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The structure and amount of actin in IMR-90 fibroblasts.

ABSTRACT: Double-labelling and peptide isolation have been used to examine the homology between the actin of IMR-90 human embryo fibroblasts and muscle actin. After separation of mixtures of [14C]carboxymethylated muscle actin and [3H]carboxymethylated proteins of IMR-90 cells of electrophoresis on sodium dodecyl sulphate-containing polyacrylamide gels, peptides were generated from the material co-migrating with actin by digestion with chymotrypsin. Peptides homologous with peptides accounting for Cys-217, Cys-256, Cys-284 and Cys-373 of muscle actin are present in this material, but no peptide homologous with a Cys-10-containing peptide was detected. From the amount of actin-derived peptides present, the actin content of IMR-90 fibroblasts was calculated to be 4.2% of the total protein of these cells.

SUBMITTER: Anderson PJ

PROVIDER: S-EPMC1186640 | biostudies-other | 1979 May

REPOSITORIES: biostudies-other

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Project description:BackgroundThe existence of a radiation bystander effect, in which non-irradiated cells respond to signals from irradiated cells, is well established. To understand early signaling and gene regulation in bystander cells, we used a bio-informatics approach, measuring global gene expression at 30 minutes and signaling pathways between 30 minutes and 4 hours after exposure to alpha-particles in IMR-90 fibroblasts.MethodsWe used whole human genome microarrays and real time quantitative PCR to measure and validate gene expression. Microarray analysis was done using BRB-Array Tools; pathway and ontology analyses were done using Ingenuity Pathway Analysis and PANTHER, respectively. We studied signaling in irradiated and bystander cells using immunoblotting and semi-quantitative image analysis.ResultsGene ontology suggested signal transduction and transcriptional regulation responding 30 minutes after treatment affected cell structure, motility and adhesion, and interleukin synthesis. We measured time-dependent expression of genes controlled by the NF-kappaB pathway; matrix metalloproteinases 1 and 3; chemokine ligands 2, 3 and 5 and interleukins 1beta, 6 and 33. There was an increased response of this set of genes 30 minutes after treatment and another wave of induction at 4 hours. We investigated AKT-GSK3beta signaling and found both AKT and GSK3beta are hyper-phosphorylated 30 minutes after irradiation and this effect is maintained through 4 hours. In bystander cells, a similar response was seen with a delay of 30 minutes. We proposed a network model where the observed decrease in phosphorylation of beta-catenin protein after GSK3beta dependent inactivation can trigger target gene expression at later times after radiation exposureConclusionsThese results are the first to show that the radiation induced bystander signal induces a widespread gene expression response at 30 minutes after treatment and these changes are accompanied by modification of signaling proteins in the PI3K-AKT-GSK3beta pathway.

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The structure and amount of actin in IMR-90 fibroblasts.

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