RESUMEN
An online tool named GEN-SNiP that identifies variations in a set of test DNA sequences with respect to a standard reference sequence is developed and deployed successfully. The tool generates a list of substitutions, insertions and deletions for each test sequences, determined by the reference sequence. In the key batch mode feature, the tool allows multiple sequences to be compared and contrasted even when small insertions and deletions are present, with results sent to the user via email. Other distinguishing features of the tool are grouping of continuous deletions or insertions in the test sequence into a single entity for better output handling, displaying of the alignment of test and reference sequence and the input sequence. The tool has been reported as unique in recent literature.
Asunto(s)
Biología Computacional/métodos , Genoma Humano/genética , Internet , Polimorfismo de Nucleótido Simple/genética , Programas Informáticos , Genoma Mitocondrial/genética , HumanosRESUMEN
HGK (hepatocyte progenitor kinase-like/germinal center kinase-like kinase) is a member of the human STE20/mitogen-activated protein kinase kinase kinase kinase family of serine/threonine kinases and is the ortholog of mouse NIK (Nck-interacting kinase). We have cloned a novel splice variant of HGK from a human tumor line and have further identified a complex family of HGK splice variants. We showed HGK to be highly expressed in most tumor cell lines relative to normal tissue. An active role for this kinase in transformation was suggested by an inhibition of H-Ras(V12)-induced focus formation by expression of inactive, dominant-negative mutants of HGK in both fibroblast and epithelial cell lines. Expression of an inactive mutant of HGK also inhibited the anchorage-independent growth of cells yet had no effect on proliferation in monolayer culture. Expression of HGK mutants modulated integrin receptor expression and had a striking effect on hepatocyte growth factor-stimulated epithelial cell invasion. Together, these results suggest an important role for HGK in cell transformation and invasiveness.
Asunto(s)
Glioblastoma/enzimología , Proteínas de Neoplasias/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Células 3T3 , Empalme Alternativo , Animales , Secuencia de Bases , Adhesión Celular/fisiología , Transformación Celular Neoplásica/genética , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Clonación Molecular , Inducción Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Fibroblastos/enzimología , Regulación Neoplásica de la Expresión Génica , Biblioteca de Genes , Genes Dominantes , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Integrinas/biosíntesis , Integrinas/genética , Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/fisiología , Sistema de Señalización de MAP Quinasas , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Invasividad Neoplásica , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Homología de Secuencia de Ácido Nucleico , Células Tumorales Cultivadas/enzimologíaRESUMEN
The gene that encodes the alpha-isoform of phosphatidylinositol 3-kinase (PIK3Ca) is frequently mutated in human cancers. We profiled the mutation status of the PIK3Ca gene in the National Cancer Institute (NCI)-60 panel of human cancer cell lines maintained by the Developmental Therapeutics Program of the NCI. Mutation hotspots on the gene were PCR amplified and sequenced, and the trace data were analyzed with software designed to detect mutations. Seven of the cell lines tested have PIK3Ca mutations: two lines derived from breast cancer, two from colon cancer, two from ovarian cancer, and one from lung cancer. BRAF and EGFR genes were normal in the PIK3Ca mutant lines. Two of the cell lines with mutant PIK3Ca also have a mutant version of the KRAS gene. The mutation status was correlated with array-based gene expression that is publicly available for the NCI-60 cell lines. We found increased expression levels for estrogen receptor (ER) and ERBB2 in PIK3Ca mutant lines. The PIK3Ca mutation status was also correlated with compound screening data for the cell lines. PIK3Ca-mutant cell lines were relatively more sensitive than PIK3Ca-normal cell lines to the ER inhibitor tamoxifen and the AKT inhibitor triciribine, among other compounds. The results provide insights into the role of mutant PIK3Ca in oncogenic signaling and allow preliminary identification of novel targets for therapeutic intervention in cancers harboring PIK3Ca mutations.
Asunto(s)
Resistencia a Antineoplásicos/genética , Perfilación de la Expresión Génica , Fosfatidilinositol 3-Quinasas/biosíntesis , Fosfatidilinositol 3-Quinasas/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Análisis Mutacional de ADN , Frecuencia de los Genes , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/genética , Ribonucleósidos/farmacología , Ribonucleósidos/uso terapéutico , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
p21-activated protein kinase (PAK) serine/threonine kinases are important effectors of Rho family GTPases and have been implicated in the regulation of cell morphology and motility, as well as in cell transformation. To further investigate the possible involvement of PAK kinases in tumorigenesis, we analyzed the expression of several family members in tumor cell lines. Here we demonstrate that PAK4 is frequently overexpressed in human tumor cell lines of various tissue origins. We also have identified serine (Ser-474) as the likely autophosphorylation site in the kinase domain of PAK4 in vivo. Mutation of this serine to glutamic acid (S474E) results in constitutive activation of the kinase. Phosphospecific antibodies directed against serine 474 detect activated PAK4 on the Golgi membrane when PAK4 is co-expressed with activated Cdc42. Furthermore, expression of the active PAK4 (S474E) mutant has transforming potential, leading to anchorage-independent growth of NIH3T3 cells. A kinase-inactive PAK4 (K350A,K351A), on the other hand, efficiently blocks transformation by activated Ras and inhibits anchorage-independent growth of HCT116 colon cancer cells. Taken together, our data strongly implicate PAK4 in oncogenic transformation and suggest that PAK4 activity is required for Ras-driven, anchorage-independent growth.