RESUMEN
The human colorectal tumor cell line LoVo has a homozygous deletion in the hMSH2 gene from exon 3 to exon 8, is deficient in mismatch repair (MMR) activity, and exhibits microsatellite instability. To determine whether the introduction of a wild type hMSH2 into LoVo restores MMR activity and stabilizes microsatellite loci, we transferred a chromosome 2 fragment containing hMSH2 into a homologous recombination-proficient chicken DT40/human hybrid (DT40 2C) and a human chromosome 2 in a mouse A9 hybrid to LoVo. Transfers of these chromosomes into LoVo resulted in LoVo both with and without a wild-type hMSH2. Complete correlation was found between the presence of the wild-type hMSH2 and hMSH2 expression, an increased stability in microsatellite loci, and competency in MMR. The hMSH2-positive LoVo hybrids also showed an increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine. This enhanced toxicity is associated with G(2) cell-cycle arrest followed by premature mitosis and cell death. These results suggest that hMSH2 may be responsible for complementing mutator and drug-resistant phenotypes in chromosome 2-transferred LoVo cells. To test whether the hMSH2 in DT40 2C cells can be modified by homologous recombination, we transfected DT40 2C with a targeting vector containing an hMSH2 exon 4 disrupted by the zeocin-resistant gene. The results showed that the hMSH2 locus in DT40 2C was efficiently targeted by an exogeneously transfected homologous sequence, suggesting that transfer of a modified hMSH2 from DT40 2C to LoVo via chromosome transfer could be used to determine the function of hMSH2.
Asunto(s)
Cromosomas Humanos Par 2 , Neoplasias Colorrectales/genética , Proteínas de Unión al ADN , Prueba de Complementación Genética , Proteínas Proto-Oncogénicas/genética , Animales , Secuencia de Bases , Muerte Celular/efectos de los fármacos , Neoplasias Colorrectales/patología , Cartilla de ADN , Fase G2/efectos de los fármacos , Marcación de Gen , Humanos , Metilnitronitrosoguanidina/farmacología , Ratones , Repeticiones de Microsatélite/genética , Proteína 2 Homóloga a MutSRESUMEN
The p16INK4a (alpha and beta form) and p15INK4b genes were analysed for homozygous deletion, hypermethylation and point mutation in B6C3F1 mouse lymphomas induced by 2',3'-dideoxycytidine or 1,3-butadiene. Although the p16INK4a-alpha gene appeared normal in DNA from 2',3'-dideoxycytidine-induced lymphomas, Southern analyses revealed homozygous deletions or rearrangements of the p16INK4a-beta and/or p15INK4b genes in four of 16 tumours. Surprisingly, two of these lymphomas showed exclusive deletions of the p16INK4a EIbeta exon. The p15INK4b promoter region was hypermethylated in two additional 2',3'-dideoxycytidine-induced lymphomas. In contrast, homozygous deletions spanning the p16INK4a and p15INK4b loci were observed in only two of 31 1,3-butadiene-induced tumours. Thus, these cyclin dependent kinase inhibitor genes may play a significant role in chemically induced mouse lymphomas and support the contention of tumour suppressor activity for the p19ARF protein encoded by the p16INK4a-beta gene. Different genetic pathways may be involved in the development of these chemically induced tumours since we have previously shown that mutations in p53 and ras genes are common in 1,3-butadiene- but not 2',3'-dideoxycytidine-induced lymphomas.
Asunto(s)
Butadienos/farmacología , Carcinógenos/farmacología , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Linfoma/genética , Neoplasias Experimentales/genética , Proteínas Supresoras de Tumor , Zalcitabina/farmacología , Animales , Islas de CpG , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Metilación de ADN , ADN de Neoplasias , Homocigoto , Linfoma/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Mutación Puntual , Polimorfismo Conformacional Retorcido-Simple , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN/métodosRESUMEN
Inherited BRCA2 mutations confer profound susceptibility to human breast and ovarian cancer. The rat and mouse Brca2 homologues share 58% and 59% identity (72% similarity), respectively, with the human BRCA2 protein. The Brca2 proteins also share a potential nuclear localization signal (human codons 3263-3269) and a highly conserved large carboxyl region (77% identity, 86% similarity between human and rodents) that may represent important functional domains. At least six of eight previously described BRC repeats have been highly conserved in rats and mice. Expression studies demonstrate an 11-12 Kb transcript with rodent tissue-specific patterns of expression consistent with human BRCA2. These results will facilitate studies of Brca2 function during normal and neoplastic development.
Asunto(s)
Proteínas de Neoplasias/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Proteína BRCA2 , Northern Blotting , Secuencia de Consenso , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas de Neoplasias/biosíntesis , Polimorfismo Genético , Ratas , Alineación de Secuencia , Distribución Tisular , Factores de Transcripción/biosíntesisRESUMEN
The genes of murine cyclin D-dependent kinase inhibitors, p15INK4b and p16INK4a, are located in a region of chromosome 4 where overlapping deletions were found in lung adenocarcinomas. The p16INK4a gene uniquely consists of alternative first exons (E1alpha and E1beta), which are spliced to exon 2 in alternative reading frames to either encode p16INK4a (alpha form) or another potential tumor suppressor, p19ARF (beta form). We examined 99 lung adenocarcinomas of C3H/HeJ x A/J F1(C3AF1) and A/J x C3H/HeJ F1(AC3F1) mouse hybrids and 18 (13 metastatic, 5 nonmetastatic) tumorigenic mouse lung epithelial cell lines for p15INK4b and p16INK4a gene inactivation. Homozygous codeletion occurred in eight of the 13 (62%) metastatic, four of the five (80%) nonmetastatic cell lines, but in only six of 99 (6%) adenocarcinomas. Neither p15INK4b nor p16INK4a gene was individually deleted in any of the tumors or cell lines, and all deletions of the p16INK4a gene extended into exon 2, which would be expected to disrupt the functions of both p16INK4a and p19ARF. We also detected no intragenic mutations of either gene in 44 tumors that displayed loss of heterozygosity at the p16INK4a locus or in any of the cell lines. Transcript levels of p16INK4a-alpha, p16INK4a-beta and p15INK4b also were examined in each of the cell lines that retained copies of these genes. Whereas an immortal mouse lung epithelial cell line (E10) and two metastatic tumor cell lines (LM1 and E9) expressed p16INK4a-beta and p15INK4b mRNA, the alpha transcript of p16INK4a was detected in only the LM1 cell line. These results suggest that both p15INK4b and p16INK4a (alpha and beta) are targets of inactivation in mouse lung tumorigenesis.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Homocigoto , Neoplasias Pulmonares/genética , Proteínas Supresoras de Tumor , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Northern Blotting , Proteínas Portadoras/biosíntesis , Deleción Cromosómica , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos , Metástasis de la Neoplasia/genética , Reacción en Cadena de la Polimerasa/métodos , Células Tumorales CultivadasRESUMEN
The BRCA1 gene is in large part responsible for hereditary human breast and ovarian cancer. Here we report the isolation of the murine Brca1 homologue cDNA clones. In addition, we identified genomic P1 clones that contain most, if not all, of the mouse Brca1 locus. DNA sequence analysis revealed that the mouse and human coding regions are 75% identical at the nucleotide level while the predicted amino acid identity is only 58%. A DNA sequence variant in the Brca1 locus was identified and used to map this gene on a (Mus m. musculus Czech II x C57BL/KsJ)F1 x C57BL/KsJ intersubspecific backcross to distal mouse chromosome 11. The mapping of this gene to a region highly syntenic with human chromosome 17, coupled with Southern and Northern analyses, confirms that we isolated the murine Brca1 homologue rather than a related RING finger gene. The isolation of the mouse Brca1 homologue will facilitate the creation of mouse models for germline BRCA1 defects.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 17 , Ratones/genética , Proteínas de Neoplasias/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Secuencia de Aminoácidos , Animales , Proteína BRCA1 , Secuencia de Bases , Northern Blotting , Southern Blotting , Neoplasias de la Mama/genética , Cruzamientos Genéticos , Cartilla de ADN , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos , Datos de Secuencia Molecular , Proteínas de Neoplasias/biosíntesis , Neoplasias Ováricas/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Homología de Secuencia de Aminoácido , Factores de Transcripción/biosíntesis , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
Loss of heterozygosity data from familial tumors suggest that BRCA1, a gene that confers susceptibility to ovarian and early-onset breast cancer, encodes a tumor suppressor. The BRCA1 region is also subject to allelic loss in sporadic breast and ovarian cancers, an indication that BRCA1 mutations may occur somatically in these tumors. The BRCA1 coding region was examined for mutations in primary breast and ovarian tumors that show allele loss at the BRCA1 locus. Mutations were detected in 3 of 32 breast and 1 of 12 ovarian carcinomas; all four mutations were germline alterations and occurred in early-onset cancers. These results suggest that mutation of BRCA1 may not be critical in the development of the majority of breast and ovarian cancers that arise in the absence of a mutant germline allele.
Asunto(s)
Neoplasias de la Mama/genética , Genes Supresores de Tumor , Mutación de Línea Germinal , Proteínas de Neoplasias/genética , Neoplasias Ováricas/genética , Factores de Transcripción/genética , Adulto , Edad de Inicio , Alelos , Proteína BRCA1 , Secuencia de Bases , Cromosomas Humanos Par 17 , Femenino , Predisposición Genética a la Enfermedad , Heterocigoto , Humanos , Persona de Mediana Edad , Datos de Secuencia MolecularRESUMEN
Using the technique of solution hybridization coupled with magnetic bead capture, we have isolated a novel homeobox-containing gene from the BRCA1 region of 17q21. This gene is the human homologue of the mouse Mox1 gene previously localized to a syntenic region of mouse chromosome 11. Multiple overlapping cDNAs of human MOX1 were identified using both a cosmid and a P1 genomic clone containing the microsatellite markers D17S750 and D17S858 which map within the BRCA1 region defined by D17S776 and D17S78. MOX1 expression was observed in a variety of normal tissues examined, including breast and ovary. Given that the gene contains a homeobox domain and has the potential to regulate growth and differentiation, MOX1 represents an attractive candidate for the BRCA1 gene. This possibility was investigated in a series of BRCA1 kindreds and primary sporadic breast tumors. No evidence for mutation was found in the coding sequence, making it unlikely that MOX1 is the BRCA1 gene. However, the widespread expression of MOX1 in non-embryonal tissues suggests a role in normal cell biology which warrants further study.