RESUMO
Transgenic mice expressing the Notch 4 intracellular domain (ICD) (Int3) in the mammary gland have two phenotypes: arrest of mammary alveolar/lobular development and mammary tumorigenesis. Notch4 signaling is mediated primarily through the interaction of Int3 with the transcription repressor/activator Rbpj. We have conditionally ablated the Rbpj gene in the mammary glands of mice expressing whey acidic protein (Wap)-Int3. Interestingly, Rbpj knockout mice (Wap-Cre(+)/Rbpj(-/-)/Wap-Int3) have normal mammary gland development, suggesting that the effect of endogenous Notch signaling on mammary gland development is complete by day 15 of pregnancy. RBP-J heterozygous (Wap-Cre(+)/Rbpj(-/+)/Wap-Int3) and Rbpj control (Rbpj(flox/flox)/Wap-Int3) mice are phenotypically the same as Wap-Int3 mice with respect to mammary gland development and tumorigenesis. In addition, the Wap-Cre(+)/Rbpj(-/-)/Wap-Int3-knockout mice also developed mammary tumors at a frequency similar to Rbpj heterozygous and Wap-Int3 control mice but with a slightly longer latency. Thus, the effect on mammary gland development is dependent on the interaction of the Notch ICD with the transcription repressor/activator Rbpj, and Notch-induced mammary tumor development is independent of this interaction.
Assuntos
Glândulas Mamárias Animais/embriologia , Neoplasias Mamárias Experimentais/genética , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Notch/fisiologia , Adenocarcinoma Papilar/genética , Adenocarcinoma Papilar/patologia , Ágar , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Viral/genética , Feminino , Proteínas de Homeodomínio/metabolismo , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Knockout , Camundongos Nus , Proteínas do Leite/genética , Proteínas de Neoplasias/genética , Gravidez , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Receptor Notch4 , Receptores Notch/química , Receptores Notch/deficiência , Receptores Notch/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Repressoras/genética , Sequências Repetidas Terminais/genética , Fatores de Transcrição HES-1 , Células Tumorais Cultivadas/citologiaRESUMO
Transgenic mice overexpressing Notch4 intracellular domain (Int3) under the control of the whey acidic protein (WAP) or mouse mammary tumor virus-long terminal repeat promoters, develop mammary tumors. Microarray analysis of these tumors revealed high levels of c-Kit expression. Gleevec is a tyrosine kinase inhibitor that targets c-Kit, platelet-derived growth factor receptors (PDGFRs) and c-Abl. This led us to speculate that tyrosine kinase receptor activity might be a driving force in the development of Int3 mammary tumors. WAP-Int3 tumor-bearing mice were treated with continuous release of Gleevec using subcutaneously implanted Alzet pumps. Phosphorylation of c-Kit, PDGFRs and c-Abl is inhibited in Int3 transgenic mammary tumors by Gleevec. Inhibition of these enzymes is associated with a decrease in cell proliferation and angiogenesis, and an induction of apoptosis. To examine the signaling mechanisms underlying Notch4/Int3 tumorigenesis, we employed small interfering RNA (siRNA) to knock down c-Kit, PDGFRs and c-Abl alone or in combination and observed the effects on soft agar growth of HC11 cells overexpressing Int3. Only siRNA constructs for c-Kit and/or PDGFR-alpha were able to inhibit HC11-Int3 colony formation in soft agar. Our data demonstrate an inhibitory effect of Gleevec on Int3-induced transformation of HC11 cells and mammary tumors and indicate an oncogenic role for c-Kit and PDGFR-alpha tyrosine kinases in the context of Int3 signaling.
Assuntos
Transformação Celular Neoplásica , Glândulas Mamárias Animais/crescimento & desenvolvimento , Neoplasias Mamárias Experimentais/patologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Notch/fisiologia , Animais , Antineoplásicos/uso terapêutico , Benzamidas , Northern Blotting , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Feminino , Mesilato de Imatinib , Imunoprecipitação , Neoplasias Mamárias Experimentais/genética , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Transgênicos , Proteínas do Leite/genética , Proteínas do Leite/metabolismo , Fosforilação , Piperazinas/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas c-kit/química , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/uso terapêutico , RNA Interferente Pequeno/farmacologia , Receptor Notch4 , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Loss of heterozygosity (LOH) on chromosome 17q21 has been detected in 30% of primary human breast tumours. The smallest common region deleted occurred in an interval between the D17S746 and D17S846 polymorphic sequences tagged sites that are located on two recombinant P1-bacteriophage clones of chromosome 17q21: 122F4 and 50H1, respectively. To identify the target gene for LOH, we defined a map of this chromosomal region. We found the following genes: JUP, FK506BP10, SC65, Gastrin (GAS) and HAP1. Of the genes that have been identified in this study, only JUP is located between D17S746 and D17S846. This was of interest since earlier studies have shown that JUP expression is altered in breast, lung and thyroid tumours as well as cell lines having LOH in chromosome 17q21. However, no mutations were detected in JUP using single-strand conformation polymorphism analysis of primary breast tumour DNAs having LOH at 17q21. We could find no evidence that the transcription promoter for JUP is methylated in tumour DNAs having LOH at 17q21. We suspect that the target gene for LOH in primary human breast tumours on chromosome 17q21 is either JUP and results in a haploinsufficiency for expression or may be an unidentified gene located in the interval between D17S846 and JUP.