RESUMO
Microdissection and differential display PCR were used to identify genes preferentially expressed in the highly proliferative terminal end buds (TEBs) in the mammary gland of 45-day-old virgin rats. One clone exhibited 87% homology to the human p190-B gene encoding a novel Rho-Gap. Using in situ hybridization, p190-B was detected in both the TEBs and the terminal ducts, with the highest expression observed in the outer layer of TEBs. During normal mammary gland development, p190-B mRNA expression was highest in the virgin mammary gland and decreased during late pregnancy and lactation. Interestingly, increased levels of p190-B mRNA relative to the normal mammary gland were seen in a subset of murine mammary tumors that appeared to be less well differentiated and potentially more aggressive. Transient transfection of a p190-B expression construct into MCF-10A human mammary epithelial cells resulted in disruption of the actin cytoskeleton, which suggests a role for p190-B in regulating the signaling pathways that influence cell migration and invasion. These results suggest that p190-B may be required for virgin mammary gland development, and its aberrant expression may occur in breast cancer.
Assuntos
Mama/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Actinas/metabolismo , Animais , Biomarcadores , Linhagem Celular , Corantes , Citoesqueleto/metabolismo , DNA Complementar , Proteínas de Ligação a DNA , Células Epiteliais/metabolismo , Feminino , Proteínas Ativadoras de GTPase , Perfilação da Expressão Gênica , Humanos , Hibridização In Situ , Glândulas Mamárias Animais/anatomia & histologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Microscopia de Fluorescência , Proteínas Nucleares/genética , Fosfoproteínas/genética , Gravidez , RNA/metabolismo , Ratos , Ratos Endogâmicos WF , Proteínas Recombinantes/metabolismo , Proteínas Repressoras , TransfecçãoRESUMO
The accuracy of immunodetection by dual parameter flow cytometry (FCM), polymerase chain reaction-mediated single strand conformation polymorphism (PCR-SSCP) and genomic sequencing to detect p53 mutations were compared. Analysis by the last two techniques was restricted to exons 5-8. Initially, 110 breast tumours were screened for p53 expression by FCM. Seventy (64%) of tumours were immunopositive. Fifteen highly immunopositive and 15 completely immunonegative tumours were selected for further analysis by PCR-SSCP and genomic sequencing. Eleven out of 15 immunopositive tumours were found to have mutation by PCR-SSCP. Genomic sequencing confirmed the presence of mutation in 10 of these 11 immunopositive tumours. Therefore, four immunopositive tumours failed to show mutation by SSCP and five by genomic sequencing. Of the 15 immunonegative tumours, one showed mutation by both PCR-SSCP and genomic sequencing and one tumour has undergone deletion of the p53 gene. Overall, immunoreactivity correlated with both PCR-SSCP and genomic sequencing in 80% of cases (24/30), and there was 96.5% (28/29) concordance between PCR-SSCP and genomic sequencing. We conclude that there is good concordance between mutations detected by PCR-SSCP and genomic sequencing, but immunochemical detection of p53 overexpression is not an absolute indicator of p53 gene mutation.