RESUMEN
Women who inherit mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are predisposed to the development of breast and ovarian cancer. We used mice with a Brca1 mutation on a BALB/cJ inbred background (BALB/cB1+/- mice) or a Brca2 genetic alteration on the 129/SvEv genetic background (129B2+/- mice) to investigate potential gene-environment interactions between defects in these genes and treatment with the highly estrogenic compound diethylstilbestrol (DES). Beginning at 3 weeks of age, BALB/cB1+/-, 129B2+/-, and wild-type female mice were fed a control diet or a diet containing 640 ppb DES for 26 weeks. DES treatment caused vaginal epithelial hyperplasia and hyperkeratosis, uterine inflammation, adenomyosis, and fibrosis, as well as oviductal smooth muscle hypertrophy. The severity of the DES response was mouse strain specific. The estrogen-responsive 129/SvEv strain exhibited an extreme response in the reproductive tract, whereas the effect in BALB/cJ and C3H/HeN(MMTV-) mice was less severe. The Brca1 and Brca2 genetic alterations influenced the phenotypic response of BALB/cJ and 129/SvEv inbred strains, respectively, to DES in the mammary gland and ovary. The mammary duct branching morphology was inhibited in DES-treated BALB/cB1+/- mice compared with similarly treated BALB/cB1+/+ littermates. In addition, the majority of BALB/cB1+/- mice had atrophied ovaries, whereas wild-type littermates were largely diagnosed with arrested follicular development. The mammary ductal architecture in untreated 129B2+/- mice revealed a subtle inhibited branching phenotype that was enhanced with DES treatment. However, no significant differences were observed in ovarian pathology between 129B2+/+ and 129B2+/- mice. These data suggest that estrogenic compounds may modulate mammary gland or ovarian morphology in BALB/cB1+/- and 129B2+/- mice. These observations are consistent with the hypothesis that compromised DNA repair processes in cells harboring Brca1 or Brca2 mutations lead to inhibited growth and differentiation compared with the proliferative response of wild-type cells to DES treatment.
Asunto(s)
Dietilestilbestrol/toxicidad , Genes BRCA1/genética , Glándulas Mamarias Animales/patología , Proteínas de Neoplasias/genética , Ovario/patología , Factores de Transcripción/genética , Animales , Proteína BRCA2 , Carcinógenos/toxicidad , Quimera , Cruzamientos Genéticos , Endometriosis/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Trompas Uterinas/efectos de los fármacos , Trompas Uterinas/patología , Femenino , Fibrosis/inducido químicamente , Marcadores Genéticos , Heterocigoto , Hipertrofia , Inflamación , Glándulas Mamarias Animales/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Transgénicos , Músculo Liso/efectos de los fármacos , Músculo Liso/patología , Ovario/efectos de los fármacos , Fenotipo , Útero/efectos de los fármacos , Útero/patología , Vagina/efectos de los fármacos , Vagina/patologíaRESUMEN
The inheritance of a mutant copy of the BRCA1 gene greatly increases a woman's lifetime risk for ovarian and breast cancer. While a homologous gene has been identified in mouse, mice carrying mutations in this gene do not display a detectable increase in tumor formation. To determine whether mutations in p53 might increase the incidence of tumors associated with the loss of BRCA1 function in mice, we have generated mice carrying mutations at both of these loci. We report here that the presence of a mutant Brca1 allele does not alter survival of either p53-/- or p53+/- mice. Although the tumor spectrum was not dramatically altered, an increased incidence of mammary tumors was observed in the Brca1+/-p53-/- mice. Four mammary tumors were seen in the Brca1+/-p53-/- group whereas only one such tumor was seen among the p53-/- control group. In addition, although the presence of a mutant Brca1 allele did not alter the survival rate or the incidence of most tumor types in the p53+/- mice, 5 of the 23 tumors isolated from the Brca1+/-p53+/- mice treated with ionizing radiation were of mammary epithelial origin, and 3 of these had lost expression of the wild-type Brca1 gene. In contrast, no such tumors were observed in the irradiated p53+/- controls. Although the number of mammary tumors observed in these animals is small, these results are suggestive of a role for BRCA1 in mammary tumor formation after exposure to specific DNA damaging agents.
Asunto(s)
Proteína BRCA1/fisiología , Neoplasias Mamarias Experimentales/etiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Proteína BRCA1/genética , Femenino , Rayos gamma , Expresión Génica , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Noqueados , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BRCA1 is a nuclear phosphoprotein that has been classified as a tumor suppressor based on the fact that women carrying a mutated copy of the BRCA1 gene are at increased risk of developing breast and ovarian cancer. The association of BRCA1 with RAD51 has led to the hypothesis that BRCA1 is involved in DNA repair. We describe here the generation and analysis of murine embryonic stem (ES) cell lines in which both copies of the murine homologue of the human BRCA1 gene have been disrupted by gene targeting. We show that exogenous DNA introduced into these BRCA1 deficient cells by electroporation is randomly integrated into the genome at a significantly higher rate than in wild type ES cells. In contrast, integration of exogenous DNA by homologous recombination occurs in BRCA1 deficient cells at a significantly lower rate than in wild type controls. When BRCA1 expression is re-established at 5-10% of normal levels by introduction of a Brca1 transgene into BRCA1 deficient ES cells, the frequency of random integration is reduced to wild type levels, although the frequency of homologous recombination is not significantly improved. These results suggest that BRCA1 plays a role in determining the response of cells to double stranded DNA breaks.
Asunto(s)
Genes BRCA1 , Recombinación Genética , Células Madre/fisiología , Animales , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/genética , Células Cultivadas , Embrión de Mamíferos , Femenino , Eliminación de Gen , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Neoplasias Ováricas/epidemiología , Neoplasias Ováricas/genética , Mapeo Restrictivo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Células Madre/citologíaRESUMEN
The breast and ovarian cancer susceptibility gene BRCA1 encodes a zinc finger protein of unknown function. Association of the BRCA1 protein with the DNA repair protein Rad51 and changes in the phosphorylation and cellular localization of the protein after exposure to DNA-damaging agents are consistent with a role for BRCA1 in DNA repair. Here, it is shown that mouse embryonic stem cells deficient in BRCA1 are defective in the ability to carry out transcription-coupled repair of oxidative DNA damage, and are hypersensitive to ionizing radiation and hydrogen peroxide. These results suggest that BRCA1 participates, directly or indirectly, in transcription-coupled repair of oxidative DNA damage.
Asunto(s)
Proteína BRCA1/fisiología , Reparación del ADN , Alelos , Animales , Proteína BRCA1/genética , Línea Celular , Daño del ADN , Peróxido de Hidrógeno , Ratones , Oxidación-Reducción , Células Madre , Timina/análogos & derivados , Timina/inmunología , Timina/metabolismo , Transcripción Genética , Rayos UltravioletaRESUMEN
A selectable system has been used to determine mutation rates within a microsatellite sequence in human cancer cell lines with or without defects in mismatch repair. A sequence consisting of 17 repeats of poly (dC-dA).poly(dT-dG) [abbreviated as (Ca)17] was inserted near the 5' end of the bacterial neomycin-resistance gene in a plasmid vector, such that the reading frame of the neo gene is disrupted. This plasmid was introduced into cancer cell lines, where it became integrated into the cellular genome. Clones with insertions or deletions of CA-repeats that restored the normal reading frame of the neo gene were selected in G418, and mutation rates were determined by fluctuation analysis. The rates of reversion in LoVo cells, which are deficient for hMSH2, were about one in a thousand per generation, which is approximately two orders of magnitude higher than in the repair-proficient HT-1080 human fibrosarcoma cell line. The mutation rates in H6 cells, which are derived from the hMLH1-deficient HCT116 line, were more heterogeneous than in LoVo, but all were considerably higher than in the repair-proficient line. Nearly all of the revertants of the repair-deficient lines had deletions of a single CA-repeat from the microsatellite sequence, whereas repair-proficient cells had a broader spectrum of mutations.
Asunto(s)
Reparación del ADN , ADN de Neoplasias/genética , Repeticiones de Microsatélite/genética , Mutagénesis/genética , Carcinoma/genética , Neoplasias del Colon/genética , Resistencia a Medicamentos/genética , Fibrosarcoma/genética , Mutación del Sistema de Lectura , Humanos , Neomicina/farmacología , Selección Genética , Expansión de Repetición de Trinucleótido , Células Tumorales CultivadasRESUMEN
BRCA1 is a nuclear phosphoprotein that is expressed in a cell cycle regulated manner in virtually all normal dividing cells. Inheritance of a mutated copy of the BRCA1 gene increases a woman's risk for developing breast and ovarian cancer (1-3). Since the tumors that arise in these individuals consistently fail to express the wild-type allele, BRCA1 is believed to encode a tumor suppressor. Loss of the remaining functional BRCA1 allele, therefore, is one of the steps leading to neoplastic transformation of some types of epithelial cells. The isolation of the murine homologue of the human BRCA1 gene opened up the possibility of using a powerful genetic approach to study the role of this gene in both normal development and tumor formation. This genetic approach involves in vitro manipulation of the genome of embryonic stem (ES) cells, stable tissue culture cell lines derived from mouse blastocysts. After introducing mutations into the murine homologue of the BRCA1 gene Brca1 in these cell lines, four groups have generated mouse lines carrying the same mutations (4-7). Surprisingly, mice carrying a single mutant Brca1 allele do not display the increased risk for breast tumors seen in humans carrying similar mutations. However, while loss of BRCA1 appears to be a one of the many events involved in tumorgenesis in humans, these mouse lines demonstrate that gene expression is essential for development; as homozygosity for each of the Brca1 mutations results in postimplantation embryonic lethality. The survival of Brca1 deficient embryos is extended by one or two days in the absence of p53 and p21 (7,8).
RESUMEN
The breast and ovarian cancer susceptibility gene, BRCA1, has been cloned and shown to encode a zinc-finger protein of unknown function. Mutations in BRCA1 account for at least 80% of families with both breast and ovarian cancer, as well as some non-familial sporadic ovarian cancers. The loss of wild-type BRCA1 in tumours of individuals carrying one nonfunctional BRCA1 allele suggests that BRCA1 encodes a tumour suppressor that may inhibit the proliferation of mammary epithelial cells. To examine the role of BRCA1 in normal tissue growth and differentiation, and to generate a potential model for the cancer susceptibility associated with loss of BRCA1 function, we have created a mouse line carrying a mutation in one Brca1 allele. Analysis of mice homozygous for the mutant allele indicate that Brca1 is critical for normal development, as these mice died in utero between 10 and 13 days of gestation (E10-E13). Abnormalities in Brca1-deficient embryos were most evident in the neural tube, with 40% of the embryos presenting with varying degrees of spina bifida and anencephaly. In addition, the neuroepithelium in Brca1-deficient embryos appeared disorganized, with signs of both rapid proliferation and excessive cell death.
Asunto(s)
Sistema Nervioso Central/embriología , Desarrollo Embrionario y Fetal/genética , Proteínas de Neoplasias/fisiología , Factores de Transcripción/fisiología , Anencefalia/embriología , Anencefalia/genética , Animales , Proteína BRCA1 , Secuencia de Bases , Epitelio/embriología , Femenino , Marcación de Gen , Genes Letales , Homocigoto , Masculino , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , ARN Mensajero/análisis , Disrafia Espinal/embriología , Disrafia Espinal/genética , Factores de Transcripción/genéticaRESUMEN
Confined placental mosaicism is found in a higher percentage of cases of unexplained intrauterine growth retardation (IUGR) than in those of normal pregnancies. To test this hypothesis of cytogenetically abnormal placental cells associated with IUGR, we identified patients in whose fetuses IUGR was suspected during the antepartum period by clinical and serial ultrasonography and Doppler examinations and confirmed by immediate neonatal physical examination. At birth placental biopsy samples and cord blood were collected and coded. Similar specimens were obtained from non-IUGR pregnancies during that period to avoid bias in evaluation of results. These specimens were processed for standard cytogenetic studies and fluorescent in situ hybridization (FISH) of interphase cells to detect any chromosomal mosaicism in a double-blind fashion. Results were obtained on 26 IUGR placentas. Of these placentas, 22 were cytogenetically normal on standard karyotype analysis and interphase fluorescent studies. Four placentas were shown to have some chromosomal aneuploidy: one with the same chromosome anomaly in multiple cells and the other with multiple single-cell aneuploidy. Of these placentas, cytogenetic and FISH studies showed one to have a monosomy 21 cell line, two placentas showed mosaicism on karyotyping, which was not confirmed by FISH, and one placenta had a suggested chromosomal instability. All placentas had normal anatomy regardless of the chromosome findings and no chromosome anomalies were seen in any of the infants. Non-IUGR placentas showed no chromosomal-confined mosaicism and all had normal findings on placental pathologic examination.