RESUMEN
Since sperm require high energy levels to perform their specialised function, it is vital that essential nutrients are available for spermatozoa when they develop, capacitate and acquire motility. However, they are vulnerable to a lack of energy and excess amounts of reactive oxygen species, which can impair sperm function, lead to immotility, acrosomal reaction impairment, DNA fragmentation and cell death. This monocentric, randomised, double-blind, placebo-controlled trial investigated the effect of 6 months of supplementation with l-carnitine, acetyl-l-carnitine and other micronutrients on sperm quality in 104 subjects with oligo- and/or astheno- and/or teratozoospermia with or without varicocele. In 94 patients who completed the study, sperm concentration was significantly increased in supplemented patients compared to the placebo (p = .0186). Total sperm count also increased significantly (p = .0117) in the supplemented group as compared to the placebo group. Both, progressive and total motility were higher in supplemented patients (p = .0088 and p = .0120, respectively). Although pregnancy rate was not an endpoint of the study, of the 12 pregnancies that occurred during the follow-up, 10 were reported in the supplementation group. In general, all these changes were more evident in varicocele patients. In conclusion, supplementation with metabolic and antioxidant compounds could be efficacious when included in strategies to improve fertility.
Asunto(s)
Antioxidantes/uso terapéutico , Astenozoospermia/tratamiento farmacológico , Varicocele/tratamiento farmacológico , Adolescente , Adulto , Antioxidantes/farmacología , Astenozoospermia/complicaciones , Suplementos Dietéticos , Método Doble Ciego , Humanos , Masculino , Persona de Mediana Edad , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Resultado del Tratamiento , Varicocele/complicaciones , Adulto JovenRESUMEN
AIM: The effectiveness of propionyl-L-carnitine (PLC) monotherapy regimen alone or in association with pulsed muscular compression was compared to the physical therapy by itself against obliterant arteriopathy Leriche Fontaine stage II. PLC is involved in cellular metabolism and is transformed into two active substances, free L-carnitine and propionyl-coenzyme A in the mitochondria, which take part in fatty acid transfer and in the citric acid cycle, respectively. METHODS: Forty-two patients with arterial disease were selected (22 males and 20 females; mean age: 62+/-8 years; 21 type 2 diabetic [DB] and 21 non-DB [NDB]). At enrollment all patients completed a symptoms questionnaire enabling both clinical and social evaluation of the impact of the arteriopathy on the quality of life. Then, patients had: routine blood samples, echo duplex scan; evaluation of the ankle/arm (Winsor) index; impedance plethysmography (Rheoscreen) to measure the crest time (CT), index of the pathological changes due to the sclerosis on the vascular wall, and measurement of walking distance by means of treadmill test. Patients were randomized in three groups, each of them composed by 14 patients (7 DB and 7 NDB): the first group was submitted to infusional PLC therapy at a dosage of 4 fl (total: 1,200 mg PLC) in 250 cc of physiological solution for 5 days a week for 4 weeks; the second group was treated with PLC in association with pulsed muscular compression therapy by Vascupump (5 sessions a week for 4 weeks); the third group was submitted only to Vascupump. RESULTS: The efficacy of both PLC and Vascupump in the treatment of the peripheral vasculopathies was confirmed. From a subjective point of view, patients referred benefits both in clinical terms, i.e. increased walking distance (average increaseaegroup I: DB 102%, NDB 118%; group II: DB 94%, NDB 193%; group III: DB 33%, NDB 67%) and of decreased intensity of the calf pain from the quality of life questionnaire (21.5 to 10.7). The instrumental parameters showed a trend towards normality, i.e decrease in CT and an increase of the Winsor index, indicators of increased peripheral blood circulation. CONCLUSION: Combined pharmaco- and physical therapy was most efficient treatment regime and best results were seen in NDB compared to the DB patients.
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Arteriopatías Oclusivas/terapia , Fármacos Cardiovasculares/uso terapéutico , Carnitina/análogos & derivados , Diabetes Mellitus Tipo 2/complicaciones , Aparatos de Compresión Neumática Intermitente , Anciano , Arteriopatías Oclusivas/complicaciones , Arteriopatías Oclusivas/fisiopatología , Fármacos Cardiovasculares/administración & dosificación , Carnitina/administración & dosificación , Carnitina/uso terapéutico , Terapia Combinada , Diabetes Mellitus Tipo 2/fisiopatología , Diabetes Mellitus Tipo 2/terapia , Femenino , Humanos , Infusiones Intravenosas , Claudicación Intermitente/etiología , Claudicación Intermitente/prevención & control , Masculino , Persona de Mediana Edad , Calidad de Vida , Recuperación de la Función , Índice de Severidad de la Enfermedad , Factores de Tiempo , Resultado del Tratamiento , CaminataRESUMEN
1-Methyl-4-phenylpyridinium ion (MPP+), an inhibitor of mitochondrial complex I, has been widely used as a neurotoxin because it elicits a severe Parkinson's disease-like syndrome with an elevation of intracellular reactive oxygen species (ROS) and apoptosis. L-carnitine plays an integral role in attenuating the brain injury associated with mitochondrial neurodegenerative disorders. The present study investigates the effects of L-carnitine against the toxicity of MPP+ in rat forebrain primary cultures. Cells in culture were treated for 24 h with 100, 250, 500 and 1000 microM MPP+ alone or co-incubated with L-carnitine. MPP+ produced a dose-related increase in DNA fragmentation as measured by cell death ELISA (enzyme-linked immunosorbent assay), an increase in the number of TUNEL (terminal dUTP nick-end labeling)-positive cells and a reduction in the mitochondrial metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). No significant effect was observed with the release of lactate dehydrogenase (LDH), indicating that cell death presumably occurred via apoptotic mechanisms. Co-incubation of MPP+ with L-carnitine significantly reduced MPP+-induced apoptosis. Western blot analyses showed that neurotoxic concentrations of MPP+ decreased the ratio of BCL-X(L) to Bax and decreased the protein levels of polysialic acid neural cell adhesion molecules (PSA-NCAM), a neuron specific marker. L-carnitine blocked these effects of MPP+ suggesting its potential therapeutic utility in degenerative disorders such as Parkinson's disease, Alzheimer's disease, ornithine transcarbamylase deficiency and other mitochondrial diseases.
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1-Metil-4-fenilpiridinio/antagonistas & inhibidores , 1-Metil-4-fenilpiridinio/toxicidad , Apoptosis/efectos de los fármacos , Carnitina/farmacología , Neuronas/efectos de los fármacos , Neuronas/patología , Fármacos Neuroprotectores , Prosencéfalo/patología , Animales , Western Blotting , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , L-Lactato Deshidrogenasa/metabolismo , Prosencéfalo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Ácidos Siálicos/metabolismo , Sales de Tetrazolio , Tiazoles , Proteína X Asociada a bcl-2/biosíntesis , Proteína bcl-X/biosíntesisAsunto(s)
Separación Celular/métodos , Células Epiteliales/citología , Biología Molecular/métodos , Ácidos Nucleicos/aislamiento & purificación , Neoplasias del Colon , ADN de Neoplasias/genética , Genes p53 , Globinas/genética , Humanos , Queratinas/genética , Glomérulos Renales , Pérdida de Heterocigocidad , Masculino , PróstataRESUMEN
BACKGROUND: Most lung cancers are attributed to smoking. These cancers have been associated with multiple genetic alterations and with the presence of preneoplastic bronchial lesions. In view of such associations, we evaluated the status of specific chromosomal loci in histologically normal and abnormal bronchial biopsy specimens from current and former smokers and specimens from nonsmokers. METHODS: Multiple biopsy specimens were obtained from 18 current smokers, 24 former smokers, and 21 nonsmokers. Polymerase chain reaction-based assays involving 15 polymorphic microsatellite DNA markers were used to examine eight chromosomal regions for genetic changes (loss of heterozygosity [LOH] and microsatellite alterations). RESULTS: LOH and microsatellite alterations were observed in biopsy specimens from both current and former smokers, but no statistically significant differences were observed between the two groups. Among individuals with a history of smoking, 86% demonstrated LOH in one or more biopsy specimens, and 24% showed LOH in all biopsy specimens. About half of the histologically normal specimens from smokers showed LOH, but the frequency of LOH and the severity of histologic change did not correspond until the carcinoma in situ stage. A subset of biopsy specimens from smokers that exhibited either normal or preneoplastic histology showed LOH at multiple chromosomal sites, a phenomenon frequently observed in carcinoma in situ and invasive cancer. LOH on chromosomes 3p and 9p was more frequent than LOH on chromosomes 5q, 17p (17p13; TP53 gene), and 13q (13q14; retinoblastoma gene). Microsatellite alterations were detected in 64% of the smokers. No genetic alterations were detected in nonsmokers. CONCLUSIONS: Genetic changes similar to those found in lung cancers can be detected in the nonmalignant bronchial epithelium of current and former smokers and may persist for many years after smoking cessation.
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Bronquios/metabolismo , Deleción Cromosómica , Daño del ADN , Fumar/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Autorradiografía , Bronquios/patología , Epitelio/metabolismo , Epitelio/patología , Femenino , Heterocigoto , Humanos , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Cese del Hábito de Fumar , Factores de TiempoRESUMEN
BACKGROUND: Carcinogenesis is a multistep process, which may begin as a consequence of chromosomal changes. Deletions in the short arm of chromosome 9 (9p) have been observed in lung carcinomas. In addition, morphologically recognizable preneoplastic lesions, frequently multiple in number, precede onset of invasive carcinomas. PURPOSE: We tested for deletions and loss of heterozygosity (LOH) at 9p loci in preneoplastic and neoplastic foci in lungs of patients with non-small-cell lung carcinomas (NSCLCs). METHODS: Seven archival, paraffin-embedded, surgically resected NSCLC specimens were selected. They were predominantly from patients with adenocarcinomas and contained multiple preneoplastic lesions, including hyperplasia, metaplasia, dysplasia, and carcinoma in situ (CIS). Fifty-three histologically identified preneoplastic and malignant lesions present in bronchi, bronchioles, and alveoli were precisely microdissected from stained tissue sections with a micromanipulator. Stromal lymphocytes were used to determine constitutional heterozygosity. The specimens were analyzed for LOH using polymerase chain reaction-based assays for polymorphism in dinucleotide repeats (microsatellite markers) in interferon alfa (IFNA) and D9S171 loci on 9p. RESULTS: All seven cases were constitutionally heterozygous for one or both microsatellite markers. Five of seven cases had LOH at one or both 9p loci in the invasive primary cancers (doubly informative cases). Four of these five cases also revealed LOH in preneoplastic foci. In the doubly informative cases, LOH was detected in five (38%) of 13 foci of hyperplasia, four (80%) of five foci of dysplasia, and three (100%) of three CIS lesions. LOH was detected in preneoplastic lesions from all regions of the respiratory tract, including bronchi, bronchioles, and alveoli, and involved five different cell types. The identical allele was lost from both the preneoplastic lesions and the corresponding tumors (12 of 12 lesions, 17 of 17 comparisons), a phenomenon we have referred to as "allele-specific mutation." Statistical analyses employing a cumulative binomial test demonstrated that the probabilities of such findings occurring by chance are 2.4 x 10(-4) and 7.6 x 10(-6), respectively. From comparisons with the previously published data on other chromosomal abnormalities in the same tissue specimens, it appears that LOH at 3p and 9p loci occurred early in the hyperplasia stage, but the ras gene point mutations were relatively late, at the CIS stage. CONCLUSIONS: LOH at 9p loci occurs at the earliest stage in the pathogenesis of lung cancer and involves all regions of the respiratory tract. LOH in NSCLC is not random but targets a specific allele in individuals. Studying preneoplastic lesions may help identify intermediate markers for risk assessment and chemoprevention.
Asunto(s)
Alelos , Carcinoma de Pulmón de Células no Pequeñas/genética , Deleción Cromosómica , Cromosomas Humanos Par 8/genética , Neoplasias Pulmonares/genética , Síndromes Paraneoplásicos/genética , Adenocarcinoma/genética , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Carcinoma de Pulmón de Células no Pequeñas/complicaciones , Femenino , Genes ras/genética , Heterocigoto , Humanos , Neoplasias Pulmonares/complicaciones , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Síndromes Paraneoplásicos/complicacionesRESUMEN
BACKGROUND: Several molecular changes, including loss of heterozygosity (i.e., deletion of one copy of allelic DNA sequences) and alterations in microsatellite DNA, have been detected early in the pathogenesis of lung cancer, even in histologically normal epithelium. In the bronchial epithelium of patients with lung cancer, we have determined the frequency, size, and patterns of molecularly abnormal clonal patches. METHODS: We studied formalin-fixed, paraffin-embedded samples from 16 surgically resected lung carcinomas (five squamous cell carcinomas, four small-cell carcinomas, six adenocarcinomas, and one large-cell carcinoma). From each carcinoma, we microdissected foci (each containing about 200 cells) of tumor tissue and equivalent samples of histologically normal and abnormal epithelium. Furthermore, multiple discontinuous foci of bronchial epithelium were analyzed from methanol-fixed samples from three additional patients with lung cancer (two with squamous cell carcinoma and one with adenocarcinoma). We used two-step polymerase chain reaction-based assays involving 12 microsatellite markers at seven chromosomal regions frequently deleted in lung cancer. RESULTS: Two hundred eighteen foci of nonmalignant bronchial epithelium (195 of histologically normal or slightly abnormal epithelium and 23 of dysplastic epithelium) were studied from the 19 surgically resected lobectomy specimens. Thirteen (68%) of the 19 specimens had at least one focus of bronchial epithelium with molecular changes. At least one molecular abnormality was detected in 32% of the 195 histologically normal or slightly abnormal foci and in 52% of the 23 dysplastic foci. Extrapolating from our two-dimensional analyses, we estimate that most clonal patches contain approximately 90 000 cells. Although, in a given individual, tumors appeared homogeneous with respect to molecular changes, the clonally altered patches of mildly abnormal epithelium were heterogeneous. CONCLUSIONS: Our findings indicate that multiple small clonal or subclonal patches containing molecular abnormalities are present in normal or slightly abnormal bronchial epithelium of patients with lung cancer.
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Bronquios/patología , Carcinoma/genética , Carcinoma/patología , Células Clonales/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Adenocarcinoma/genética , Adenocarcinoma/patología , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/patología , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Pequeñas/patología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Epitelio/patología , Humanos , Pérdida de Heterocigocidad , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Retinoic acid plays an important role in lung development and differentiation, acting primarily via nuclear receptors encoded by the retinoic acid receptor-beta (RARbeta) gene. Because receptor isoforms RARbeta2 and RARbeta4 are repressed in human lung cancers, we investigated whether methylation of their promoter, P2, might lead to silencing of the RARbeta gene in human lung tumors and cell lines. METHODS: Methylation of the P2 promoter from small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cell lines and tumor samples was analyzed by the methylation-specific polymerase chain reaction (PCR). Expression of RARbeta2 and RARbeta4 was analyzed by reverse transcription-PCR. Loss of heterozygosity (LOH) was analyzed by PCR amplification followed by electrophoretic separation of PCR products. Statistical differences were analyzed by Fisher's exact test with continuity correction. RESULTS: The P2 promoter was methylated in 72% (63 of 87) of SCLC and in 41% (52 of 127) of NSCLC tumors and cell lines, and the difference was statistically significant (two-sided P:<.001). By contrast, in 57 of 58 control samples, we observed only the unmethylated form of the gene. Four tumor cell lines with unmethylated promoter regions expressed both RARbeta2 and RARbeta4. Four tumor lines with methylated promoter regions lacked expression of these isoforms, but demethylation by exposure to 5-aza-2'-deoxycytidine restored their expression. LOH at chromosome 3p24 was observed in 100% (13 of 13) of SCLC lines and 67% (12 of 18) of NSCLC cell lines, and the difference was statistically significant (two-sided P: =.028). CONCLUSIONS: Methylation of the RARbeta P2 promoter is one mechanism that silences RARbeta2 and RARbeta4 expression in many lung cancers, particularly SCLC. Chemical demethylation is a potential approach to lung cancer therapy.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Pequeñas/metabolismo , Pérdida de Heterocigocidad , Neoplasias Pulmonares/metabolismo , Regiones Promotoras Genéticas , Receptores de Ácido Retinoico/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Pequeñas/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Metilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
BACKGROUND: The recently identified RASSF1 locus is located within a 120-kilobase region of chromosome 3p21.3 that frequently undergoes allele loss in lung and breast cancers. We explored the hypothesis that RASSF1 encodes a tumor suppressor gene for lung and breast cancers. METHODS: We assessed expression of two RASSF1 gene products, RASSF1A and RASSF1C, and the methylation status of their respective promoters in 27 non-small-cell lung cancer (NSCLC) cell lines, in 107 resected NSCLCs, in 47 small-cell lung cancer (SCLC) cell lines, in 22 breast cancer cell lines, in 39 resected breast cancers, in 104 nonmalignant lung samples, and in three breast and lung epithelial cultures. We also transfected a lung cancer cell line that lacks RASSF1A expression with vectors containing RASSF1A complementary DNA to determine whether exogenous expression of RASSF1A would affect in vitro growth and in vivo tumorigenicity of this cell line. All statistical tests were two-sided. RESULTS: RASSF1A messenger RNA was expressed in nonmalignant epithelial cultures but not in 100% of the SCLC, in 65% of the NSCLC, or in 60% of the breast cancer lines. By contrast, RASSF1C was expressed in all nonmalignant cell cultures and in nearly all cancer cell lines. RASSF1A promoter hypermethylation was detected in 100% of SCLC, in 63% of NSCLC, in 64% of breast cancer lines, in 30% of primary NSCLCs, and in 49% of primary breast tumors but in none of the nonmalignant lung tissues. RASSF1A promoter hypermethylation in resected NSCLCs was associated with impaired patient survival (P =.046). Exogenous expression of RASSF1A in a cell line lacking expression decreased in vitro colony formation and in vivo tumorigenicity. CONCLUSION: RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung and breast cancers through hypermethylation of its promoter region.
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Neoplasias de la Mama/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Pequeñas/genética , Metilación de ADN , Genes Supresores de Tumor , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas , Proteínas Supresoras de Tumor , Adulto , Anciano , Islas de CpG , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
We investigated preneoplastic lesions associated with lung cancer to determine at what stage in lung carcinogenesis K-ras mutations appear. We selected six archival lung cancer resection cases that had ras mutations. We precisely microdissected 74 relevant areas from paraffin-embedded sections. K-ras mutations at codons 12, 13, and 61 were determined by the designed restriction fragment length polymorphism method using mismatched nested primers and confirmed by direct sequencing. All samples of invasive and metastatic cancers had K-ras mutations, as did four of five lesions of noninvasive cancer. Mutations were detected in only 1 of 12 dysplastic lesions and were absent from hyperplastic and normal-appearing cells. In all cases, the specific point mutations and the mutational pattern in the tumors, metastases, and the corresponding noninvasive lesions were identical. These results indicate that K-ras mutations arise relatively late in the pathogenesis of lung cancer and may be associated with the appearance of the malignant phenotype.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Genes ras/genética , Neoplasias Pulmonares/genética , Mutación Puntual/genética , Lesiones Precancerosas/genética , Codón/genética , Humanos , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Aberrant methylation of CpG islands acquired in tumor cells in promoter regions is one method for loss of gene function. We determined the frequency of aberrant promoter methylation (referred to as methylation) of the genes retinoic acid receptor beta-2 (RARbeta), tissue inhibitor of metalloproteinase 3 (TIMP-3), p16INK4a, O6-methylguanine-DNA-methyltransferase (MGMT), death-associated protein kinase (DAPK), E-cadherin (ECAD), p14ARF, and glutathione S-transferase P1 (GSTP1) in 107 resected primary non-small cell lung cancers (NSCLCs) and in 104 corresponding nonmalignant lung tissues by methylation-specific PCR. Methylation in the tumor samples was detected in 40% for RARbeta, 26% for TIMP-3, 25% for p16INK4a, 21% for MGMT, 19% for DAPK, 18% for ECAD, 8% for p14ARF, and 7% for GSTP1, whereas it was not seen in the vast majority of the corresponding nonmalignant tissues. Moreover, p16INK4a methylation was correlated with loss of p16INK4a expression by immunohistochemistry. A total of 82% of the NSCLCs had methylation of at least one of these genes; 37% of the NSCLCs had one gene methylated, 22% of the NSCLCs had two genes methylated, 13% of the NSCLCs had three genes methylated, 8% of the NSCLCs had four genes methylated, and 2% of the NSCLCs had five genes methylated. Methylation of these genes was correlated with some clinicopathological characteristics of the patients. In comparing the methylation patterns of tumors and nonmalignant lung tissues from the same patients, there were many discordancies where the genes methylated in nonmalignant tissues were not methylated in the corresponding tumors. This suggests that the methylation was occurring as a preneoplastic change. We conclude that these findings confirm in a large sample that methylation is a frequent event in NSCLC, can also occur in smoking-damaged nonmalignant lung tissues, and may be the most common mechanism to inactivate cancer-related genes in NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Metilación de ADN , Neoplasias Pulmonares/genética , Regiones Promotoras Genéticas/genética , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Reguladoras de la Apoptosis , Cadherinas/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Proteínas Quinasas Asociadas a Muerte Celular , Femenino , Genes p16/genética , Gutatión-S-Transferasa pi , Glutatión Transferasa/genética , Humanos , Inmunohistoquímica , Isoenzimas/genética , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , O(6)-Metilguanina-ADN Metiltransferasa/genética , Reacción en Cadena de la Polimerasa/métodos , Proteínas/genética , Receptores de Ácido Retinoico/genética , Factores de Riesgo , Tasa de Supervivencia , Inhibidor Tisular de Metaloproteinasa-3/genética , Proteína p14ARF Supresora de TumorRESUMEN
To identify the major tumor suppressor gene (TSG) loci involved in the pathogenesis of lung cancer, we have conducted a high-resolution (10 cM), genome-wide search of loss of heterozygosity (LOH). Thirty-six lung cancer cell lines [14 small cell lung cancers (SCLCs) and 22 non-SCLCs (NSCLCs)] and their matched control DNAs were analyzed using 399 fluorescent microsatellite markers from the ABI Prism linkage mapping set v.2 on an ABI 377 sequencer/genotyper. Overall, 22 different regions with more than 60% LOH were identified: (a) 13 regions with a preference for SCLC; (b) 7 regions with a preference for NSCLC; and (c) 2 regions affecting both SCLC and NSCLC. The chromosomal arms with the most frequent LOH were 1p, 3p, 4p, 4q, 5q, 8p, 9p (p16), 9q, 10p, 10q, 13q (Rb), 15q, 17p (p53), 18q, 19p, Xp, Xq. In addition, new homozygous deletions were found at 2p23, 8q24, 18q11, and Xq22. On average, 34% (SCLC) to 36% (NSCLC) of markers showed allele loss in individual tumors, with an average size of subchromosomal region of loss of five to six markers (50-60 cM). Whereas SCLC and NSCLC had different regions of frequent LOH (hot spots), and NSCLC had more of these regions (n = 22) than SCLC (n = 17), in all other parameters (fractional allelic loss, number of breakpoints, and number of microsatellite alterations), SCLC and NSCLC were not significantly different. Clustering analysis revealed correlations between LOH on different chromosomes that suggest previously unknown genetic interactions for lung cancer development. We conclude that (a) in lung cancer cell lines, at least 17-22 chromosomal regions with frequent allele loss are involved, suggesting that the same number of putative TSGs are inactivated; (b) SCLC and NSCLC frequently undergo different specific genetic alterations; and (c) clusters of TSGs are likely to be inactivated together. Overall, these data provide global estimates of the extent of genetic changes leading to lung cancer and will be useful for the positional cloning of new TSGs and for the identification of multiple new biomarkers for translational research.
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Alelos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Pequeñas/genética , Genoma Humano , Neoplasias Pulmonares/genética , Análisis por Conglomerados , ADN de Neoplasias/genética , Femenino , Genes Supresores de Tumor/genética , Marcadores Genéticos , Homocigoto , Humanos , Pérdida de Heterocigocidad/genética , Masculino , Familia de Multigenes/genética , Células Tumorales CultivadasRESUMEN
Allele loss and loss of expression of fragile histidine triad (FHIT), a putative tumor suppressor gene located in chromosome region 3p14.2, are frequent in several types of cancers. Tumor-acquired methylation of promoter region CpG islands is one method for silencing tumor suppressor genes. We investigated 5' CpG island methylation of the FHIT gene in 107 primary non-small cell lung cancer (NSCLC) samples and corresponding nonmalignant lung tissues, 39 primary breast carcinomas, as well as in 49 lung and 22 breast cancer cell lines by a methylation-specific PCR assay. In addition, we analyzed brushes from the bronchial epithelium of 35 heavy smokers without cancer. FHIT methylation was detected in 37% of primary NSCLCs, 31% of primary breast cancers, and 65% of lung and 86% of breast cancer cell lines. The frequency of methylation in small cell and NSCLC cell lines were identical. Methylation was found in 9% of the corresponding nonmalignant lung tissues and in 17% of bronchial brushes from heavy cigarette smokers. FHIT methylation was significantly correlated with loss of FHIT mRNA expression by Northern blot analysis in lung cancer cell lines and with loss of Fhit expression in NSCLC and breast tumors by immunostaining. We conclude that methylation of FHIT is a frequent event in NSCLC and breast cancers and is an important mechanism for loss of expression of this gene. Methylation of FHIT commences during lung cancer pathogenesis and may represent a marker for risk assessment.
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Ácido Anhídrido Hidrolasas , Azacitidina/análogos & derivados , Neoplasias de la Mama/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Islas de CpG , Metilación de ADN , Silenciador del Gen , Neoplasias Pulmonares/genética , Proteínas de Neoplasias , Proteínas/genética , Adulto , Anciano , Anciano de 80 o más Años , Antimetabolitos Antineoplásicos/farmacología , Azacitidina/farmacología , Biomarcadores de Tumor/genética , Northern Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Islas de CpG/genética , Decitabina , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Humanos , Pérdida de Heterocigocidad , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Expression of some members of the cadherin family is reduced in several human tumors, and CDH13 (H-cadherin), located on chromosome 16q24.2-3, may function as a tumor suppressor gene. In human tumors, loss of expression of many tumor suppressor genes occurs by aberrant promoter region methylation. We examined the methylation status of the CDH13 promoter in breast and lung cancers and correlated it with mRNA expression using methylation-specific PCR and reverse transcription-PCR. Methylation was frequent in primary breast tumors (18 of 55, 33%) and cell lines (7 of 20, 35%). In lung cancers, methylation was present more frequently in non-small cell lung cancer tumors (18 of 42, 43%) and cell lines (15 of 30, 50%) than in small cell lung cancer cell lines (6 of 30, 20%; P = 0.03). Only the methylated or unmethylated forms of the gene were present in most (73 of 80, 91%) tumor cell lines. CDH13 expression was present in 24 of 30 (80%) of nonmethylated tumor lines. All 18 methylated lines tested lacked expression irrespective of whether the unmethylated form was present, confirming biallelic inactivation in methylated lines. Gene expression was restored in all five methylated cell lines tested after treatment with the demethylating agent 5'-aza-2-deoxycytidine. Our results demonstrate frequent aberrant methylation of CDH13 in breast and lung cancers accompanied by loss of gene expression, although expression may occasionally be lost by other mechanisms.
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Neoplasias de la Mama/genética , Cadherinas/genética , Metilación de ADN , Neoplasias Pulmonares/genética , Neoplasias de la Mama/metabolismo , Cadherinas/biosíntesis , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Pequeñas/metabolismo , ADN de Neoplasias/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Genes Supresores de Tumor , Humanos , Pérdida de Heterocigocidad , Neoplasias Pulmonares/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
Although gallbladder carcinoma is one of the most frequent neoplasms in Chile, there is limited information about the molecular changes involved in its pathogenesis. We investigated the incidence of ras gene mutations and loss of heterozygosity (LOH) at the following genes/loci: p53, DCC, rb, 5q 3p, 8p, and 9p. We precisely microdissected 194 relevant areas from paraffin-embedded microslides from 25 gallbladder carcinomas and their accompanying nonneoplastic lesions (which were present in 15 cases) from patients in Chile. The specimens were analyzed by PCR-based assays for LOH, and we designed a RFLP method for ras mutations and immunohistochemistry for p53 protein overexpression. We determined that LOH at p53 (91%), 9p (50%), 8p (44%) and DCC (31%) are frequent events and that LOH at p53, 9p, and DCC are early events, while ras mutations and LOH at 3p, rb, and 5q occurred occasionally. LOH at p53 occurred more frequently and earlier than protein overexpression. The mean number of mutations present in invasive carcinomas was 2.1, and in six cases, LOH at the p53 gene was the sole mutation detected. The same allele was lost in 61 (93%) of 71 nonneoplastic foci as in the corresponding invasive carcinomas for all four mutations studied. The odds of this occurring by chance are approximately 4 x 10(-15). Although clonality cannot be excluded, allelic loss appears to be highly directed, but the mechanism for allele-specific mutations remains to be determined.
Asunto(s)
Alelos , Deleción Cromosómica , Cromosomas Humanos , Neoplasias de la Vesícula Biliar/genética , Mutación , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma/cirugía , Secuencia de Bases , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Pequeñas/patología , Carcinoma de Células Pequeñas/cirugía , Chile/epidemiología , Epitelio/patología , Vesícula Biliar/patología , Neoplasias de la Vesícula Biliar/epidemiología , Neoplasias de la Vesícula Biliar/patología , Neoplasias de la Vesícula Biliar/cirugía , Genes de Retinoblastoma , Genes p53 , Genes ras , Marcadores Genéticos , Humanos , Metaplasia , Invasividad Neoplásica , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Allelic losses on the short arm of chromosome 8 (8p) have been reported as frequent events in several cancers, including lung. However, no comprehensive mapping analysis of chromosome 8p in lung cancer tumors has been performed, and no data are available about the stage at which these abnormalities occur during the multistage development of lung cancer. Using 26 microsatellite markers, we mapped the chromosome 8 regions frequently deleted in lung cancer in 13 small cell carcinoma and 17 non-small cell lung carcinoma cell lines and in 68 microdissected archival primary lung tumors (22 small cell lung carcinomas, 25 squamous cell carcinomas, and 21 adenocarcinomas). We also studied the role of 8p deletions in lung cancer pathogenesis by examining 95 microdissected normal epithelium and preneoplastic samples from 11 surgically resected squamous cell lung carcinomas and from 58 bronchoscopy biopsy samples obtained from 31 current and former smokers. High frequencies of deletions at 8p21-23 regions were detected in lung cancer cell lines and in primary lung tumors. Deletions commenced early during the multistage development of lung cancer at the hyperplasia/metaplasia stage in cancer patients and in smokers without cancer. Allelic deletions persisted for up to 48 years after smoking cessation. There was a progressive increase of the overall 8p21-23 loss of heterozygosity frequency and in the size of the deleted region with increasing severity of histopathological preneoplastic changes. In epithelial samples from resected squamous cell lung carcinomas, we compared the presence of loss of heterozygosity at 8p21-23 with deletions at chromosomes 3p and 9p. Of interest, the pattern of deletions was not random, and 8p21-23 allelic losses always followed 3p deletions and usually followed 9p deletions. We conclude that 8p21-23 deletions are frequent and early events in the pathogenesis of lung carcinomas.
Asunto(s)
Cromosomas Humanos Par 8 , Pérdida de Heterocigocidad , Neoplasias Pulmonares/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Bronquios/fisiología , Deleción Cromosómica , Mapeo Cromosómico , Epitelio/fisiología , Femenino , Genes Supresores de Tumor , Humanos , Masculino , Persona de Mediana Edad , Fumar/genéticaRESUMEN
Allelotyping studies suggest that allelic losses at one or both arms of chromosome 4 are frequent in several tumor types, but information about breast cancer is scant. A recent comparative genomic hybridization analysis revealed frequent losses of chromosome 4 in breast carcinomas. In an effort to more precisely locate the putative tumor suppressor gene(s) on chromosome 4 involved in the pathogenesis of breast carcinomas, we performed loss of heterozygosity studies using 19 polymorphic microsatellite markers. After precise microdissection of archival surgical cases, we analyzed DNA obtained from 44 breast carcinomas for loss of heterozygosity. In addition, DNA from tumor cell lines derived from 14 of these 44 breast carcinomas were also analyzed. We observed deletions of chromosome 4 at multiple sites in both tumor cell lines and breast carcinomas. The deletions in cell lines and their corresponding tumors were extensive in nature, whereas they were more localized in noncultured breast carcinomas. The localized deletions in the noncultured breast carcinomas clearly defined four nonoverlapping regions of frequent deletions: 4q33-34 (76%); 4q25-26 (63%); 4p15.1-15.3 (57%); and 4p16.3 (50%). Our results suggest that there may be multiple putative tumor suppressor genes, located on both arms of chromosome 4, whose inactivation is important in the pathogenesis of breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma/genética , Cromosomas Humanos Par 4/genética , Eliminación de Gen , Alelos , Neoplasias de la Mama/patología , Carcinoma/patología , Mapeo Cromosómico , ADN de Neoplasias/genética , Femenino , Genes Supresores de Tumor , Humanos , Pérdida de Heterocigocidad , Repeticiones de Microsatélite , Células Tumorales CultivadasRESUMEN
We investigated the aberrant promoter methylation profile of bladder cancers and correlated the data with clinicopathological findings. The methylation status of 10 genes was determined in 98 surgically resected bladder cancers, and we calculated the median methylation index (MI), a reflection of the methylated fraction of the genes tested. Methylation frequencies of the genes tested in bladder cancers were 36% for CDH1, 35% for RASSF1A and APC, 29% for CDH13, 16% for FHIT, 15% for RAR beta, 11% for GSTP1, 7% for p16(INK4A), 4% for DAPK, and 2% for MGMT. Methylation of four of the individual genes (CDH1, RASSF1A, APC, and CDH13) and the MI were significantly correlated with several parameters of poor prognosis (tumor grade, growth pattern, muscle invasion, tumor stage, and ploidy pattern). Methylation of CDH1, FHIT, and a high MI were associated with shortened survival. CDH1 methylation positive status was independently associated with poor survival in multivariate analyses. Our results suggest that the methylation profile may be a potential new biomarker of risk prediction in bladder cancer.
Asunto(s)
Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/patología , Metilación de ADN , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/cirugía , Carcinoma de Células Transicionales/cirugía , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Regiones Promotoras Genéticas , Factores de Riesgo , Neoplasias de la Vejiga Urinaria/cirugíaRESUMEN
To study the molecular abnormalities involved in the multistage development of cervical carcinoma (CC), we investigated the presence of oncogenic human papillomavirus (HPV) sequences, loss of heterozygosity (LOH), and microsatellite alterations at several genes/loci at 3p (3p14.2 at the FHIT gene, 3p14.3-21.1, 3p21, and 3p22-24.2), 9p21, RB and P53, and P53 gene point mutations in precisely microdissected archival tissues from 20 CCs and their accompanying precursor lesions (cervical intraepithelial neoplasia, CIN; n = 40) and normal epithelia (n = 20). In all HPV-positive cases (90% of CCs), HPV sequences were detected as the earliest appearing molecular change or simultaneously with other changes. LOH at any 3p region was found in 70% of CCs, and 3p14.2 (FHIT gene/FRA3B fragile site) (56%) and 3p21 (57%) were the most frequent 3p sites of loss. LOH at some 3p region was in the CIN I stage, and the 3p deletions in precursor CIN lesions were smaller than the 3p losses found in the associated invasive CC. LOH at the other regions studied and P53 gene mutations were less frequent and later events. Microsatellite alterations were detected in 35% of CCs, and identical abnormalities were detected in the associated precursor lesions. Although infection with oncogenic HPV strains is the earliest and most frequent molecular event, progressive deletions at one or more 3p regions (particularly at 3p14.2, and 3p21) are also frequent events occurring early in the pathogenesis of CC.
Asunto(s)
Ácido Anhídrido Hidrolasas , Carcinoma de Células Escamosas/genética , Deleción Cromosómica , Cromosomas Humanos Par 3 , Proteínas de Neoplasias , Neoplasias del Cuello Uterino/genética , Carcinoma de Células Escamosas/virología , ADN Viral/análisis , Femenino , Genes de Retinoblastoma/genética , Genes p53/genética , Heterocigoto , Humanos , Repeticiones de Microsatélite/genética , Mutación , Papillomaviridae/genética , Proteínas/genética , Estudios Retrospectivos , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/virologíaRESUMEN
We evaluated primary lung cancers, tumor cell lines, and preneoplastic bronchial lesions for molecular genetic abnormalities in the candidate tumor suppressor gene FHIT, which spans the FRA3B fragile site at 3p14.2. 3p14.2 allele loss was very frequent in 32 lung cancer cell lines [100% of small cell lung cancer and 88% of non-small cell lung cancer (NSCLC)] and 108 primary NSCLC cancers (45%), with numerous breakpoints indicating involvement of several distinct regions in the FRA3B site. 3p14 allele loss was least frequent in the adenocarcinoma subtype and occurred at the relatively late carcinoma in situ stage of preneoplastic bronchial lesions found in NSCLC patients. Homozygous deletions within the FHIT/FRA3B region were found in 6 of 135 (4.4%) thoracic cancer cell lines. Northern blot showed low or absent FHIT expression in most thoracic cancer cell lines tested, whereas reverse transcription-PCR showed that 59-62% exhibited aberrant FHIT transcripts but nearly always (93-100%) also expressing the wild-type transcripts. Aberrant transcripts included precise deletions of FHIT exons, insertion of non-FHIT sequences between exons and insertions replacing exons. Complete open reading frame single-strand conformational polymorphism analysis of 102 lung cancer cDNAs revealed only one nonsplicing mutation. Normal cells including bronchial epithelium, lung, and trachea expressed wild-type FHIT transcript and a variant transcript deleted for exon 8 but not the other aberrant transcripts, arguing against exon 8-deleted FHIT transcripts being tumor specific. Our findings support the conclusion that FHIT/FRA3B abnormalities are associated with lung cancer pathogenesis but that FHIT abnormalities differ from the types of mutations and lack of wild-type transcript found in classic tumor suppressor genes, and functional studies are needed to define the role of FHIT in thoracic tumorigenesis.