RESUMEN

Female transgenic mice that constitutively overexpress the transcription factor ATF3 in the basal epithelium of the mammary gland develop mammary carcinomas with high frequency, but only if allowed to mate and raise pups early in life. This transgenic mouse model system reproduces some features of human breast cancer in that about 20% of human breast tumor specimens exhibit overexpression of ATF3 in the tumor cells. The ATF3-induced mouse tumors are phenotypically similar to mammary tumors induced by overexpression of activating Wnt/ß-catenin pathway genes. We now show that the Wnt/ß-catenin pathway is indeed activated in ATF3-induced tumors. ß-catenin is transcriptionally up-regulated in the tumors, and high levels of nuclear ß-catenin are seen in tumor cells. A reporter gene for Wnt/ß-catenin pathway activity, TOPGAL, is up-regulated in the tumors and several downstream targets of Wnt signaling, including Ccnd1, Jun, Axin2 and Dkk4, are also expressed at higher levels in ATF3-induced tumors compared to mammary glands of transgenic females. Several positive-acting ligands for this pathway, including Wnt3, Wnt3a, Wnt7b, and Wnt5a, are significantly overexpressed in tumor tissue, and mRNA for Wnt3 is about 5-fold more abundant in transgenic mammary tissue than in non-transgenic mammary tissue. Two known transcriptional targets of ATF3, Snai1 and Snai2, are also overexpressed in the tumors, and Snail and Slug proteins are found to be located primarily in the nuclei of tumor cells. In vitro knockdown of Atf3 expression results in significant decreases in expression of Wnt7b, Tcf7, Snai2 and Jun, suggesting that these genes may be direct transcriptional targets of ATF3 protein. By chromatin immunoprecipitation analysis, both ATF3 and JUN proteins appear to bind to a particular subclass of AP-1 sites upstream of the transcriptional start sites of each of these genes.

Asunto(s)

Factor de Transcripción Activador 3/genética , Neoplasias Mamarias Animales/etiología , Transducción de Señal , Factor de Transcripción Activador 3/efectos adversos , Animales , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Mamarias Experimentales , Ratones , Ratones Transgénicos , Proteína Oncogénica p65(gag-jun)/metabolismo , Transcripción Genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismo

RESUMEN

Werner syndrome (WS) is a rare, segmental progeroid syndrome caused by defects in the WRN gene, which encodes a RecQ helicase. WRN has roles in many aspects of DNA metabolism including DNA repair and recombination. In this study, we exploited two different recombination assays previously used to describe a role for the structure-specific endonuclease ERCC1-XPF in mitotic and targeted homologous recombination. We constructed Chinese hamster ovary (CHO) cell lines isogenic with the cell lines used in these previous studies by depleting WRN using shRNA vectors. When intrachromosomal, mitotic recombination was assayed in WRN-depleted CHO cells, a hyperrecombination phenotype was observed, and a small number of aberrant recombinants were generated. Targeted homologous recombination was also examined in WRN-depleted CHO cells using a plasmid-chromosome targeting assay. In these experiments, loss of WRN resulted in a significant decrease in nonhomologous integration events and ablation of recombinants that required random integration of the corrected targeting vector. Aberrant recombinants were also recovered, but only from WRN-depleted cells. The pleiotropic recombination phenotypes conferred by WRN depletion, reflected in distinct homologous and nonhomologous recombination pathways, suggest a role for WRN in processing specific types of homologous recombination intermediates as well as an important function in nonhomologous recombination.

Asunto(s)

Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/fisiología , Mitosis , RecQ Helicasas/genética , RecQ Helicasas/fisiología , Recombinación Genética , Animales , Células CHO , Cromosomas/ultraestructura , Cricetinae , Cricetulus , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Humanos , Ratones , Fenotipo , ARN Interferente Pequeño/metabolismo , RecQ Helicasas/metabolismo , Helicasa del Síndrome de Werner

RESUMEN

Hybrids between distinct Xiphophorus species have been utilized for over 70 years to study melanoma and other neoplasms that can develop spontaneously in hybrid offspring. Genetic linkage mapping has proven to be important in delineating genomic areas that harbor oncogenes and tumor suppressors. Within this report, two parallel backcrosses have been utilized to generate a genetic linkage map for Xiphophorus fishes. Isozyme/allozyme, RFLP and PCR-based mapping techniques, including AP-PCR/RAPDs and microsatellite loci were utilized. The derived linkage map provides a total of 403 mapped polymorphisms distributed among 24 linkage groups, representative of 24 acro- and telocentric chromosome pairs. Genomic coverage is approximately one marker per 5.8 cM. Detailed genotypic analysis of the utilized hybrids revealed two areas of the genome that show significant segregation distortion. Loci within the linkage group harboring the sex determining locus (LG 24) and an autosomal linkage group (LG 21) show highly significant deviations from Mendelian expectations. This phenomenon is not present in a hybrid cross that utilizes a different backcross hybrid progenitor species. The derived map with sequence-tagged markers provides a framework for physical map generation, large-scale genomic sequencing and will further enable cross-genome comparisons of vertebrate genomes.