RESUMEN
Chromosomal INstability (CIN), a hallmark of cancer, refers to cells with an increased rate of gain or loss of whole chromosomes or chromosome parts. CIN is linked to the progression of tumors with poor clinical outcomes such as drug resistance. CIN can give tumors the diversity to resist therapy, but it comes at the cost of significant stress to tumor cells. To tolerate this, cancer cells must modify their energy use to provide adaptation against genetic changes as well as to promote their survival and growth. In this study, we have demonstrated that CIN induction causes sensitivity to metabolic stress. We show that mild metabolic disruption that does not affect normal cells, can lead to high levels of oxidative stress and subsequent cell death in CIN cells because they are already managing elevated stress levels. Altered metabolism is a differential characteristic of cancer cells, so our identification of key regulators that can exploit these changes to cause cell death may provide cancer-specific potential drug targets, especially for advanced cancers that exhibit CIN.
Asunto(s)
Inestabilidad Cromosómica , Neoplasias/metabolismo , Estrés Fisiológico/genética , Animales , Animales Modificados Genéticamente , Apoptosis/genética , Daño del ADN , Drosophila melanogaster , Embrión no Mamífero , Glutatión/metabolismo , Peroxidación de Lípido/genética , Neoplasias/genética , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK-green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.
Asunto(s)
Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Drosophila melanogaster/citología , Integrinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transactivadores , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Adhesión Celular , Proteínas del Citoesqueleto/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Integrinas/genética , Datos de Secuencia Molecular , Músculos/anomalías , Músculos/citología , Músculos/embriología , Músculos/metabolismo , Mutación/genética , Fenotipo , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Transducción de Señal , Alas de Animales/anomalías , Alas de Animales/citología , Alas de Animales/embriología , Alas de Animales/metabolismo , beta CateninaRESUMEN
Mutations in kakapo were recovered in genetic screens designed to isolate genes required for integrin-mediated adhesion in Drosophila. We cloned the gene and found that it encodes a large protein (>5,000 amino acids) that is highly similar to plectin and BPAG1 over the first 1,000-amino acid region, and contains within this region an alpha-actinin type actin-binding domain. A central region containing dystrophin-like repeats is followed by a carboxy domain that is distinct from plectin and dystrophin, having neither the intermediate filament-binding domain of plectin nor the dystroglycan/syntrophin-binding domain of dystrophin. Instead, Kakapo has a carboxy terminus similar to the growth arrest-specific protein Gas2. Kakapo is strongly expressed late during embryogenesis at the most prominent site of position-specific integrin adhesion, the muscle attachment sites. It is concentrated at apical and basal surfaces of epidermal muscle attachment cells, at the termini of the prominent microtubule bundles, and is required in these cells for strong attachment to muscles. Kakapo is also expressed more widely at a lower level where it is essential for epidermal cell layer stability. These results suggest that the Kakapo protein forms essential links among integrins, actin, and microtubules.
Asunto(s)
Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila , Drosophila/genética , Drosophila/metabolismo , Genes de Insecto , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Proteínas de Microfilamentos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Adhesión Celular/genética , Adhesión Celular/fisiología , Clonación Molecular , Proteínas del Citoesqueleto/química , Cartilla de ADN/genética , Drosophila/citología , Distrofina/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos/química , Proteínas de Filamentos Intermediarios/genética , Modelos Biológicos , Datos de Secuencia Molecular , Músculos/citología , Músculos/embriología , Músculos/metabolismo , Mutación , Filogenia , Plectina , Homología de Secuencia de AminoácidoRESUMEN
We reported the identification of a new family of DNA-binding proteins from our characterization of the dead ringer (dri) gene of Drosophila melanogaster. We show that dri encodes a nuclear protein that contains a sequence-specific DNA-binding domain that bears no similarity to known DNA-binding domains. A number of proteins were found to contain sequences homologous to this domain. Other proteins containing the conserved motif include yeast SWI1, two human retinoblastoma binding proteins, and other mammalian regulatory proteins. A mouse B-cell-specific regulator exhibits 75% identity with DRI over the 137-amino-acid DNA-binding domains of these proteins, indicating a high degree of conservation of this domain. Gel retardation and optimal binding site screens revealed that the in vitro sequence specificity of DRI is strikingly similar to that of many homeodomain proteins, although the sequence and predicted secondary structure do not resemble a homeodomain. The early general expression of dri and the similarity of DRI and homeodomain in vitro DNA-binding specificity compound the problem of understanding the in vivo specificity of action of these proteins. Maternally derived dri product is found throughout the embryo until germ band extension, when dri is expressed in a developmentally regulated set of tissues, including salivary gland ducts, parts of the gut, and a subset of neural cells. The discovery of this new, conserved DNA-binding domain offers an explanation for the regulatory activity of several important members of this class and predicts significant regulatory roles for the others.
Asunto(s)
Secuencia Conservada , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Drosophila , Proteínas de Homeodominio/aislamiento & purificación , Proteínas Nucleares/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Proteínas de Unión al ADN/genética , Drosophila melanogaster/metabolismo , Proteínas de Homeodominio/genética , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
We have found that the repressor of 186 lytic transcription, CI, represses transcription of the late control gene B, with no involvement of the B protein itself. In clone studies we showed that CI repressed transcription from the B promoter and that temperature inactivation of CIts led to B derepression. We conclude that CI repressor directly represses transcription of the B gene and, with prophage induction, it is probable that the inactivation of the CI repressor not only derepresses early lytic transcription, but also derepresses B gene transcription, leading to the activation of transcription from the late promoters.