RESUMEN

National Toxicology Program (NTP) inhalation studies demonstrated that cumene significantly increased the incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. Cumene or isopropylbenzene is a component of crude oil used primarily in the production of phenol and acetone. The authors performed global gene expression analysis to distinguish patterns of gene regulation between cumene-induced tumors and normal lung tissue and to look for patterns based on the presence or absence of K-ras and p53 mutations in the tumors. Principal component analysis segregated the carcinomas into groups with and without K-ras mutations, but failed to separate the tumors based on p53 mutation status. Expression of genes associated with the Erk MAP kinase signaling pathway was significantly altered in carcinomas with K-ras mutations compared to tumors without K-ras mutations or normal lung. Gene expression analysis also suggested that cumene-induced carcinomas with K-ras mutations have greater malignant potential than those without mutations. In addition, significance analysis of function and expression (SAFE) demonstrated expression changes of genes regulated by histone modification in carcinomas with K-ras mutations. The gene expression analysis suggested the formation of alveolar/bronchiolar carcinomas in cumene-exposed mice typically involves mutation of K-ras, which results in increased Erk MAP kinase signaling and modification of histones.

Asunto(s)

Derivados del Benceno/toxicidad , Genes ras/genética , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal/genética , Adenocarcinoma Bronquioloalveolar/inducido químicamente , Adenocarcinoma Bronquioloalveolar/genética , Adenocarcinoma Bronquioloalveolar/patología , Animales , Femenino , Regulación Neoplásica de la Expresión Génica , Inmunohistoquímica , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos , Transducción de Señal/fisiología

RESUMEN

Tsc-22 is a novel tumor suppressor gene that represents a new class of transcription factors that has transcriptional repressor activity. We found Tsc-22 downregulation in livers from B6C3F1 mice following treatment for 2 weeks with carcinogenic doses of the antianxiety drug oxazepam (2500 ppm) or the peroxisome proliferator Wyeth-14,643 (500 ppm) but not with two other carcinogens such as o-nitrotoluene or methyleugenol or three noncarcinogens including p-nitrotoluene, eugenol, or acetaminophen. The expression of Tsc-22 was also repressed in B6C3F1 mouse liver tumors that were induced by several chemicals from 2-year carcinogenicity studies as well as in spontaneous liver tumors. To identify potential Tsc-22 target genes in mouse liver, we transfected small interference RNA (SiRNA) designed to inhibit Tsc-22 into murine liver BNL-CL.2 cells. We selected two potential transcriptional targets of Tsc-22, growth arrest and DNA damage-inducible gene 45 beta (Gadd45b) and leucine zipper, putative tumor suppressor 2 (Lzts2) to test based on our previous complementary DNA microarray studies, showing that expression of these cancer-associated genes was increased when Tsc-22 was repressed. SiRNA treatment of BNL-CL.2 cells with Tsc-22 oligonucleotides but not nonspecific oligonucleotides decreased RNA and protein expression of Tsc-22 by 80-90%, while expression of Gadd45b gene, but not Lzts2, was increased over time after an initial decrease. Treatment of these cells with oxazepam for 48 h also resulted in decreased Tsc-22 and increased Gadd45b expression. These data provide evidence that Tsc-22 is a suppressor of Gadd45b expression, which may contribute to an early antiapoptotic response.

Asunto(s)

Antígenos de Diferenciación/metabolismo , Carcinógenos/toxicidad , Regulación Neoplásica de la Expresión Génica/genética , Genes Supresores de Tumor/efectos de los fármacos , Neoplasias Hepáticas Experimentales/inducido químicamente , Proteínas Represoras/metabolismo , Animales , Antígenos de Diferenciación/genética , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Hígado/efectos de los fármacos , Hígado/metabolismo , Neoplasias Hepáticas Experimentales/genética , Masculino , Ratones , Ratones Endogámicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxazepam/farmacología , ARN Interferente Pequeño/farmacología , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Factor de Crecimiento Transformador beta1/farmacología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

Lung cancer is the leading cause of cancer death worldwide, and the need to develop better diagnostic techniques and therapies is urgent. Mouse models have been utilized for studying carcinogenesis of human lung cancers, and many of the major genetic alterations detected in human lung cancers have also been identified in mouse lung tumors. The importance of mouse models for understanding human lung carcinogenic processes and in developing early diagnostic techniques, preventive measures and therapies cannot be overstated. In this report, the major known molecular alterations in lung tumorigenesis of mice are reviewed and compared to those in humans.

Asunto(s)

Adenocarcinoma/genética , Modelos Animales de Enfermedad , Neoplasias Pulmonares/genética , Adenocarcinoma/diagnóstico , Animales , Diagnóstico Precoz , Regulación Neoplásica de la Expresión Génica , Genes ras/genética , Humanos , Neoplasias Pulmonares/diagnóstico , Ratones , Mutación , Especificidad de la Especie , Proteína p53 Supresora de Tumor/genética

RESUMEN

1,3-Butadiene and chloroprene are multisite carcinogens in B6C3F1 mice with the strongest tumor response being the induction of lung neoplasms in females. Incidence of brain tumors in mice exposed to 1,3-butadiene was equivocal. This article reviews the efforts of our laboratory and others to uncover the mechanisms of butadiene and chloroprene induced lung and brain tumor responses in the B6C3F1 mouse. The formation of lung tumors by these chemicals involved mutations in the K-ras cancer gene and loss of heterozygosity in the region of K-ras on distal chromosome 6, while alterations in p53 and p16 were implicated in brain tumorigenesis.

Asunto(s)

Neoplasias Encefálicas/genética , Butadienos/toxicidad , Carcinógenos/toxicidad , Cloropreno/toxicidad , Genes Relacionados con las Neoplasias/genética , Neoplasias Pulmonares/genética , Mutagénesis/efectos de los fármacos , Alelos , Animales , Neoplasias Encefálicas/inducido químicamente , Butadienos/administración & dosificación , Carcinógenos/administración & dosificación , Cloropreno/administración & dosificación , Cromosomas de los Mamíferos/efectos de los fármacos , Aductos de ADN/efectos de los fármacos , Aductos de ADN/metabolismo , Femenino , Genes ras , Humanos , Exposición por Inhalación , Pérdida de Heterocigocidad/efectos de los fármacos , Neoplasias Pulmonares/inducido químicamente , Masculino , Ratones

RESUMEN

A transplacental carcinogenicity study was conducted by exposing pregnant Swiss (CD-1) mice to 0, 50, 100, 200, or 300 mg of 3'-azido-3'-deoxythymidine (AZT)/kg bw/day, through a 18 to 19-day gestation [National Toxicology Program, NIH Pub. No. 04-4458, 2004]. The incidences of alveolar/bronchiolar adenomas and carcinomas, in the 200 and 300 mg/kg male treatment groups, were significantly greater than that of the controls. In the present study, we evaluated the benign and malignant lung neoplasms from this bioassay for point mutations, in the K-ras and p53 cancer genes that are often mutated in human lung tumors. K-ras and p53 mutations were detected by cycle sequencing of polymerase chain reaction-amplified DNA, isolated from formalin-fixed, paraffin-embedded neoplasms. K-ras mutations were detected in 25 of 38 (66%) of the AZT-induced lung tumors, and the predominant mutations were codon 12 G-->T transversions. p53 mutations were detected in 32 of 38 (84%) of the AZT-induced lung tumors, with the predominant mutations being exon 8, codon 285 A-->T transversions, and exon 6, codon 198 T-->A transversions. No K-ras or p53 mutations were detected in five tumors, examined from control mice. The patterns of mutations identified in the lung tumors suggest that incorporation of AZT or its metabolites into DNA, oxidative stress, and genomic instability may be the contributing factors to the mutation profile and development of lung cancer in these mice.

Asunto(s)

Fármacos Anti-VIH/toxicidad , Genes p53/genética , Genes ras/genética , Inhibidores de la Transcriptasa Inversa/toxicidad , Zidovudina/toxicidad , Animales , Femenino , Neoplasias Pulmonares/genética , Intercambio Materno-Fetal , Ratones , Ratones Endogámicos , Mutación , Embarazo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

This study was performed to characterize the gene expression profile and to identify the major carcinogenic pathways involved in rat peritoneal mesothelioma (RPM) formation following treatment of Fischer 344 rats with o-nitrotoluene (o-NT) or bromochloracetic acid (BCA). Oligo arrays, with over 20,000 target genes, were used to evaluate o-NT- and BCA-induced RPMs, when compared to a non-transformed mesothelial cell line (Fred-PE). Analysis using Ingenuity Pathway Analysis software revealed 169 cancer-related genes that were categorized into binding activity, growth and proliferation, cell cycle progression, apoptosis, and invasion and metastasis. The microarray data were validated by positive correlation with quantitative real-time RT-PCR on 16 selected genes including igf1, tgfb3 and nov. Important carcinogenic pathways involved in RPM formation included insulin-like growth factor 1 (IGF-1), p38 MAPkinase, Wnt/beta-catenin and integrin signaling pathways. This study demonstrated that mesotheliomas in rats exposed to o-NT- and BCA were similar to mesotheliomas in humans, at least at the cellular and molecular level.

Asunto(s)

Regulación Neoplásica de la Expresión Génica/genética , Mesotelioma/genética , Neoplasias Peritoneales/genética , Transducción de Señal/genética , Acetatos/toxicidad , Animales , Línea Celular , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/genética , Integrinas/genética , Masculino , Mesotelioma/inducido químicamente , Mesotelioma/patología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Neoplasias Peritoneales/inducido químicamente , Neoplasias Peritoneales/patología , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Tolueno/análogos & derivados , Tolueno/toxicidad , Proteínas Wnt/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética

RESUMEN

Treatment with 8-methoxypsoralen (8-MOP) and ultraviolet radiation (primarily UVA), called PUVA therapy, has been used to treat different chronic skin diseases but led to a significant increased risk for skin cancer. The National Toxicology Program (NTP) performed a study in mice treated with PUVA that showed a significant increase in squamous cell carcinomas of the skin. In the present study, we evaluated the protein expression of p53 and PCNA and DNA mutations of p53 and H-ras genes in both hyperplastic and neoplastic squamous cell lesions from the NTP study. By immunohistochemical staining, protein expression of both p53 and PCNA was detected in 3/16 (19%) of hyperplastic lesions and 14/17 (82%) of SCCs in groups treated with both 8-MOP and UVA. The mutation frequency of p53 in SCCs from mice administered 8-MOP plus UVA was 15/17 (88%) with a predominant distribution of mutations in exon 6 (14/15 - 93%). No H-ras mutations were detected in the hyperplastic lesions/tumors. The mutagenic effect of PUVA on the p53 tumor suppressor gene may lead to a conformational modification and inactivation of the p53 protein, which are considered critical steps in PUVA-induced skin carcinogenesis. The p53 mutational frequency and patterns from our study were different from those reported in human PUVA-type tumors.

Asunto(s)

Carcinoma de Células Escamosas/genética , Genes p53 , Genes ras , Mutación , Terapia PUVA/efectos adversos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Neoplasias Cutáneas/genética , Animales , Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/patología , Análisis Mutacional de ADN , ADN de Neoplasias/análisis , Hiperplasia/genética , Hiperplasia/metabolismo , Hiperplasia/patología , Técnicas para Inmunoenzimas , Metoxaleno/toxicidad , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos Ultravioleta/efectos adversos

RESUMEN

Inbred strains of mouse show various susceptibilities to spontaneous and chemical-induced lung tumorigenesis. Genetic analyses have revealed that lung tumor susceptibilities of inbred mouse strains are governed by quantitative trait loci (QLTs) located on multiple chromosomes. A major lung tumor resistance QLT, designated pulmonary adenoma resistance 2 (Par2), was mapped to the mouse chromosome 18 independently by several groups and accounted for up to 60% phenotype variance between susceptible A/J and more resistant BALB/c strains. The authors recently conducted studies to positionally clone the Par2 gene. This review summarizes the effort and progress towards the identification of Par2 candidates.

Asunto(s)

Adenoma/genética , Predisposición Genética a la Enfermedad , Neoplasias Pulmonares/genética , Sitios de Carácter Cuantitativo , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Expresión Génica , Ratones , Datos de Secuencia Molecular , Polimorfismo Genético

RESUMEN

The nervous system of the B6C3F1 mouse has rarely been a target for chemical carcinogenesis in the National Toxicology Program (NTP) bioassays. However, 6 malignant gliomas and 2 neuroblastomas were observed in B6C3F1 mice exposed to 625 ppm 1,3-butadiene (NTP technical reports 288 and 434). These mouse brain tumors were evaluated with regard to the profile of genetic alterations that are observed in human brain tumors. Alterations in the p53 tumor suppressor gene were common. Missense mutations were observed in 3/6 malignant gliomas and 2/2 neuroblastomas and were associated with loss of heterozygosity. Most of the mutations occurred in exons 5-8 of the p53 gene and were G-->A transitions, and did not involve CpG sites. Loss of heterozygosity at the Ink4a/Arf gene locus was observed in 5/5 malignant gliomas and 1/1 neuroblastoma, while the PTEN(phosphatase and tensin homologue) gene locus was unaffected by deletions. One of 2 neuroblastomas had a mutation in codon 61 of H-ras, while H-ras mutations were not observed in the malignant gliomas examined. Only 1 brain tumor has been reported from control mice of over 500 NTP studies. This malignant glioma showed no evidence of alterations in the p53 gene or K- and H-ras mutations. It is likely that the specific genetic alterations observed were induced or selected for by 1,3-butadiene treatment that contributed to the development of mouse brain tumors. The observed findings are similar in part to the genetic alterations reported in human brain tumors.

Asunto(s)

Neoplasias Encefálicas/genética , Butadienos/toxicidad , Carcinógenos/toxicidad , Glioma/genética , Neuroblastoma/genética , Animales , Neoplasias Encefálicas/inducido químicamente , Neoplasias Encefálicas/patología , Codón , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Exones , Femenino , Eliminación de Gen , Glioma/inducido químicamente , Glioma/patología , Inmunohistoquímica , Pérdida de Heterocigocidad , Masculino , Ratones , Ratones Endogámicos , Mutación Missense , Neuroblastoma/inducido químicamente , Neuroblastoma/patología , Reacción en Cadena de la Polimerasa , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética

RESUMEN

Previously we demonstrated that the mouse liver tumor response to the non-genotoxic carcinogens oxazepam and Wyeth-14,643 involved more differences than similarities in changes in early gene expression. In this study we used quantitative real-time PCR and oligonucleotide microarray analysis to identify genes that were up- or down-regulated in mouse liver early after treatment with different known carcinogens, including oxazepam (125 and 2500 p.p.m.), o-nitrotoluene (1250 and 5000 p.p.m.) and methyleugenol (75 mg/kg/day), or the non-carcinogens p-nitrotoluene (5000 p.p.m.), eugenol (75 mg/kg/day) and acetaminophen (6000 p.p.m.). Starting at 6 weeks of age, mice were treated with the different compounds for 2 weeks in the diet, at which time the livers were collected. First, expression of 12 genes found previously to be altered in liver after 2 weeks treatment with oxazepam and/or Wyeth-14,643 was examined in livers from the various chemical treatment groups. These gene expression changes were confirmed for the livers from the oxazepam-treated mice in the present study, but were not good early markers for all the carcinogens in this study. In addition, expression of 20 842 genes was assessed by oligonucleotide microarray [n = 4 livers/group, 2 hybridizations/liver (with fluor reversals)] and the results were analyzed using the Rosetta Resolver System and GeneSpring software. The analyses revealed that several cancer-related genes, including Fhit, Wwox, Tsc-22 and Gadd45b, were induced or repressed in unique patterns for specific carcinogens and not altered by the non-carcinogens. The data indicate that even if the tumor response, including molecular alterations, is similar, such as for oxazepam and methyleugenol, early gene expression changes appear to be carcinogen specific and seem to involve apoptosis and cell cycle-related genes.

Asunto(s)

Acetaminofén/farmacología , Carcinógenos/farmacología , Eugenol/análogos & derivados , Eugenol/farmacología , Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Tolueno/análogos & derivados , Tolueno/farmacología , Animales , Femenino , Masculino , Ratones , Hibridación de Ácido Nucleico , Oxazepam/farmacología , Reacción en Cadena de la Polimerasa

RESUMEN

Loss of expression of the death-associated protein (DAP)-kinase gene by aberrant promoter methylation may play an important role in cancer development and progression. The purpose of this investigation was to determine the commonality for inactivation of the DAP-kinase gene in adenocarcinomas induced in mice by chronic exposure to mainstream cigarette smoke, the tobacco carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and vinyl carbamate, and the occupational carcinogen methylene chloride. The timing for inactivation was also determined in alveolar hyperplasias that arise in lung cancer induced in the A/J mouse by NNK. The DAP-kinase gene was not expressed in three of five NNK-induced lung tumor-derived cell lines or in a spontaneously arising lung tumor-derived cell line. Treatment with 5-aza-2'-deoxycytidine restored expression; dense methylation throughout the DAP-kinase CpG island detected by bisulfite sequencing supported methylation as the inactivating event in these cell lines. Methylation-specific PCR detected inactivation of the DAP-kinase gene in 43% of tumors associated with cigarette smoke, a frequency similar to those reported in human non-small cell lung cancer. In addition, DAP-kinase methylation was detected in 52%, 60%, and 50% of tumors associated with NNK, vinyl carbamate, and methylene chloride, respectively. Methylation was observed at similar prevalence in both NNK-induced hyperplasias and adenocarcinomas (46% versus 52%), suggesting that inactivation of this gene is one pathway for tumor development in the mouse lung. Bisulfite sequencing of both premalignant and malignant lesions revealed dense methylation, substantiating that this gene is functionally inactivated at the earliest histological stages of adenocarcinoma development. This study is the first to use a murine model of cigarette smoke-induced lung cancer and demonstrate commonality for inactivation by promoter hypermethylation of a gene implicated in the development of this disease in humans.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Carcinógenos/toxicidad , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/genética , Nitrosaminas/toxicidad , Fumar/efectos adversos , Uretano/análogos & derivados , Animales , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Islas de CpG , Metilación de ADN/efectos de los fármacos , Proteínas Quinasas Asociadas a Muerte Celular , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen/efectos de los fármacos , Hiperplasia , Pulmón/patología , Neoplasias Pulmonares/inducido químicamente , Cloruro de Metileno/toxicidad , Ratones , Ratones Endogámicos A , Regiones Promotoras Genéticas/efectos de los fármacos , Uretano/toxicidad

RESUMEN

In this study, we performed systematic candidate gene analyses of the Pulmonary adenoma resistance 2 locus. Differential gene expression in lung tissues and nucleotide polymorphisms in coding regions between A/J and BALB/cJ mice were examined using reverse transcription-PCR and direct sequencing. Although not all genes in the interval were analyzed at this moment due to the recent database updating, we have found that the Pol iota gene, encoding the DNA polymerase iota, contains 25 nucleotide polymorphisms in its coding region between A/J and BALB/cJ mice, resulting in a total of ten amino acid changes. Primer extension assays with purified BALB/cJ and A/J proteins in vitro demonstrate that both forms of Pol iota are active but that they may differ in substrate discrimination, which may affect the formation of Kras2 mutations in mouse lung tumors. Altered expression of POL iota protein and an amino acid-changing nucleotide polymorphism were observed in human lung cancer cells, suggesting a possible role in the development of lung cancer. Thus, our data support the Pol iota gene as a modifier of lung tumorigenesis by altering DNA polymerase activity.

Asunto(s)

Adenoma/genética , ADN Polimerasa Dirigida por ADN/genética , Inmunidad Innata , Neoplasias Pulmonares/genética , Polimorfismo de Nucleótido Simple/genética , Adenoma/inducido químicamente , Adenoma/inmunología , Empalme Alternativo , Secuencia de Aminoácidos , Animales , ADN Polimerasa Dirigida por ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Pulmón/metabolismo , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos A , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas p21(ras) , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Células Tumorales Cultivadas , Proteínas ras , ADN Polimerasa iota

RESUMEN

Genotoxic carcinogens exert their tumorigenic effects in part by inducing genomic instability. We recently showed that loss of heterozygosity (LOH) on chromosome 12 associates significantly with the induction of chromosome instability (CIN) by the likely human lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and vinyl carbamate (VC) during mouse lung carcinogenesis. Here, we demonstrate the carcinogen specificity of this event and its effect on lung tumor evolution. LOH on chromosome 12 was observed in 45% of NNK-induced, 59% of VC-induced, 58% of aflatoxin B1 (AFB1)-induced, 14% of N-ethyl-N-nitrosourea (ENU)-induced and 12% of spontaneous lung adenocarcinomas. The frequency of LOH in each of the carcinogen-induced groups, except ENU, was significantly higher than in the spontaneous group (P<0.001). Deletion mapping revealed four potential candidate regions of 1-4 centiMorgans suspected to contain targeted tumor suppressor genes, with at least one expected to have a role in CIN. The relationship between LOH on chromosome 12 and additional chromosomal alterations occurring during lung tumor progression was also examined. LOH on chromosomes 1 and 14 were moderately frequent during malignant progression in tumors from all treatment groups, occurring in 21-35 and 18-33% of tumors. However, these alterations showed significant concurrence with LOH on chromosome 12 in VC-, NNK- and AFB1-induced tumors (P<0.05). The results suggest that a carcinogen-selective mechanism of lung cancer induction involves the frequent inactivation of genes on chromosome 12, including a stability gene that evidently promotes the evolutionary selection of additional chromosomal alterations during malignant progression.

Asunto(s)

Adenocarcinoma/genética , Carcinógenos/toxicidad , Pérdida de Heterocigocidad/genética , Neoplasias Pulmonares/genética , Pulmón/patología , Uretano/análogos & derivados , Adenocarcinoma/inducido químicamente , Animales , Inestabilidad Cromosómica , Deleción Cromosómica , Mapeo Cromosómico , Cruzamientos Genéticos , Etilnitrosourea/toxicidad , Pulmón/efectos de los fármacos , Neoplasias Pulmonares/inducido químicamente , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Uretano/toxicidad

RESUMEN

Based on long-term toxicity and carcinogenicity studies in B6C3F1 mice conducted by the National Toxicology Program, 2,2-Bis(bromomethyl)-1,3-propanediol (BMP) and tetranitromethane (TNM) have been identified as carcinogens. Following 2 yr of exposure to 312, 625, or 1,250 ppm BMP in feed, or exposure to 0.5 or 2 ppm TNM by inhalation, increased incidences of lung neoplasms were observed in B6C3F1 mice at all exposure concentrations compared to unexposed mice. The present study characterizes genetic alterations in the K-ras protooncogene in BMP- and TNM-induced lung neoplasms, respectively, and compares the findings to spontaneous lung neoplasms from corresponding control mice. The frequencies of the K-ras mutations were 57% (29/51) in BMP-induced lung neoplasms compared to 15% (3/20) in lung neoplasms from dosed feed control mice, and 54% (14/26) in TNM-induced lung neoplasms compared to 60% (3/5) in lung neoplasms from inhalation control mice. G --> A transitions at the second base of the K-ras codon 12 (GGT --> GAT) were the most frequent pattern of K-ras mutations identified in BMP-induced (20/29) and TNM-induced lung neoplasms (13/14), which differed from the mutational patterns identified in the lung neoplasms from unexposed control mice. These results indicate that mutations in the K-ras gene are involved in B6C3F1 lung carcinogenesis following BMP- and TNM-exposure, and the high frequency and specificity of the ras mutation profile in lung neoplasms (G --> A transition) may be due to in vivo genotoxicity by the parent compounds or their metabolites.

Asunto(s)

Genes ras/genética , Neoplasias Pulmonares/genética , Mutación Missense , Mutación Puntual , Animales , Codón/genética , ADN/análisis , Análisis Mutacional de ADN , Dieta , Relación Dosis-Respuesta a Droga , Femenino , Retardadores de Llama/toxicidad , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos , Oxidantes/toxicidad , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Glicoles de Propileno/toxicidad , Tetranitrometano/toxicidad

RESUMEN

We have performed oligonucleotide array analysis on various murine lung tissues [normal lungs, lung adenomas, and lung adenocarcinomas (ACs)] using Affymetrix U74Av2 GeneChips to examine the complex genetic changes occurring during lung carcinogenesis. Analysis yielded 20 novel genes differentially expressed in both lung adenomas and ACs versus normal lungs, including the tumor suppressor APC2 and the oncogene Ros 1. In addition, 50 genes were found to be differentially expressed in lung adenomas versus lung ACs, including the differentiation factor Hox C6, the oncogene Ets 2, and the Ras nuclear transport factor, nuclear transport factor 2. To understand the potential relationship between genes expressed in murine lung tumors and its relationship to altered gene expression observed during embryogenesis and postnatal development, tissues from embryonic lungs and from lungs of mice up to 4 weeks following birth were examined using Affymetrix U74Av2 GeneChips. From this analysis, approximately 1300 genes were determined to exhibit differential expression in fetal lung versus postnatal lung. When we compared lung adenomas, lung ACs, and normal lung parenchyma, 24 developmentally regulated genes were found aberrantly expressed in lung tumors; these included the cell cycle control factor CDC5, the cellular differentiation factor TEA domain 4, and the proapoptotic factor BNIP 2. Finally, we compared the murine lung tumor gene expression data to the expression of genes in human lung cancer, in order to assess the relevance of murine lung cancer models in the study of human AC formation. When the 17 human lung ACs and six human lung large cell carcinomas were examined, it was found that 13 of the 17 human lung ACs clustered tightly together in a pattern that was different from the remaining four human lung ACs and six large cell carcinomas, which exhibited a different pattern. Interestingly, the mouse lung adenomas appeared similar to 13 clustered ACs, while mouse lung ACs appeared more similar in pattern to the group consisting of four ACs and six large-cell carcinomas (LCCs). Nevertheless, when compared with the combined human ACs, 39 genes with similar expression changes in murine lung tumors and human ACs/LCCs were identified, such as the oncogene-related BCL7B, the cell cycle regulator CDK4, and the proapoptotic Endophilin B1. Overall, we have determined, for the first time, the expression profiles during murine lung tumor progression and have established, at the molecular level, an association between murine lung tumorigenesis and lung development. We have also attempted to compare the expression profiles found in mouse lung cancers and those in human lung ACs.

Asunto(s)

Adenocarcinoma/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Pulmón/crecimiento & desarrollo , Adenoma/genética , Animales , Animales Recién Nacidos , Perfilación de la Expresión Génica/métodos , Genes Relacionados con las Neoplasias/genética , Genes Relacionados con las Neoplasias/fisiología , Genes Supresores de Tumor/fisiología , Humanos , Pulmón/embriología , Pulmón/metabolismo , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos A , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos

RESUMEN

The present study characterized the immunohistochemical localization of beta-catenin protein in hepatocellular neoplasms and hepatoblastomas in B6C3F(1) mice exposed to diethanolamine (DEA) for 2 years and evaluated genetic alterations in the Catnb and H-ras genes which are known to play important roles in the pathogenesis of liver malignancies. Genomic DNA was isolated from paraffin sections of each liver tumor. Catnb exon 2 (corresponds to exon 3 in human) genetic alterations were identified in 18/18 (100%) hepatoblastomas from DEA exposed mice. Deletion mutations (15/18, 83%) were identified more frequently than point mutations (6/18, 33%) in hepatoblastomas. Eleven of 34 (32%) hepatocellular adenomas and carcinomas from DEA treated mice had mutations in exon 2 of the beta-catenin gene, while only 1 of 10 spontaneous neoplasms had a deletion mutation of codon 5-6. Common to all liver neoplasms (hepatocellular adenomas, carcinomas and hepatoblastomas) was membrane staining for the beta-catenin protein, while cytoplasmic and nuclear staining was observed only in hepatoblastomas. The lack of H-ras mutations in hepatocellular neoplasms and hepatoblastomas suggests that the ras signal transduction pathway is not involved in the development of liver tumors following DEA exposure which is different from that of spontaneous liver tumors that often contain H-ras mutations.

Asunto(s)

Carcinógenos/toxicidad , Proteínas del Citoesqueleto/genética , Etanolaminas/toxicidad , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/genética , Transactivadores/genética , Adenoma de Células Hepáticas/inducido químicamente , Adenoma de Células Hepáticas/genética , Adenoma de Células Hepáticas/metabolismo , Animales , Proteínas del Citoesqueleto/metabolismo , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/genética , Femenino , Genes ras/efectos de los fármacos , Inmunohistoquímica , Neoplasias Hepáticas Experimentales/metabolismo , Masculino , Ratones , Mutación , Polimorfismo Conformacional Retorcido-Simple , Factores de Tiempo , Transactivadores/metabolismo , beta Catenina

RESUMEN

In this study we used liver neoplasms induced by several chemical carcinogens to investigate potential nuclear targets associated with beta-catenin/Wnt signaling and potential membrane-associated beta-catenin binding partners. Strong expression of cyclin D1, in a pattern similar to that observed previously for beta-catenin, was observed by Western analysis for all five hepatoblastomas examined regardless of treatment. Increased expression of cyclin D1 was also detected in 12 of 35 (34%) hepatocellular neoplasms. Ten of 15 tumors (67%) that had mutations in the Catnb gene had upregulation of cyclin D1, while only 2 of 20 tumors (10%) without Catnb mutations had increased cyclin D1 expression. Immunohistochemical analysis confirmed strong expression of cyclin D1 in most nuclei of hepatoblastomas and scattered nuclear staining in hepatocellular tumors that had Catnb mutations. Increased c-Jun expression was observed in 19 of 30 (63%) hepatocellular tumors and all hepatoblastomas, although upregulation was not completely correlated with Catnb mutation. C-Myc expression was not increased in the tumors. Reduced expression of E-cadherin, which interacts with beta-catenin at the membrane, was observed in some tumors, but this did not correlate with Catnb mutation. Expression of the epidermal growth factor receptor, which may have a role in beta-catenin tyrosine phosphorylation, was lower in some tumors than in normal tissue depending on chemical treatment. The results provide evidence that increased expression of cyclin D1 and c-Jun may provide an advantage during tumor progression and in the transition from hepatocellular neoplasms to hepatoblastomas. Moreover, it is likely increased cyclin D1 expression results at least in part from Catnb mutation, beta-catenin accumulation, and increased Wnt signaling.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Proteínas del Citoesqueleto/genética , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Proteínas de Neoplasias/metabolismo , Transactivadores/genética , Proteínas de Pez Cebra , Adenoma de Células Hepáticas/inducido químicamente , Adenoma de Células Hepáticas/genética , Adenoma de Células Hepáticas/metabolismo , Animales , Cadherinas/metabolismo , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Ciclina D1/metabolismo , Receptores ErbB/metabolismo , Genes ras/genética , Hepatoblastoma/inducido químicamente , Hepatoblastoma/genética , Hepatoblastoma/metabolismo , Técnicas para Inmunoenzimas , Neoplasias Hepáticas Experimentales/inducido químicamente , Ratones , Ratones Endogámicos , Mutación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Wnt , beta Catenina

RESUMEN

We hypothesized that the mouse liver tumor response to non-genotoxic carcinogens would involve some common early gene and protein expression changes that could ultimately be used to predict chemical hepatocarcinogenesis. In order to identify a panel of genes to test, we analyzed global differences in gene and protein expression in livers from B6C3F1 mice following dietary treatment with two rodent carcinogens, the benzodiazepine anti-anxiety drug oxazepam (2500 p.p.m.) and the hypolipidemic agent Wyeth (Wy)-14,643 (500 p.p.m.) compared with livers from untreated mice. Male mice were exposed for 2 weeks and 1, 3 or 6 months to oxazepam or Wy-14,643 in an age-matched study design. By histopathological evaluation, no liver preneoplastic foci or tumors were detected at 6 months in treated or control groups. By cDNA microarray analysis [NIEHS Mouse Chip (8700 genes); n = 3 individual livers/group, four hybridizations/sample], expression of 36 genes or 220 genes were changed relative to control livers following 6 months of oxazepam or Wy-14,643 treatment, respectively. To obtain a more comprehensive picture of gene/protein expression changes, we also conducted a proteomics study by 2D-gel electrophoresis followed by matrix assisted laser desorption/ionization-mass spectrometry on cytoplasmic, nuclear, and microsomal subcellular fractions of the same liver samples utilized for the cDNA microarray analysis. Real-time PCR, western blot analysis and immunohistochemistry were utilized for validation and to expand the results to other time points. Cyp2b20, growth arrest- and damage-inducible gene beta (Gadd45beta), tumor necrosis factor alpha-induced protein 2 and insulin-like growth factor binding protein 1 (Igfbp5) genes and proteins were upregulated by oxazepam, and Cyp2b20, Cyclin D1, proliferating cell nuclear antigen, Igfbp5, Gadd45beta and cell death-inducing DNA fragmentation factor alpha subunit-like effector A exhibited higher expression after Wy-14,643 treatment. Most of these genes/proteins were also deregulated at 2 weeks. There appeared to be more distinct than common changes in the expression of carcinogenesis-related genes/proteins between the two compounds, suggesting that the major carcinogenic pathways are different for these compounds and may be distinct for different chemical classes.

Asunto(s)

Carcinógenos , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Hepáticas Experimentales/inducido químicamente , Oxazepam/toxicidad , Pirimidinas/toxicidad , Animales , Secuencia de Bases , Cartilla de ADN , Neoplasias Hepáticas Experimentales/genética , Masculino , Ratones , Reacción en Cadena de la Polimerasa

RESUMEN

Lung tumors from AC3F1 mice treated with aflatoxin B(1) (AFB(1)), were examined for loss of alleles, point mutations and hypermethylation of CpG sites within the promoters of the two genes in the Ink4a/Arf gene locus. Loss of microsatellite alleles in the Ink4a/Arf region occurred in 22 of 74 (30%) AFB(1)-induced lung tumors. Fifty-one of 61 (83%) tumors had at least partial methylation of CpG sites within the p16Ink4a promoter-exon 1alpha region. At least partial methylation of CpG sites was observed in 43 of 49 (88%) tumors analyzed for p19Arf promoter hypermethylation, with methylation of identified transcription factor binding sites or consensus sequences occurring in 21 tumors (DMP1/Ets in two tumors, CTCF in four tumors, E2F in three tumors, Sp1 in 16 tumors). Two tumors contained point mutations in the p19Arf promoter. Nuclear staining for p19(Arf) was decreased by 80-100% in 41 of 71 (58%) tumors. The concordance between p19Arf molecular perturbations and altered protein expression was 63%. However, upon comparing p19Arf promoter perturbations (i.e. methylation of functional transcription factor binding sites and point mutations) and altered p19(Arf) expression, the concordance was 86%, suggesting a mechanism for changes in protein expression in some tumors. There was an absence of a mutually exclusive relationship between disruption of p53 and p19(Arf), since the concordance was 62%. Similarly, no evidence was found of inverse relationships between perturbation of p16Ink4a and p19(Arf) (43% concordance) or p16Ink4(a) and p53 (37% concordance), suggesting that inactivation of these genes occurs independently and provides evidence that, although these genes may participate in cooperative cellular pathways, they also have functions in independent pathways that are important in mouse lung tumorigenesis.

Asunto(s)

Aflatoxina B1/farmacología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/genética , Proteína p14ARF Supresora de Tumor/genética , Animales , Islas de CpG/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Pérdida de Heterocigocidad/genética , Ratones , Regiones Promotoras Genéticas/genética , Proteína p53 Supresora de Tumor/genética

RESUMEN

Recent evidence suggests that genome-wide allelic imbalances are inducible by carcinogens and may occur as cells adapt to carcinogenic exposure during tumorigenesis. We investigated the role of carcinogenic exposure on global and selected loss of heterozygosity (LOH) during mouse lung adenocarcinogenesis. Tumor induction by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) or vinyl carbamate (VC) resulted in a significant overall increase in the number of chromosomes affected by LOH per tumor when compared with spontaneous lung tumors. Allelic loss on chromosome 12 occurred at a frequency of 35% and 40% in NNK- and VC-induced tumors, respectively, compared with 8.3% in spontaneous tumors (P < 0.01). In contrast, spontaneous lung adenocarcinomas displayed LOH on chromosome 4 at a frequency of 77%, whereas a frequency of only 36% (P < 0.001) was observed in tumors induced by NNK. Sixty-four percent of the VC-induced tumors displayed LOH on chromosome 4. In addition, allelic losses on chromosomes 12 and 14 were significantly associated with an increase in chromosomal instability, suggesting that genes inactivated on these chromosomes may contribute to this effect. The results from this study demonstrate that genotoxic carcinogens increase chromosome instability, as evidenced by a significant increase in global LOH frequency, and significantly alter the selection of chromosomal alterations during lung tumor development.

Asunto(s)

Adenocarcinoma/genética , Carcinógenos/toxicidad , Pérdida de Heterocigocidad/efectos de los fármacos , Neoplasias Pulmonares/genética , Nitrosaminas/toxicidad , Uretano/análogos & derivados , Uretano/toxicidad , Adenocarcinoma/inducido químicamente , Animales , Neoplasias Pulmonares/inducido químicamente , Ratones , Ratones Endogámicos A , Ratones Endogámicos C3H