RESUMEN
Although the process of mammary tumorigenesis requires multiple genetic events, it is unclear to what extent carcinogenesis proceeds through preferred secondary pathways following a specific initiating oncogenic event. Similarly, the extent to which established mammary tumors remain dependent on individual mutations for maintenance of the transformed state is unknown. Here we use the tetracycline regulatory system to conditionally express the human c-MYC oncogene in the mammary epithelium of transgenic mice. MYC encodes a transcription factor implicated in multiple human cancers. In particular, amplification and overexpression of c-MYC in human breast cancers is associated with poor prognosis, although the genetic mechanisms by which c-MYC promotes tumor progression are poorly understood. We show that deregulated c-MYC expression in this inducible system results in the formation of invasive mammary adenocarcinomas, many of which fully regress following c-MYC deinduction. Approximately half of these tumors harbor spontaneous activating point mutations in the ras family of proto-oncogenes with a strong preference for Kras2 compared with Hras1. Nearly all tumors lacking activating ras mutations fully regressed following c-MYC deinduction, whereas tumors bearing ras mutations did not, suggesting that secondary mutations in ras contribute to tumor progression. These findings demonstrate that c-MYC-induced mammary tumorigenesis proceeds through a preferred secondary oncogenic pathway involving Kras2.
Asunto(s)
Adenocarcinoma/fisiopatología , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Experimentales/fisiopatología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas/genética , Infecciones por Retroviridae/fisiopatología , Infecciones Tumorales por Virus/fisiopatología , Animales , Femenino , Genes ras , Humanos , Péptidos y Proteínas de Señalización Intracelular , Virus del Tumor Mamario del Ratón/genética , Virus del Tumor Mamario del Ratón/fisiología , Ratones , Ratones Transgénicos , Mutagénesis , Ornitina Descarboxilasa/genética , Proteínas/genética , Proteínas Proto-Oncogénicas p21(ras) , ARN Mensajero/metabolismo , Proteínas ras , Proteinas GADD45RESUMEN
The observation that normal pathways of differentiation and development are invariably altered during the process of carcinogenesis implies an intrinsic relationship between these processes. This relationship is particularly evident in the breast, as exemplified by the existence of endocrine risk factors for breast cancer that are related to the timing of normal developmental events. Understanding the mechanisms by which normal developmental events alter breast cancer risk is a central focus of our laboratory. Herein, we describe three approaches being taken in our laboratory toward defining the molecular basis of this relationship. These include: determining the roles played by the tumor suppressor genes, BRCA1 and BRCA2, in the normal differentiation and development of the breast; studying the function of three novel protein kinases identified in our laboratory in mammary epithelial development; and defining the molecular and cellular changes that occur in the breast as a result of reproductive events known to influence breast cancer risk.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Mama/crecimiento & desarrollo , Reproducción , Animales , Proteína BRCA2 , Neoplasias de la Mama/etiología , Diferenciación Celular/fisiología , Femenino , Genes BRCA1 , Genes Supresores de Tumor , Humanos , Proteínas de Neoplasias/genética , Factores de Riesgo , Factores de Transcripción/genéticaRESUMEN
The receptor for the subgroup A avian sarcoma and leukosis viruses [ASLV(A)] is the cellular glycoprotein Tva. A soluble form of Tva, sTva, was produced and purified with a baculovirus expression system. Using this system, 7 to 10 mg of purified sTva per liter of cultured Sf9 cells was obtained. Characterization of the carbohydrate modification of sTva revealed that the three N glycosylation sites in sTva were differentially utilized; however, the O glycosylation common to Tva produced in mammalian and avian cells was not observed. Purified sTva demonstrates significant biological activity, specifically blocking infection of avian cells by ASLV(A) with a 90% inhibitory concentration of approximately 25 pM. A quantitative enzyme-linked immunosorbent assay, developed to assess the binding of sTva to ASLV envelope glycoprotein, demonstrates that sTva has a high affinity for EnvA, with an apparent dissociation constant of approximately 0.3 nM. Once they are bound, a very stable complex is formed between EnvA and sTva, with an estimated complex half-life of 6 h. The soluble receptor protein described here represents a valuable tool for analysis of the receptor-envelope glycoprotein interaction and for structural analysis of Tva.
Asunto(s)
Alpharetrovirus/metabolismo , Receptores Virales/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Proteínas Aviares , Baculoviridae , Línea Celular , Datos de Secuencia Molecular , Receptores Virales/análisis , Receptores Virales/genéticaRESUMEN
OBJECTIVE: To compare molecular assays for characteristic chromosomal translocations with standard histopathologic and cytogenetic analysis in the differential diagnosis of pediatric soft tissue sarcomas. DESIGN: Blinded comparison with histopathologic diagnosis. SETTING: Tertiary care children's hospital. PATIENTS: A total of 79 soft tissue sarcoma patients with frozen tumor tissue and histopathologic slides available for review. METHODS: The RNA from the tumors was assayed by the reverse transcriptase-polymerase chain reaction. These assays detect PAX3-FKHR and PAX7-FKHR chimeric transcripts in alveolar rhabdomyosarcoma, EWS-FLI1 and EWS-ERG chimeric transcripts in Ewing's sarcoma, and EWS-WT1 chimeric transcripts in desmoplastic small round cell tumor. MAIN OUTCOME MEASURES: The polymerase chain reaction findings were compared with cytogenetic and histopathologic results. RESULTS: These assays detected chimeric transcripts in all cases in which translocations were found by standard cytogenetics as well as additional cases without cytogenetically detectable translocations. PAX3-FKHR or PAX7-FKHR fusions were present in 18 of 21 alveolar rhabdomyosarcomas, two of 30 embryonal rhabdomyosarcomas, and one of seven undifferentiated sarcomas. EWS-FLI1 or EWS-ERG fusions were detected in six of eight Ewing's sarcomas and one of seven undifferentiated sarcomas. The EWS-WT1 fusion was found in three of three desmoplastic small round cell tumors. CONCLUSIONS: Molecular assays for specific gene fusions provide a genetic approach to the differential diagnosis of soft tissue sarcomas. The genetic categories correspond closely to the standard histopathologic categories. The polymerase chain reaction assays for chimeric transcripts are useful tools for the rapid and objective assessment of pediatric soft tissue sarcomas.
Asunto(s)
Sarcoma/genética , Sarcoma/patología , Factores de Transcripción , Translocación Genética/genética , Secuencia de Bases , Niño , Proteínas de Unión al ADN , Diagnóstico Diferencial , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Neoplásico/análisis , ADN Polimerasa Dirigida por ARN , Proteínas Recombinantes de Fusión , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Sarcoma de Células Pequeñas/genética , Sarcoma de Células Pequeñas/patologíaRESUMEN
Although the t(2;13)(q35;q14) translocation has been found in most cases of the pediatric cancer alveolar rhabdomyosarcoma, several cases have been reported with a variant t(1;13)(p36;q14) translocation. Our findings indicate that this t(1;13) rearranges PAX7 on chromosome 1 and fuses it to FKHR on chromosome 13. This fusion results in a chimeric transcript consisting of 5' PAX7 and 3' FKHR regions, which is similar to the 5' PAX3-3' FKHR transcript formed by the t(2;13). The 5' PAX3 and PAX7 regions encode related DNA binding domains, and therefore we postulate that these translocations create similar chimeric transcription factors that alter expression of a common group of target genes.