilate (300 mg) and rivaroxaban (40 mg) associated or not with clarithromycin. Peak plasma concentration and area under the curve (AUC) were compared across the three ABCB1 genotypes. The effect of clarithromycin on dabigatran or rivaroxaban pharmacokinetics was assessed. Results Interindividual coefficients of variation for AUC were 77% for dabigatran and 51% for rivaroxaban. ABCB1 genotype did not significantly affect drug pharmacokinetics: AUC ratios between mutant-allele carriers and wild-type volunteers were 1.27 (95% confidence interval [CI] 0.84-1.92) and 1.20 (95% CI 0.96-1.51) for dabigatran and rivaroxaban, respectively. Clarithromycin coadministration led to a two-fold increase in both drugs' AUC, irrespective of ABCB1 genotype: ratios of geometric means were 2.0 (95% CI 1.15-3.60) and 1.94 (95% CI 1.42-2.63) for dabigatran and rivaroxaban, respectively. Conclusions ABCB1 genotype is not a significant determinant of interindividual variability in dabigatran and rivaroxaban pharmacokinetics. The levels of one drug did not predict the levels of the other. Coadministration of a P-gp/CYP3A4 inhibitor with dabigatran or rivaroxaban may warrant caution in patients at risk of overexposure.

[Rapamycine and mTOR inhibitors: from bench to bedside]. / Rapamycine et inhibition de mTOR: des voies de signalisation aux applications cliniques.

Rapamycin is a macrocyclic lactone with antifungal and antibiotic properties isolated from Streptomyces hygroscopicus during the 70's. Studies of rapamycin properties in yeast led to the discovery of TOR (Target Of Rapamycin) and its mammalian analogue, mTOR. mTOR is a central regulator of cell growth and proliferation in response to environmental stimuli such as growth factors or nutrients. There are two proteins that have been shown to be regulated by mTOR in response to a broad range of mitogenic stimuli. The translation regulation induced by mTOR is mediated by the p70 S6 kinase activation and the 4E-BP1 inhibition. Both proteins participate in the regulation of translation process and growth in cells stimulated by either mitogens or hormones. Antiproliferative effects of rapamycin and analogues have been demonstrated on numerous cell types, explaining the development of these drugs in clinical practice: as immunosuppressive drugs in solid organ transplantation, in oncology for the treatment of various types of cancer, and for the prevention of restenosis after coronary angioplasty. Rapamycin is a potent immunosuppressive drug used in solid organ transplantation for the prevention of acute rejection. In oncology these antiproliferative effects are evaluated in several types of cancers. Rapamycin is now widely used for coating stents to reduce post-stenting restenosis phenomenon after coronary angioplasty. Finally, rapamycin is now evaluated in various diseases characterized by proliferative disorders.

[Vitamin K epoxide reductase: Fresh blood for oral anticoagulant therapies]. / La vitamine K époxyde réductase: du sang neuf dans les traitements anticoagulants oraux.

INTRODUCTION: Vitamin K epoxide reductase complex subunit I (VKORC1) is a key enzyme in the vitamin K cycle, cofactor required for the activation of vitamin K-dependent clotting factors. EXEGESIS: VKORC1 recycles vitamin K 2,3 epoxide back to active vitamin K hydroquinone, an important factor for the carboxylation step of clotting factors. VKORC1 is the target enzyme of inhibition by oral anticoagulants or anti-vitamin K (warfarin, acenocoumarol). CONCLUSION: We show here the clinical consequences of genetic variations of VKORC1 during VKA therapy.

Inhibition of CYP1A2 and CYP3A4 by oltipraz results in reduction of aflatoxin B1 metabolism in human hepatocytes in primary culture.

Dithiolethiones are thought to act as potent chemoprotective agents against aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat by inducing glutathione S-transferases (GSTs). To determine whether these antioxidants can be similarly effective in human beings, we have investigated metabolism of AFB1, in primary human hepatocytes with or without pretreatment by oltipraz (OPZ), a synthetic derivative of the natural 1,2-dithiole-3-thione. Aflatoxin M1 (AFM1), glutathione conjugates of AFB1 oxides (AFBSGs), and unchanged AFB1 were quantitated in cultures derived from eight human liver donors. Parenchymal cells obtained from the three GST M1-positive livers metabolized AFB1 to AFM1 and to AFBSGs derived from the isomeric exo-and endo-8,9-oxides, whereas no AFBSGs were formed in the GST M1-null cells. Pretreatment of the cells with 3-methylcholanthrene or rifampicin, inducers of CYP1A2 and CYP3A4, respectively, caused a significant increase in AFB1 metabolism. Although OPZ induced GST A2, and to a lesser extent GST A1 and GST M1, it decreased formation of AFM1 and AFBSG, which involves CYP1A2 and CYP3A4. Inhibition by OPZ of AFB1 metabolism by reducing CYP1A2 and CYP3A4 was also demonstrated by decreased activity of their monooxygenase activities toward ethoxyresorufin and nifedipine, respectively. The significant inhibition by OPZ of human recombinant yeast CYP1A2 and CYP3A4 was also shown. These results demonstrate that AFBSG can be formed by GST M1-positive human hepatocytes only, and suggest that chemoprotection with OPZ is due to an inhibition of activation of AFB1, in addition to a GST-dependent inactivation of the carcinogenic exo-epoxide.

Comparison of mouse and human colon tumors with regard to phase I and phase II drug-metabolizing enzyme systems.

Since human colorectal tumors are insensitive to most chemotherapeutic agents, there is a need for the discovery of new drugs that would show activity against this disease. In an attempt to better appreciate the relevance of a widely used mouse colon tumor (colon adenocarcinoma Co38) as a screening model for human colorectal tumors, we compared the main phase I and phase II drug-metabolizing enzyme systems in both tumoral and nontumoral colon tissues. The following enzymes were assayed by Western blot: cytochromes P-450 (1A1/A2, 2B1/B2, 2C, 2E1, and 3A), epoxide hydrolase, and glutathione-S-transferases (GST-alpha, -mu, and -pi). The activities of the following enzymes or cofactors were determined by spectrophotometric or fluorometric assays: total cytochrome P-450, 1-chloro-2,4-dinitrobenzene-GST, selenium-independent glutathione peroxidase, 3,4-dichloronitrobenzene-GST, ethacrynic acid-GST, total glutathione, epoxide hydrolase, UDP-glucuronosyltransferase, beta-glucuronidase, sulfotransferase, and sulfatase. Results obtained by Western blot showed that mouse colon adenocarcinoma Co38 did not express any of the probed cytochromes P-450, whereas human colorectal tumors expressed only low levels of cytochrome P-450 3A. GST-alpha and GST-pi were detected in all tumoral and nontumoral tissues of both species. The neutral GST-mu was expressed in all murine tissues investigated and was found to be polymorphic in human tissues. For human peritumoral and tumoral colorectal tissues there was no significant difference between GST isoenzyme levels, whereas mouse colon adenocarcinoma Co38 had a lower expression of GST-mu and GST-pi, compared to normal mouse colon. Enzymatic activities for glutathione peroxidase, 3,4-dichloronitrobenzene-GST, and ethacrynic acid-GST confirmed the Western blot results for GST-alpha, GST-mu, and GST-pi, respectively. Total GSH levels were similar between murine and human tumors but were 3-fold higher in human tumors than in peritumoral tissues, whereas they were 7-fold lower in mouse colon tumor Co38, compared to normal mouse colon. Epoxide hydrolase was not expressed in either mouse colon adenocarcinoma Co38 or normal mouse colon tissues, whereas it was expressed in human colon peritumoral and tumoral tissues at similar levels. No significant difference was observed between human tumors and peritumoral tissues for UDP-glucuronosyltransferase, beta-glucuronidase, sulfotransferase, and sulfatase. For murine colon tissues, the conjugation pathways (UDP-glucuronosyltransferase and sulfotransferase) were lower in colon adenocarcinoma Co38, whereas the converse was observed for the corresponding hydrolytic enzymes (beta-glucuronidase and sulfatase).(ABSTRACT TRUNCATED AT 400 WORDS)

High levels of DNA adducts in human colon are associated with colorectal cancer.

Colon cancer is one of the most frequent causes of cancer death in western countries. Epidemiological studies suggest that colorectal cancer can be attributed, at least in part, to carcinogens and mutagens present in the diet and/or the environment. The covalent binding of these xenobiotics or their reactive metabolites to DNA is believed to initiate this chemical carcinogenesis. In the present study, using a 32P-postlabeling method, we investigated DNA adduct levels in control colons from patients without colorectal adenocarcinoma and in nontumoral and tumoral tissues from patients with colorectal adenocarcinoma. Our results show that the DNA adduct level is significantly higher (P < 0.001) in nontumoral than in control or tumoral colon samples. For the first time, we demonstrate in humans that the presence of numerous adducts in colonic mucosa is associated with colorectal cancer, a finding in agreement with the importance of chemical factors in causing this disease; therefore, after confirmation of the link between DNA adducts and colorectal cancer, the measurement of DNA adduct levels in colon samples could constitute a useful approach to the early detection of colorectal cancer.

Detection of plasma tumor DNA in head and neck squamous cell carcinoma by microsatellite typing and p53 mutation analysis.

Recent arguments have suggested that tumor DNA in cancer patients could be found in plasma, but different points remain unclear. Using a series of 117 head and neck squamous cell carcinoma tumors, our goals for this study were: (a) to quantify the amount of plasma DNA; (b) to evaluate the presence of plasma tumor DNA; and (c) to analyze the clinical relevance of tests based on plasma DNA analyses. Low levels of plasma DNA were found in most samples, but all were successfully amplified. Two different methods were used to detect tumor-specific genetic alterations: (a) microsatellite instability at UT5085 with an established sensitivity of 1:500; and (b) p53 mutation screening. Of the 117 tumors typed at UT5085, 65 demonstrated bandshifts (55%). Plasma and tumor DNA a showed similar alteration in only one case among these samples, and the prevalence of tumor DNA in plasma was estimated to be <2% using microsatellite analysis. Tumor DNA was detected in plasma at a higher prevalence (2 of 11 cases) when using p53 mutant allele-specific amplification. These results showed that in plasma, tumor DNA is largely diluted by normal DNA. By comparison with previously published studies, the prevalence of microsatellite alterations in plasma in this series of head and neck squamous cell carcinomas is very low, despite the fact that a large series of tumors was analyzed. To explain this discrepancy, we analyzed the possibility of PCR artifacts as suspected by the presence of loss of heterozygosity in two plasma DNA samples without a similar tumor DNA alteration. When DNA concentrations were under the threshold of detection (<100 ng/ml), we demonstrated that PCR artifacts could occur at random, and, if misinterpreted, these false genetic alterations could artificially enhance the frequency of plasma DNA alterations. This may have been suspected in previously published series, but it has never been discussed before. Microsatellite analysis on plasma DNA is difficult to interpret and can frequently be misleading. Plasma DNA should be analyzed with very sensitive and specific methods such as mutant allele-specific amplification, which excludes artifacts but requires specific optimization that is probably not compatible with routine and clinical use.

Isolation of the human cytochrome P-450 IIC8 gene: multiple glucocorticoid responsive elements in the 5' region.

The 5' portion of the cytochrome P-450 IIC8 gene has been isolated from a human genomic library. A 3.5 kb genomic fragment including 2477 bp of 5' flanking region and exon 1 of the gene was sequenced. S1 nuclease mapping and specific primer extension experiments localized the transcription start site 23 bp upstream from the ATG. Two and three putative TATA and CAAT boxes were identified together with seven core motifs for glucocorticoid responsive elements, an interesting feature in this constitutive P-450 subfamily.

Purification of a new cytochrome P-450 from human liver microsomes.

Using a classical methodology of purification consisting of three chromatographic steps (Octyl-Sepharose, DEAE-cellulose, CM-cellulose) we have purified a new cytochrome P-450 from human liver microsomes. It was called cytochrome P-450(9). It has been proven to be different from all precedingly purified human liver microsomal cytochrome P-450 isozymes by its immunological and electrophoretical properties. It does not cross-react with any rat liver cytochrome P-450 and anti-cytochrome P-450(9) does not recognize rat liver microsomes; thus this cytochrome P-450(9) is specific to humans. This cytochrome P-450 isozyme exists in low amounts in human liver microsomes and exhibits an important quantitative polymorphism. In reconstituted system, cytochrome P-450(9) is able to hydroxylate all substrates tested but is not specific of any; its exact role in xenobiotic metabolism in man remains to be elucidated.

Presence of proteins recognized by mammalian cytochrome P-450 antibodies in Euglena gracilis.

We have attempted to probe three microsomal cytochrome P-450 isozymes in Euglena gracilis using immunochemical methods. They cross-react with anti-rat cytochrome P4502C11, cytochrome P4502E, and cytochrome P4502B. Activities of alkoxyphenoxazone dealkylation have been tested in living cells. In untreated cultures, the amount of proteins recognized by anti-cytochrome P4502C11 or anti-cytochrome P4502E is high. Phenobarbital treatment increased the levels of microsomal proteins recognized by antibody to cytochrome P4502B, as well as dealkylases of pentoxyresorufin, but decreased the level of proteins recognized by anti-cytochrome P450C11 or cytochrome P4502E. These results suggest that these unicellular algae may contain different isozymes of microsomal cytochromes P-450, comparable to those in mammalian liver. They are cytochrome P-450 equivalents of mammalian isoenzymes 2C, 2E and 2B. However, we could not demonstrate ethanol induction of cytochrome P-450 equivalent to isoenzyme 2E. Its role in xeno- or endobiotic metabolism remains to be elucidated.

p53 alterations predict tumor response to neoadjuvant chemotherapy in head and neck squamous cell carcinoma: a prospective series.

PURPOSE: The tumor suppressor gene p53 plays a crucial role in cell cycle control and apoptosis in response to DNA damages. p53 gene mutations and allelic losses at 17p are one of the most common genetic alterations in primary head and neck squamous cell carcinoma (HNSCC). Alterations of the p53 gene have been shown to contribute to carcinogenesis and drug resistance. PATIENTS AND METHODS: In this prospective series, patients with HNSCC were treated with cisplatin-fluorouracil neoadjuvant chemotherapy. p53 status was characterized in 106 patients with HNSCC (p53 mutations, allelic losses at p53 locus, and plasma anti-p53 antibodies) to determine the existence of a relationship between p53 gene status and response to neoadjuvant chemotherapy. RESULTS: Exons 4 to 9 of the p53 gene were analyzed, and mutations were found in 72 of 106 patients with HNSCC. p53 mutations were associated with loss of heterozygosity at chromosome 17p (P <.001). The prevalence of p53-mutated tumors was higher in the group of patients with nonresponse to neoadjuvant chemotherapy than in the group of responders (81% v 61%, respectively; P <.04). When compiling p53 mutations and anti-p53 antibodies in plasma, the correlation between p53 status and response to chemotherapy was significant (87% v 57%, respectively; P =.003). A multivariate analysis showed that p53 status is an independent predictive factor of response to chemotherapy. CONCLUSION: This prospective study suggests that p53 status may be a useful indicator of response to neoadjuvant chemotherapy in HNSCC.