RESUMEN
A variable (GT)(n) repeat in the 5'-regulatory region of N-methyl-D-aspartate GRIN2A subtype has recently been identified and associated with psychiatric disorders. In this study, we examined the association of this polymorphism with alcohol dependence. Subject-control analysis included 206 alcohol-dependent and 168 control subjects. Average observed repeat numbers and genotype distributions were significantly different (P-value = 0.001) in alcohol-dependent subjects versus control subjects. Short alleles were significantly less frequent among alcohol-dependent subjects (odds ratio = 0.58, P-value = 7 × 10(-4)). These results could be replicated in an independent sample of 116 alcohol-dependent subjects. For the first time, a significant association was identified between this polymorphism and alcoholism.
Asunto(s)
Alcoholismo/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Receptores de N-Metil-D-Aspartato/genética , Adulto , Estudios de Casos y Controles , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana EdadRESUMEN
A high resistance and heterogeneous response to conventional anti-cancer chemotherapies characterize malignant cutaneous melanoma, the most aggressive and deadly form of skin cancer. Withaferin A (WFA), a withanolide derived from the medicinal plant Withania somnifera, has been reported for its anti-tumorigenic activity against various cancer cells. For the first time, we examined the death-inducing potential of WFA against a panel of four different human melanoma cells and investigated the cellular mechanisms involved. WFA induces apoptotic cell death with various IC50 ranging from 1.8 to 6.1 µM. The susceptibility of cells toward WFA-induced apoptosis correlated with low Bcl-2/Bax and Bcl-2/Bim ratios. In all cell lines, the apoptotic process triggered by WFA involves the mitochondrial pathway and was associated with Bcl-2 down regulation, Bax mitochondrial translocation, cytochrome c release into the cytosol, transmembrane potential (ΔΨm) dissipation, caspase 9 and caspase 3 activation and DNA fragmentation. WFA cytotoxicity requires early reactive oxygen species (ROS) production and glutathione depletion, the inhibition of ROS increase by the antioxidant N-acetylcysteine resulting in complete suppression of mitochondrial and nuclear events. Altogether, these results support the therapeutic potential of WFA against human melanoma.
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Apoptosis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Witanólidos/farmacología , Proteína X Asociada a bcl-2/metabolismo , Animales , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Fragmentación del ADN/efectos de los fármacos , Regulación hacia Abajo , Humanos , Melanoma/tratamiento farmacológico , Melanoma/patología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Witanólidos/uso terapéuticoRESUMEN
BACKGROUND/AIMS: Liver pathology induced by chemotherapy (steatosis or vascular injury) is known to increase the liver's sensitivity to ischemia/ reperfusion (I/R) injury, thereby increasing morbidity and mortality after liver resection. Our aim was to assess whether ischemic preconditioning (IP) reduces I/R injury to livers with chemotherapy-induced pathology. METHODS: We analyzed a series of livers from patients treated with chemotherapy for colorectal cancer who underwent IP (n=30) or not (n=31) before hepatectomy. All but one of the livers exhibited chemotherapy-induced steatosis and/ or peliosis before the I/R insult. RESULTS: Necrosis was less frequent (p=0.038) in livers with IP than in the others. IP had no influence on apoptosis as assessed by terminal transferase uridyl nick-end labeling (TUNEL) assay or caspase-3, -8 and -9 expression. IP induced a twofold increase in B-cell leukemia/ lymphoma 2 (Bcl-2; p<0.05), which was localized to hepatocytes of centrolobular and peliotic areas and colocalized with the autophagy protein beclin-1 in livers with IP, suggesting their coordinated role in autophagy. Increased expression of the phosphorylated Bcl-2 was observed in preconditioned livers and was associated with a decreased immunoprecipitation of beclin-1 and the increased expression of light chain 3 type II (LC3-II). The increased number of autophagic vacuoles seen by electron microscopy confirmed an association of autophagy in chemotherapy-injured livers following IP. However, the differences in protein expression were not reflected in postresection liver-injury tests or measure of patient morbidity. CONCLUSIONS: IP is associated with a reduction in necrosis of hepatocytes already damaged by chemotherapy and an activation of autophagy. Bcl-2 and beclin-1 could be major targets in the regulation of cell death during I/R injury.
Asunto(s)
Precondicionamiento Isquémico , Hígado/irrigación sanguínea , Hígado/patología , Anciano , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia , Beclina-1 , Neoplasias Colorrectales/tratamiento farmacológico , Femenino , Humanos , Hígado/lesiones , Hígado/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/cirugía , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Necrosis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Daño por Reperfusión/prevención & controlRESUMEN
Prior studies have associated 677C-T Methylenetetrahydrofolate reductase (MTHFR) gene polymorphism with decreased enzymatic activity and modified homocysteine regulation. This study determines and compares MTHFR 677C-T distribution and examines its consequences on homocysteine metabolism and alcohol dependence in alcoholic patients classified according to the Babor and Lesch typologies. MTHFR TT genotype was more prevalent in AD patients with milder alcohol dependence (Babor type A) and with Lesch type 3, associated with depression. MTHFR TT was also associated with hyperhomocysteinemia. Determining MTHFR 677C-T genotype, folate and vitamin B12 levels could assist physicians in identifying type 3 patients and improve addictions management.
Asunto(s)
Alcoholismo/clasificación , Alcoholismo/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Polimorfismo Genético/genética , Adulto , Alcoholismo/epidemiología , Trastorno Depresivo/epidemiología , Femenino , Genotipo , Homocisteína/sangre , Humanos , Masculino , Persona de Mediana Edad , Vitamina B 12/sangreRESUMEN
BACKGROUND: To establish a new experimental model of human hepatocellular carcinoma by orthotopic implantation of tumoral cells with its subsequent removal, to generate and modulate circulating tumoral cells. MATERIALS AND METHODS: Three human hepatoma cell lines (HepG2, PLC/PRF, and Mahlavu) were orthotopically implanted under the Glisson's capsule of the left lateral lobe of the liver in a total of 56 non-obese diabetic/severe combined immunodeficiency mice. Tumor removal was performed 30 d after injection, and a laparotomy without tumor removal was done in control mice. Generation of circulating cells was monitored by flow cytometry using fluorescein isothiocyanate-conjugated anti-HLA antibody. RESULTS: In 26 mice implanted with Mahlavu cells, 20 developed a unique tumor allowing a resection (77%), which was technically feasible in 80% of cases. The overall perioperative mortality was 30% (3/10) after resection; no mortality was observed in the control group. The circulating tumoral cells decreased dramatically after resection of the tumor as compared with control mice. CONCLUSION: This new model is feasible and may be an interesting useful tool to study the hepatocellular carcinoma metastatic process and is consistent with the human clinical practice.
Asunto(s)
Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Trasplante de Neoplasias , Animales , Carcinoma Hepatocelular/sangre , Línea Celular Tumoral , Humanos , Inyecciones , Neoplasias Hepáticas/sangre , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
BACKGROUND: Hyperhomocysteinemia is frequently observed in alcohol-dependent subjects, in particularly in those with marked withdrawal symptoms. The common C677T transition on the methylenetetrahydrofolate reductase (MTHFR) gene influences homocysteinemia. Our objective was to study the prevalence of the MTHFR C677T polymorphism in alcohol-dependent subjects and the influence of this polymorphism on symptoms associated with alcoholism. METHODS: MTHFR C677T polymorphism was determined in 93 control subjects and 242 alcohol-dependent subjects. Serum homocysteine, folate and vitamin B12 levels together with hepatic biological parameters were determined in the control and alcohol-dependent subjects. RESULTS: Hyperhomocysteinemia is frequently observed in alcohol-dependent subjects, particularly in those with marked withdrawal symptoms. Alcohol-dependent subjects showed a significant decrease in MTHFR 677TT prevalence (9%, 21/242) compared to controls (18%, 17/93) (p<0.02). The relative risk estimated as an odds ratio for alcoholism in subjects with the TT genotype is 0.42 (odd ratio 95% confidence interval, 0.21-0.83). Moreover, drinkers with TT genotype presented lower values for markers of alcohol misuse (p<0.05), better liver function tests, a lower frequency of relapses and no marked withdrawal symptoms as assessed by the Lesch typology. CONCLUSION: MTHFR 677TT genotype could play a protective role against alcohol dependence. Moreover, when subjects with MTHFR 677TT genotype become dependent to alcohol, they seem to constitute a subgroup of alcoholic patients with a decreased risk for developing neurotoxic withdrawal symptoms and hepatic toxicity.
Asunto(s)
Alcoholismo/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Polimorfismo Genético/genética , Consumo de Bebidas Alcohólicas/genética , Neuropatía Alcohólica/genética , Alcoholismo/sangre , Alcoholismo/enzimología , Grupos Control , Femenino , Ácido Fólico/sangre , Deficiencia de Ácido Fólico/sangre , Deficiencia de Ácido Fólico/genética , Frecuencia de los Genes , Predisposición Genética a la Enfermedad/genética , Genotipo , Homocisteína/sangre , Humanos , Hiperhomocisteinemia/sangre , Hiperhomocisteinemia/genética , Hepatopatías Alcohólicas/genética , Masculino , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Persona de Mediana Edad , Riesgo , Vitamina B 12/sangreRESUMEN
Overexpression of T-cadherin (T-cad) transcripts occurs in approximately 50% of human hepatocellular carcinomas (HCCs). To elucidate T-cad functions in HCC, we examined T-cad protein expression in normal and tumoral human livers and hepatoma cell lines and investigated its influence on invasive potential of HCC using RNA interference silencing of T-cad expression in Mahlavu cells. Whereas T-cad expression was restricted to endothelial cells (EC) from large blood vessels in normal livers, it was up-regulated in sinusoidal EC from 8/15 invasive HCCs. Importantly, in three of them (38%) T-cad was detected in tumor cells within regions in which E-cadherin expression was absent. Among six hepatoma cell lines, only Mahlavu expressed T-cad but not E-cadherin. T-cad exhibited a globally punctuate distribution in quiescent Mahlavu and additionally it concentrated at the leading edge of migrating cells. Matrigel invasion assay revealed that Mahlavu possess a high invasive potential that was significantly inhibited by T-cad silencing. Wound healing and random motility assays demonstrated that inhibition of T-cad expression in Mahlavu significantly reduced their motility. We propose that T-cad expression in tumor cells might occur by cadherin-switching during epithelial-mesenchymal transition and may represent an additional mechanism contributing to HCC metastasis.
Asunto(s)
Cadherinas/genética , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Hígado/fisiología , Animales , Técnicas de Cultivo de Célula , División Celular , Línea Celular Tumoral , Movimiento Celular , Cartilla de ADN , Células Endoteliales/fisiología , Fibroblastos/fisiología , Hepatocitos/fisiología , Humanos , Hígado/citología , Invasividad Neoplásica , ARN Interferente Pequeño/genética , Conejos , Transcripción Genética , Transfección , Cicatrización de HeridasRESUMEN
This study assessed the possibility to build a prognosis predictor, based on microarray gene expression measures, in stage II and III colon cancer patients. Tumour (T) and non-neoplastic mucosa (NM) mRNA samples from 18 patients (nine with a recurrence, nine with no recurrence) were profiled using the Affymetrix HGU133A GeneChip. The k-nearest neighbour method was used for prognosis prediction using T and NM gene expression measures. Six-fold cross-validation was applied to select the number of neighbours and the number of informative genes to include in the predictors. Based on this information, one T-based and one NM-based predictor were proposed and their accuracies were estimated by double cross-validation. In six-fold cross-validation, the lowest numbers of informative genes giving the lowest numbers of false predictions (two out of 18) were 30 and 70 with the T and NM gene expression measures, respectively. A 30-gene T-based predictor and a 70-gene NM-based predictor were then built, with estimated accuracies of 78 and 83%, respectively. This study suggests that one can build an accurate prognosis predictor for stage II and III colon cancer patients, based on gene expression measures, and one can use either tumour or non-neoplastic mucosa for this purpose.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/secundario , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Perfilación de la Expresión Génica , Marcadores Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Humanos , Mucosa Intestinal , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Pronóstico , Sensibilidad y EspecificidadRESUMEN
Ischemia triggers an inflammatory response that precipitates cell death during reperfusion. Several studies have shown that tissues are protected by ischemic preconditioning (IP) consisting of 10 min of ischemia followed by 10 min of reperfusion just before ischemia. The molecular basis of this protective effect is poorly understood. We used cDNA arrays (20K) to compare global gene expression in liver biopsies from living human liver donors who underwent IP (n=7) or not (n=7) just before liver devascularization. Microarray data were analyzed using pairedt test with a type I error rate fixed at alpha = 2.5 10(6) (Bonferroni correction). We found that 60 genes were differentially expressed (36 over- and 24 underexpressed in preconditioning group). After IP, the most significantly overexpressed gene was IL-1Ra. This was confirmed by immunoblotting. Differentially expressed were genes involved in apoptosis (NOD2, ephrin-A1, and calpain) and in the carbohydrate metabolism. A significant increase in the amount of the anti-apoptotic protein Bcl-2 in preconditioned livers but no change in the cleavage of procaspase-3, -8, and -9 was observed. We also observed an increase in the amount in the inducible nitric oxide synthase. Therefore, the benefits of IP may be associated with the overproduction of IL-1Ra, Bcl-2, and NO countering the proinflammatory and proapoptotic effects generated during ischemia-reperfusion.
Asunto(s)
Regulación de la Expresión Génica , Precondicionamiento Isquémico , Hígado/patología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Daño por Reperfusión , Sialoglicoproteínas/metabolismo , Adulto , Apoptosis , Biopsia , Western Blotting , Carbohidratos/química , Caspasa 3 , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , ADN Complementario/metabolismo , Efrina-A1/metabolismo , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Inflamación , Proteína Antagonista del Receptor de Interleucina 1 , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Reperfusión , Factores de TiempoRESUMEN
Methionine depletion in the human cell line CCRF-CEM through the action of recombinant methioninase (rMETase), a methionine-cleaving enzyme, was previously demonstrated to produce a strong cytotoxic synergistic effect with fluorouracil (FUra) throughout a broad range of concentrations of FUra and rMETase, including subcytotoxic levels of rMETase. Potentiation was associated with a decrease in free thymidylate synthase from preexisting levels. To further investigate the action of rMETase on CCRF-CEM cells, in the present study we explored the effects of rMETase as a single agent on DNA methylation levels and DNA synthesis, which may be changed as a result of deprivation of methionine. Cells treated with rMETase under subcytotoxic conditions contained significantly lower levels of genomic methylated DNA than did control cells, as demonstrated by incorporation of the methyl radical of [methyl-(3)H]S-adenosylmethionine in DNA and by use of methylation-sensitive arbitrarily primed PCR. DNA hypomethylation produced by rMETase was of similar magnitude as that produced with the DNA methyltransferase inhibitor 5-azacytidine. Cells exposed to rMETase synthesized significantly more DNA than did untreated cells. Incorporation of [6-3H]thymidine and [6-3H]2'-deoxyuridine in these cells was augmented over that in control by mean factors of 1.78 and 2.36, respectively. Increased 3H nucleoside incorporation resulted in greater numbers of nuclear grains as demonstrated by autoradiography. The increase in DNA synthesis induced by rMETase is likely to result from enhancement of DNA repair because it was not accompanied by differences in cell cycle phase distribution or in total DNA content as determined by flow cytometry. We hypothesize that potentiation of FUra cytotoxicity by rMETase may result from increased inhibition of thymidylate synthase, together with DNA hypomethylation and enhanced DNA repair that could be involved in cell responses to drug-induced damage.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Liasas de Carbono-Azufre/farmacología , Metilación de ADN/efectos de los fármacos , ADN de Neoplasias/biosíntesis , Leucemia de Células T/tratamiento farmacológico , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Proteínas Recombinantes/farmacología , Células Tumorales CultivadasRESUMEN
Most gastrointestinal stromal tumors (GISTs) contain activating mutations of the proto-oncogene c-kit. The GNNK- isoform of c-kit has a greater oncogenic potential than the GNNK+ isoform. We studied tumors from 29 patients with GIST, 19 of whom had c-kit mutations, and compared them to normal cells and HMC-1 mast cell line. c-kit transcripts were quantified by real-time PCR. The ratios of GNNK-/+ isoforms and of wild-type/mutant alleles were determined by RT-PCR and fluorometric quantification. On average, GISTs contained 1.9 times more c-kit transcripts than the HMC-1 cell line and GISTs with c-kit mutations contained 2.8 times more c-kit transcripts than those without (P=0.003). The median GNNK-/+ isoform ratios in GISTs with and without c-kit mutations were 4.4 and 4.1, respectively, and there was no difference in the GNNK-/+ ratios between the GISTs and the control samples. Both mutant and wild-type alleles of c-kit were expressed in similar amounts in 13/15 mutant GISTs. The oncogenic effects of KIT in GISTs are not related to the higher expression level of the GNNK- isoform. The high expression level of both mutated and wild-type allele transcripts of c-kit suggests that interactions between spontaneously activated and normal c-kit receptors are important in GIST tumorigenesis.
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Regulación Neoplásica de la Expresión Génica/genética , Mutación/genética , Proteínas Proto-Oncogénicas c-kit/genética , Neoplasias Gástricas/genética , Adulto , Anciano , Secuencia de Bases , Cartilla de ADN , Femenino , Humanos , Inmunohistoquímica , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Proto-Oncogenes Mas , ARN Mensajero/genética , Neoplasias Gástricas/patología , Células del Estroma/patología , Transcripción GenéticaRESUMEN
Many studies have associated chromosomal deletions in the 16q24 region with human cancers, including hepatocellular carcinoma. A more limited region around the microsatellite D16S402 has been shown implicated in the metastatic spread of hepatocellular carcinoma, prostate cancer, and Wilms' tumors. It is likely that one or more tumor suppressor genes are located in this 16q24 area. We used SYBR Green reagents to quantify, by real-time quantitative RT-PCR, the production of mRNA for 13 genes mapping to 16q24. The locations of these genes were determined from published human genome sequencing data. We studied mRNA levels in 10 liver tumor tissues, 10 nontumor liver tissues, five hepatoma cell lines, and in isolated hepatocytes. Results were compared with those for loss of heterozygosity observed in the D16S402 region and recurrence. No down-regulation was observed in tumor tissues. Two genes were consistently overexpressed: OKL38 and CDH13. CDH13, which functions in cell-cell adhesion, seems to be involved in liver carcinogenesis. However, no relationship was observed between the expression of this gene and changes in the D16S402 microsatellite or tumor recurrence. None of the other genes tested seemed to be a good candidate for a major tumor suppressor gene in liver carcinogenesis. Our results suggest that additional unknown genes involved in carcinogenesis are located in the 16q24 area.
Asunto(s)
Carcinoma Hepatocelular/genética , Deleción Cromosómica , Cromosomas Humanos Par 16/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Adolescente , Adulto , Anciano , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Mapeo Cromosómico , Cartilla de ADN/química , ADN de Neoplasias/análisis , Genes Supresores de Tumor , Humanos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Pérdida de Heterocigocidad/genética , Repeticiones de Microsatélite/genética , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
WFDC1 is a recently isolated human gene identified as a tumour suppressor gene candidate. It is not known whether alterations in this gene are associated with human cancers. The WFDC1 gene maps in human chromosome 16q24, an area of frequent loss of heterozygosity (LOH) in several tumour types, in particular in hepatocellular carcinoma (HCC). We investigated its role in 46 European HCC by means of the detection of LOH at the WFDC1 locus. We describe here an assay for the detection of loss of heterozygosity at this locus using two dinucleotide repeat polymorphisms identified in WFDC1 introns, with a combined informativity of 86%. LOH was observed in 4/40 informative HCC samples. We further investigated the role of WFDC1 as a tumour suppressor gene candidate in five hepatocellular cell lines and in tumours exhibiting LOH by means of mutation, promoter methylation and gene expression analysis. In HCC samples showing LOH, no mutation of the remaining allele was observed. No significant up or down gene expression was observed in tumour samples comparatively to normal liver and gene expression did not seem related to promoter methylation. These results suggest a minor role, if any, of WFDC1 in hepatocarcinogenesis. However, this approach might be useful for investigating the role of this candidate tumour suppressor gene in other tumour types.
Asunto(s)
Carcinoma Hepatocelular/genética , Genes Supresores de Tumor , Neoplasias Hepáticas/genética , Pérdida de Heterocigocidad/genética , Proteínas/genética , Células Cultivadas , Cromosomas Humanos Par 16/genética , Metilación de ADN , Análisis Mutacional de ADN , Repeticiones de Dinucleótido/genética , Hepatocitos , Humanos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Regiones Promotoras Genéticas , Proteínas/metabolismo , Células Tumorales CultivadasRESUMEN
Gastrointestinal stromal tumors are the most common mesenchymal neoplasms of the digestive tract. These tumors express the c-kit receptor tyrosine kinase, and many have activating mutations in the juxtamembrane region coded by the exon 11 of KIT. Detection of these mutations has prognostic and therapeutic impact. The aim of the study was to compare a new detection method by length analysis of polymerase chain reaction products (LAPP) to direct sequencing. The detection of either deletion or insertion mutations within the exon 11 of KIT was performed on genomic DNA extracted from 40 paraffin-embedded samples from 38 patients. Double-strand direct sequencing revealed a mutation in 25 of 40 samples. In two additional samples, a mutation was suspected but could not be determined by sequencing. LAPP revealed a mutation in 27 samples, corresponding to the 25 determined and 2 suspected samples. One of these latter samples contained three different alleles. Mutations corresponded to either deletions (n = 24) or insertion (n = 1) and had the same size with sequencing and LAPP. Our results show that LAPP is as accurate and more sensitive than direct sequencing for the detection of deletion or insertion mutations of exon 11 of KIT in gastrointestinal stromal tumors.
Asunto(s)
Exones/genética , Neoplasias Gastrointestinales/genética , Polimorfismo de Longitud del Fragmento de Restricción , Proteínas Proto-Oncogénicas c-kit/genética , Sarcoma/genética , Secuencia de Bases , Análisis Mutacional de ADN , Neoplasias Gastrointestinales/química , Neoplasias Gastrointestinales/patología , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Proteínas Proto-Oncogénicas c-kit/análisis , Reproducibilidad de los Resultados , Sarcoma/química , Sarcoma/secundario , Sensibilidad y Especificidad , Células del Estroma/química , Células del Estroma/patologíaRESUMEN
Nucleoside Reverse Transcriptase Inhibitors (NRTIs) used for the treatment of HIV-1 inhibit the replication of mitochondrial DNA (mtDNA), which may contribute to severe mitochondrial toxicity including lipodystrophy, through the inhibition of polymerase gamma (POLG). Polymorphisms of POLG could explain the variation in mitochondrial toxicity in HIV-1-infected patients. We explored the relationship between selected polymorphisms of POLG and lipodystrophy related to NRTIs. We studied single nucleotide polymorphisms (SNP) at three amino acid residues (R1142, E1143 and R1146) and the CAG repeats of POLG in a case-control study including HIV-1 treated patients with lipodystrophy (n=69) and 2 controls (without lipodystrophy) per case matched by age, race and sex (n=138). Compared with matched controls, the polymorphisms in E1143 were significantly more frequent in case patients with lipodystrophy (aOR=4.7; p=0.048), and this was associated with a significant decrease of mtDNA in PBMC. In addition, among the parameters tested, the conditional logistic regression showed that the lipodystrophy has a strong link with E1143 polymorphisms, associated with D4T treatment (aOR=9.29, p=0.002). In conclusion, patients harbouring the changes of E1143 in the catalytic site of POLG exhibit a 4-fold increased risk to develop lipodystrophy than HIV-1 treated patients who do not have changes in E1143 and this risk can increase if the patient presenting the SNP received D4T. These could be due to decreased content of mtDNA in PBMC in these patients. Therefore, the toxicity of NRTIs leading to lipodystrophy in some HIV-1 infected patients could be explained in part by the occurrence of POLG polymorphisms.
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Fármacos Anti-VIH/efectos adversos , ADN Polimerasa Dirigida por ADN/genética , Infecciones por VIH/tratamiento farmacológico , Lipodistrofia/inducido químicamente , Polimorfismo de Nucleótido Simple , Inhibidores de la Transcriptasa Inversa/efectos adversos , Adulto , Fármacos Anti-VIH/uso terapéutico , Estudios de Casos y Controles , ADN Polimerasa gamma , ADN Mitocondrial/análisis , ADN Mitocondrial/efectos de los fármacos , Femenino , Frecuencia de los Genes , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Adulto JovenRESUMEN
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Cirrhosis caused by hepatitis B virus, hepatitis C virus or chronic alcohol intake is associated with major risk. Systematic screening for HCC of asymptomatic patients with cirrhosis is needed for earlier detection of small tumors requiring treatment (liver transplantation, surgical resection, percutaneous techniques). The recommended screening strategy among cirrhotic patients is based on regular liver ultrasonography associated with serum alpha-fetoprotein (AFP) assay. As the performance of AFP is not satisfactory, additional tumoral markers are proposed (des-gamma-carboxyprothrombin, glycosylated AFP-L3 fraction). Currently, diagnosis of HCC in cirrhotic patients includes non-invasive tests (imaging after contrast administration, AFP assay); diagnostic biopsy is performed when imaging is limited. After treatment, tumor recurrence is assessed by regular follow-up (AFP assay and imaging). Despite the lack of accurate markers, recent developments in genomic and proteomic approaches will allow the discovery of new biomarkers for primary tumors, as well as for recurrence. This review summarizes the current state of biomarkers for screening, diagnosis and follow-up of HCC, and highlights new perspectives in the field.
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Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Antígenos de Neoplasias/metabolismo , Epigénesis Genética , Genómica , Glicosilación , Humanos , Hígado/diagnóstico por imagen , Cirrosis Hepática/complicaciones , Modelos Biológicos , Ultrasonografía , alfa-Fetoproteínas/metabolismoRESUMEN
Understanding the molecular mechanisms underlying fatty liver disease (FLD) in humans is of major importance. We used high-density oligonucleotide microarrays (22.3 K) to assess the mechanisms responsible for the development of human liver steatosis. We compared global gene expression in normal (n=9) and steatotic (n=9) livers without histological signs of inflammation or fibrosis. A total of 34 additional human samples including normal (n=11), steatosis (n=11), HCV-related steatosis (n=4) or steatohepatitis associated with alcohol consumption (n=4) or obesity (n=4) were used for immunohistochemistry or quantitative real-time PCR studies. With unsupervised classification (no gene selection), all steatotic liver samples clustered together. Using step-down maxT multiple testing procedure for controlling the Family-Wise Error-Rate at level 5%, 110 cDNAs (100 over- and 10 underexpressed) were found to be differentially expressed in steatotic and normal livers. Of them were genes involved in mitochondrial phosphorylative and oxidative metabolism. The mean ratio of mitochondrial DNA to nuclear DNA content was higher in liver steatosis compared to normal liver biopsies (1.12+/-0.14 vs 0.67+/-0.10; P=0.01). An increased expression of genes involved in inflammation (IL-1R family, TGFB) was also observed and confirmed by quantitative RT-PCR or immunochemistry. In steatohepatitis, an increase of the protein expression of mitochondrial antigens, IL-1R1, IGF2 and TGFB1 was also observed, interleukin 1 receptor being always strongly expressed in steatohepatitis linked to alcohol or obesity. In conclusion, mitochondrial alterations play a major role in the development of steatosis per se. Activation of inflammatory pathways is present at a very early stage of steatosis, even if no morphological sign of inflammation is observed.
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Hígado Graso/genética , Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , ADN Mitocondrial/genética , Hígado Graso/patología , Humanos , Inmunohistoquímica , Mitocondrias Hepáticas/metabolismo , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Highly active antiretroviral therapy (HAART) can cause mitochondrial toxicity. The concentration of mitochondrial DNA (mtDNA) in peripheral blood cells has been reported to be a marker of this toxicity. However, these observations are controversial and were drawn from small series. Thus, we analysed the value of blood mtDNA as a marker of mitochondrial toxicity in a large cohort of human immunodeficiency virus (HIV)-infected out-patients during routine clinical evaluations. Real-time quantitative PCR was used to determine the mtDNA to nuclear DNA (nDNA) ratio in peripheral blood mononuclear cells from 157 consecutive HIV-1-infected patients (13 naive, 144 receiving HAART) and 30 HIV-1-uninfected patients. The mtDNA to nDNA ratio was significantly lower in both groups of HIV-infected patients than in the control group. No significant difference was observed between treated and naive HIV-infected patients. Lactataemia was significantly lower in controls than in the group of HIV-treated patients. None of the treated patients had lactataemia >5 mmol/l or bicarbonates <20 mmol/l. Triglyceride levels were significantly higher in the HAART-treated patients than in the nontreated patients. Clinical symptoms of lipodystrophy were observed in 62 HAART-treated patients. These symptoms were not associated with an abnormal mtDNA to nDNA ratio or plasma triglyceride concentration. The mtDNA to nDNA ratio was lower in DDI/D4T-treated patients than in AZT/3TC-treated patients. In conclusion, there are no obvious links between the mtDNA to nDNA ratio in peripheral mononuclear cells and any clinical symptoms or lactate level. Thus, the mtDNA to nDNA ratio in leukocytes does not seem to be an accurate marker of mild and/or long-term mitochondrial toxicity.
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Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Terapia Antirretroviral Altamente Activa/efectos adversos , ADN Mitocondrial/sangre , VIH-1 , Mitocondrias/efectos de los fármacos , Adulto , Femenino , Síndrome de Lipodistrofia Asociada a VIH/etiología , Humanos , Lactatos/sangre , Lípidos/sangre , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
Microsatellite instability (MSI) seems to be a rare event in hepatocarcinogenesis and might actually be associated with the progression of hepatocellular carcinoma (HCC) in which the liver is often the site of chronic hepatitis or cirrhosis. The aim of this work was to define the MSI phenotype in HCC affecting exclusively normal livers to avoid slippage errors due to cirrhosis. One hundred and sixty-four patients with HCC affecting non-cirrhotic livers were operated on in our hospital between 1984 and 2001. We analyzed 37 patients selected for low alcohol consumption and the absence of HBV or HCV infection. All the livers were histologically normal. MSI was analyzed according to the criteria defined during the conference consensus workshop for colorectal cancer. High MSI (MSI-H > 30%) was found in 6 (16%) and low MSI (MSI-L < 30%) in 10 (27%) of the 37 HCCs. None of the 10 microsatellite markers tested were altered in the remaining 21 tumors (57%). Immunohistochemistry showed that normal amounts of hMLH1 and hMSH2 were present both in MSI-H and in MSI-L HCCs. MSI-H was significantly associated with more aggressive histological tumor features and a shorter median delay before recurrence. Thus, we have found a small subgroup of HCC tumors which can be considered as a new clinical/histological entity.
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Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Repeticiones de Microsatélite/genética , Proteínas Adaptadoras Transductoras de Señales , Carcinoma Hepatocelular/cirugía , Proteínas Portadoras , Neoplasias Colorrectales/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Supervivencia sin Enfermedad , Fibrosis/complicaciones , Estudios de Seguimiento , Humanos , Neoplasias Hepáticas/cirugía , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Mutagénesis , Proteínas de Neoplasias/genética , Proteínas Nucleares , Proteínas Proto-Oncogénicas/genética , Factores de TiempoRESUMEN
Methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folate metabolism, plays a major role in the provision of methyl groups for DNA methylation and in the production of dTMP for DNA synthesis. Different polymorphisms have been described for this enzyme, the most studied being the C677T, which has been shown to be associated with predisposition to colorectal cancer in patients who consume a high alcohol diet. The aim of this study was to determine whether the MTHFR polymorphism is related to hepatocellular carcinoma (HCC) in patients with alcoholic cirrhosis. MTHFR genotypes were determined in 300 liver transplant patients, 72 of whom had alcoholic cirrhosis with HCC and 122 of whom had alcoholic cirrhosis without HCC. The remaining patients were transplanted for HCC on normal liver (n = 27) or viral cirrhosis with HCC (n = 49) or without HCC (n = 30). We also tested 80 healthy subjects. Among the group of patients transplanted for alcoholic cirrhosis, the frequency of MTHFR variants CC versus CT and TT was significantly higher in patients with HCC than in patients without macroscopic evidence of HCC (P = 0.02). This difference was not observed between patients with and without HCC developed either on viral cirrhosis or on non-cirrhotic liver. If we considered all the patients transplanted for HCC, the MTHFR CC genotype was significantly higher in patients who had developed HCC on alcoholic cirrhosis rather than on viral cirrhosis (P = 0.002) or on non-cirrhotic livers (P = 0.02). The relative risk for HCC in subjects with alcoholic cirrhosis and the CC genotype was 2.03. These results suggest that the MTHFR CC genotype increases the risk to develop HCC in patients who consume a high alcohol diet.