ABSTRACT
Exposure of human leukaemia MOLT-4 cells to ionizing irradiation led to apoptosis, which was detected by flow cytometric analysis and degradation of the nuclear lamina. The multiple signalling pathways triggered by either membrane or DNA damage play a critical role in radiation-induced apoptosis. The response to DNA damage is typically associated with the p53 protein accumulation. In this study, we proved that the transcriptionally active p53 variant occurs in the MOLT-4 cells and its abundance alteration is triggered in the gamma-irradiated cell population concomitantly with phosphorylation at both the serine-392 and serine-15 residues. The p21 upregulation followed the p53 phosphorylation process in irradiated MOLT-4 cells.
Subject(s)
Gamma Rays , Leukemia/metabolism , Phosphoserine/metabolism , T-Lymphocytes/metabolism , Tumor Suppressor Protein p53/metabolism , Apoptosis/radiation effects , Cell Line, Tumor , Cell Survival/radiation effects , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , Dose-Response Relationship, Radiation , Flow Cytometry , Humans , Phosphorylation/radiation effects , T-Lymphocytes/radiation effects , Up-Regulation/radiation effectsABSTRACT
PURPOSE: To determine whether radiation-induced changes in protein abundance can be correlated with their differential gene expression in a murine fibroblast L929 cell line. MATERIALS AND METHODS: L929 cells were irradiated with 6 Gy. Cell lysates were collected at different points in time (20 min, 12, 24, 36, 48 and 72 h). The extracted proteins were separated by two-dimensional gel electrophoresis and quantified using computerized image analysis. Proteins exhibiting a differential expression equal to or more than twofold were identified by mass spectrometry following trypsin digestion. From these, 10 proteins characterized by large changes of radiation-induced abundance were selected in order to measure their corresponding gene expression using RTQ-PCR (real-time quantitative polymerase chain reaction). RESULTS: Up to 15-fold changes in the abundance of these 10 proteins were associated with no detectable changes more than twofold on the gene expression level. However, one gene (VEGF-D) showed a significant (p=0.005) up-regulation (1.8-fold). CONCLUSIONS: Deducing protein abundance from mRNA expression levels and vice versa appears to be of limited use. Furthermore, examination of transcriptional and translational changes provides different but complementary information.
Subject(s)
Protein Biosynthesis/radiation effects , Transcription, Genetic/radiation effects , Animals , Cell Line , Gene Expression Regulation/radiation effects , Glycolysis/radiation effects , Intracellular Signaling Peptides and Proteins , Mice , Protein Folding , Proteins/genetics , RNA-Binding Proteins , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/radiation effectsABSTRACT
In this study we examined the protein expression profiles in X-irradiated L929 cells to get insight into how mammalian cells respond to radiation-induced cell damage. L929 cells were irradiated with the dose of 6 Gy and cell lysates were collected at different time intervals (20 min, 12, 24, 36, 48 and 72 h). The extracted proteins were separated by 2-DE and quantified using computerized image analysis. Proteins exhibiting significant abundance alterations when comparing irradiated to unirradiated cells were identified by mass spectrometry. Using the proteomics approach we detected 47 proteins that exhibited a significant radiation-induced increase or decrease in the course of 72 h. From this group of spots 28 proteins were identified by mass spectrometry and of these 24 proteins exhibited minimally 2-fold differences in mean abundance values in comparison to controls. The identified proteins represent diverse sets of proteins participating either in protective and reparative cell responses or in induction of apoptosis and oncogenesis. The results document that proteomics is a useful method for unravelling the molecular mechanisms involved in cell reaction to ionizing radiation.
