RESUMEN
The insulin-like growth factor 2 gene (IGF2) is an imprinting gene, which mediates cell growth and apoptosis. The loss of imprinting (LOI) of IGF2 has been associated with the development of cancer. In the present study, loss LOI of IGF2 in lung cancer was analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in combination with DNA sequencing of samples collected by laser capture microdissection. The status of each sample was assigned as imprinting when PCR-RFLP revealed only one band or sequence with a single peak; otherwise, the case was classified as LOI. LOI was identified in eight out of 13 adenocarcinoma cases (62%), but was not detected in any of the nine squamous cell carcinoma cases (0%). These results suggest that IGF2 LOI is involved in the molecular pathogenesis of lung adenocarcinoma, but not squamous cell carcinoma, and that LOI may be detected through increased IGF2 expression levels.
RESUMEN
Extracellular signal-regulated kinase (ERK) signalling plays a central role in various biological processes, including cell migration, but it remains unknown what factors directly regulate the strength and duration of ERK activation. We found that, among the B56 family of protein phosphatase 2A (PP2A) regulatory subunits, B56γ1 suppressed EGF-induced cell migration on collagen, bound to phosphorylated-ERK, and dephosphorylated ERK, whereas B56α1 and B56ß1 did not. B56γ1 was immunolocalized in nuclei. The IER3 protein was immediately highly expressed in response to costimulation of cells with EGF and collagen. Knockdown of IER3 inhibited cell migration and enhanced dephosphorylation of ERK. Analysis of the time course of PP2A-B56γ1 activity following the costimulation showed an immediate loss of phosphatase activity, followed by a rapid increase in activity, and this activity then remained at a stable level that was lower than the original level. Our results indicate that the strength and duration of the nuclear ERK activation signal that is initially induced by ERK kinase (MEK) are determined at least in part by modulation of the phosphatase activity of PP2A-B56γ1 through two independent pathways.
Asunto(s)
Movimiento Celular , Núcleo Celular/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína Fosfatasa 2/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Colágeno/farmacología , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Humanos , Cinética , Proteínas de la Membrana/metabolismo , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 2/química , Transporte de Proteínas/efectos de los fármacosRESUMEN
To ascertain the implications of loss of imprinting (LOI) of the insulin-like growth factor II gene (IGF2) for carcinogenesis, the precise frequency of LOI in colorectal carcinoma was examined using a laser capture microdissection method, and compared to the matched normal colorectal mucosa. LOI was examined by PCR-restriction fragment length polymorphism in combination with direct sequencing. The status was assigned as imprinting when PCR-RFLP showed only one band or sequence with a single peak, otherwise cases were classified as LOI. LOI was found in 13 of 24 informative cases of carcinoma (54%), which was higher than the ratios reported previously. LOI was also found in the normal colorectal mucosae in 14 cases (58%). The LOIs in carcinomas and in the normal mucosae were closely correlated: 10 of 13 LOI-positive carcinomas showed LOI in the matched normal mucosae. These results suggest that LOI of IGF2 in colorectal carcinoma and LOI in the background mucosa play important roles in carcinogenesis.
Asunto(s)
Neoplasias Colorrectales/genética , Impresión Genómica/genética , Factor II del Crecimiento Similar a la Insulina/genética , Microdisección/métodos , Sitios de Unión/genética , Neoplasias Colorrectales/patología , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Rayos Láser , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Little is known about the functions of two different beta5 integrins: repeated-FNK (FNKFNK764-769) and single-FNK (FNK764-766) amino acid sequences in the cytoplasmic domain. We examined whether they occurred as germ line mutations or somatic mutations associated with neoplastic transformation, and whether there were functional alterations. Out of six cultured cell lines, only KATO-III cells had the single-FNK beta5 sequence. The single-FNK beta5 was found in 9 out of 79 patients with colon carcinoma, but no somatic mutations were detected in cancerous tissues. CHO cells were transformed with expression vectors containing single-FNK or repeated-FNK beta5 cDNA, which were derived from KATO-III cells. CHO cells transfected with single-FNK and repeated-FNK showed similar adhesiveness to, and proliferative activity on, vitronectin substrates.
