ABSTRACT
The tumor suppressor p53 is a nucleocytoplasmic shuttling protein that accumulates in the nucleus of cells exposed to various cellular stresses. One important role of nuclear p53 is to mobilize a stress response by transactivating target genes such as the p21(Waf1) gene. In this study, we investigated more closely the localization of p53 in cells following various stresses. Immunocytochemistry of fixed human fibroblasts treated with either UV light, the kinase and transcription inhibitor DRB or the proteasome inhibitor MG132 revealed abundant p53 localized to the nucleus. When cells treated with UV or DRB were permeabilized prior to fixation to allow soluble proteins to diffuse, the nuclear p53 signal was abolished. However, in cells treated with MG132, residual p53 localized to distinct large foci. Furthermore, nucleolin co-localized with p53 to these foci, suggesting that these foci were nucleolar structures. Interestingly, the MDM2 protein was found to co-localize with p53 to nucleolar structures following proteasome inhibition. Our results suggest that the p53 proteins accumulating in the nucleus following UV-irradiation or blockage of transcription are freely soluble and, thus, should be able to roam the nucleus to ensure high occupancy of p53 binding sites. However, inhibition of proteasome activity may be a unique stress in that it leads to the sequestering of p53 proteins to the nucleolus, thereby blunting the p53-mediated transactivation of target genes.
Subject(s)
Cell Nucleolus/metabolism , Nuclear Proteins , Transcription, Genetic/physiology , Tumor Suppressor Protein p53/metabolism , Blotting, Northern , Cell Nucleolus/chemistry , Cell Nucleolus/radiation effects , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , Cysteine Endopeptidases , Cysteine Proteinase Inhibitors/pharmacology , Dichlororibofuranosylbenzimidazole/pharmacology , Diffusion , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression/drug effects , Gene Expression/physiology , Humans , Leupeptins/pharmacology , Multienzyme Complexes/antagonists & inhibitors , Phosphoproteins/analysis , Phosphoproteins/metabolism , Proteasome Endopeptidase Complex , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , RNA Polymerase II/antagonists & inhibitors , RNA, Messenger/analysis , RNA-Binding Proteins/analysis , RNA-Binding Proteins/metabolism , Solubility , Transcription, Genetic/drug effects , Tumor Suppressor Protein p53/analysis , Ultraviolet Rays , NucleolinABSTRACT
In this study, we explored what effect inhibitors of the 26S proteasome have on cell cycle distribution and induction of apoptosis in human skin fibroblasts and colon cancer cells differing in their p53 status. We found that proteasome inhibition resulted in nuclear accumulation of p53. This was surprising because it is thought that the degradation of p53 is mediated by cytoplasmic 26S proteasomes. Nuclear accumulation of p53 was accompanied by the induction of both p21WAF1 mRNA and protein as well as a decrease in cells entering S phase. Interestingly, cells with compromised p53 function showed a marked increase in the proportion of cells in the G2-M phase of the cell cycle and an attenuated induction of apoptosis after proteasome inhibition. Taken together, our results suggest that proteasome inhibition results in nuclear accumulation of p53 and a p53-stimulated induction of both G1 arrest and apoptosis.
Subject(s)
Apoptosis/physiology , Cysteine Endopeptidases/metabolism , Interphase/physiology , Multienzyme Complexes/metabolism , Tumor Suppressor Protein p53/physiology , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Apoptosis/drug effects , Cell Nucleus/metabolism , Colonic Neoplasms , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/analysis , Cyclins/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Fibroblasts/cytology , Fibroblasts/enzymology , G1 Phase/drug effects , G1 Phase/physiology , G2 Phase/drug effects , G2 Phase/physiology , Humans , Interphase/drug effects , Mitosis/drug effects , Mitosis/physiology , Proteasome Endopeptidase Complex , Skin/cytology , Tumor Cells, CulturedABSTRACT
BACKGROUND: Preclinical atherosclerosis is associated with increased endothelial cell (EC) expression of leukocyte adhesion molecules (LAMs), which mediate monocyte adhesion during atherogenesis. Identification of cell-surface LAMs may uniquely allow assessment of endothelial function, but there are no in vivo methods for detecting LAMs. We tested a new microbubble designed to bind to and allow specific ultrasound detection of intercellular adhesion molecule-1 (ICAM-1). METHODS AND RESULTS: A perfluorobutane gas-filled lipid-derived microsphere with monoclonal antibody to ICAM-1 covalently bound to the bubble shell was synthesized. Bubbles with either nonspecific IgG or no protein on the shell were synthesized as controls. Coverslips of cultured human coronary artery ECs were placed in a parallel-plate perfusion chamber and exposed to 1 of the 3 microbubble species, followed by perfusion with culture medium. Experiments were performed with either normal or interleukin-1beta-activated ECs overexpressing ICAM-1, and bubble adherence was quantified with epifluorescent videomicroscopy. There was limited adherence of control bubbles to normal or activated ECs, whereas a 40-fold increase in adhesion occurred when anti-ICAM-1-conjugated bubbles were exposed to activated ECs compared with normal ECs (8.1+/-3.5 versus 0.21+/-0.09 bubbles per cell, respectively, P<0.001). Although diminished, this difference persisted even after perfusion at higher wall shear rates. CONCLUSIONS: A gas-filled microbubble with anti-ICAM-1 antibody on its shell specifically binds to activated ECs overexpressing ICAM-1. Diagnostic ultrasound in conjunction with targeted contrast agents has the unique potential to characterize cell phenotype in vivo.
