ABSTRACT
A fluorescent-based high-throughput screening (HTS) assay for small molecules that inhibit the interaction of MdmX with p53 was developed and applied to identify new inhibitors. The assay evaluated the MdmX-p53 interaction by detecting the quenching of the fluorescence of green fluorescent protein (GFP) fused to the MdmX protein, after its interaction with a p53 peptide labeled with a fluorescence quencher. In this report, the developed HTS assay was applied to about 40 000 compounds, and 255 hit compounds that abrogated the GFP quenching were selected. Next, the obtained hits were reevaluated by other assays. First, their effects on the diffusion time of a fluorescently-labeled p53 peptide after incubation with the MdmX protein were tested by measuring the diffusion time using fluorescence correlation spectroscopy, and six stable hit compounds with IC(50) values less than 5 µM were selected. Next, we further confirmed their inhibition of the MdmX-p53 interaction by surface plasmon resonance. To indicate the efficacy of the hit compound as a candidate anticancer drug, we showed that the hit compound triggered apoptosis after p53 and p21 accumulation in cultured MV4;11 leukemia cells. Thus, the new HTS assay is effective for obtaining novel MdmX-p53 interaction inhibitors that are valuable as candidate compounds for cancer treatment.
Subject(s)
High-Throughput Screening Assays/methods , Proto-Oncogene Proteins c-mdm2/metabolism , Small Molecule Libraries , Spectrometry, Fluorescence , Tumor Suppressor Protein p53/metabolism , Binding, Competitive/drug effects , Cell Line, Tumor , Drug Screening Assays, Antitumor/methods , Humans , Protein Binding/drug effectsABSTRACT
We evaluated the cytotoxicity of surfactants in human cells. Synthetic surfactants showed different cytotoxicity levels depending on their structures. The cytotoxicity of commercial washing products was determined mainly by the contents of surfactants. All of them induced premature senescence in normal cells, but not in tumor-derived or immortalized cells, under sublethal conditions. Residual surfactants might be a risk factor for skin aging.
Subject(s)
Cellular Senescence/drug effects , Skin Aging/drug effects , Skin/drug effects , Skin/pathology , Surface-Active Agents/toxicity , Cell Line , Collagenases/drug effects , Collagenases/metabolism , Fibronectins/drug effects , Fibronectins/metabolism , Humans , Plasminogen Activator Inhibitor 1/metabolism , Surface-Active Agents/administration & dosageABSTRACT
Pregnancy-specific glycoproteins (PSGs) comprise a family of highly similar polypeptides encoded by 11 transcriptionally active genes that compactly cluster on band 19q13.2. All members of the PSG family were found to be markedly up-regulated by addition of 5-bromodeoxyuridine in HeLa cells. Similarly, all of the members were markedly up-regulated during replicative senescence in normal human fibroblasts. Promoter analysis of the PSG1, 4, and 11 genes in HeLa cells did not reveal a cis-regulatory element responsive to 5-bromodeoxyuridine in their 5'-flanking sequences. These results suggest that the PSG genes are regulated at a level of higher order chromatin structure besides by a signal of pregnancy.
Subject(s)
Cell Proliferation , Cellular Senescence , Pregnancy-Specific beta 1-Glycoproteins/metabolism , Bromodeoxyuridine/metabolism , DNA Damage , Gene Expression Regulation , Genotype , HeLa Cells , Humans , Oxidative Stress/drug effects , Phenotype , Pregnancy-Specific beta 1-Glycoproteins/genetics , Promoter Regions, Genetic , RNA, Messenger/metabolism , Time Factors , TransfectionABSTRACT
Ectopic genes transferred to cells are temporally expressed, although this phenomenon has not yet been well characterized. We found that 5-bromodeoxyuridine dramatically increased transient expression of ectopic genes in human cells. This effect was elicited by adding 5-bromodeoxyuridine prior to or after transfection. No promoter specificity was observed. Real time PCR analysis showed an approximately 2-fold increase in mRNA levels. Since 5-bromodeoxyuridine decondenses heterochromatin and changes the nuclear envelope, these changes might affect transcriptional and post-transcriptional events in the gene expression of plasmids.
Subject(s)
Antimetabolites/pharmacology , Bromodeoxyuridine/pharmacology , Gene Expression/drug effects , Actins/genetics , DNA/biosynthesis , DNA/genetics , Genes, Reporter/drug effects , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Humans , Luciferases/biosynthesis , Luciferases/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic/drug effects , Vimentin/geneticsABSTRACT
The heterogeneous nuclear ribonucleoprotein C1/C2 is one of the most abundant proteins in the nucleus, and shown to have roles in cellular differentiation and proliferation through post-transcriptional regulations of certain mRNA species. We studied its role in stress response using siRNA mediated knockdown approach in HeLa cells. Upon transient transfection with plasmid encoding siRNA, the cells showed increased sensitivities to various chemical agents, namely H(2)O(2, )paraquat, camptothecin, ICRF-193 and halogenated deoxyuridines. These results demonstrate that hnRNP C1/C2 is involved in maintenance of cellular homeostasis besides cellular differentiation and proliferation.
Subject(s)
Gene Expression Regulation , Heterogeneous-Nuclear Ribonucleoprotein Group C/metabolism , RNA, Small Interfering/metabolism , Antimetabolites/metabolism , Antineoplastic Agents/metabolism , Bromodeoxyuridine/metabolism , Camptothecin/metabolism , Diketopiperazines , Enzyme Inhibitors/metabolism , HeLa Cells , Herbicides/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group C/genetics , Humans , Hydrogen Peroxide/metabolism , Oxidants/metabolism , Paraquat/metabolism , Piperazines/metabolism , RNA, Small Interfering/geneticsABSTRACT
Immortal SVts8 cells that express thermolabile SV40 T antigen exhibit a senescence-like phenomenon upon inactivation of the T antigen. By using a cDNA subtractive hybridization technique, RAB27B, a member of the RAB GTPase family, was found to be up-regulated in senescent SVts8 cells. The up-regulation of RAB27B depends on the p53 gene. Enhanced expression was also observed in replicative senescence in normal human fibroblasts.
Subject(s)
Cellular Senescence/physiology , Fibroblasts/physiology , Up-Regulation/physiology , rab GTP-Binding Proteins/biosynthesis , Cell Line, Transformed , Fibroblasts/cytology , Gene Expression Profiling , Humans , Tumor Suppressor Protein p53/metabolism , rab GTP-Binding Proteins/genetics , rab27 GTP-Binding ProteinsABSTRACT
To analyze the relationship between resistance to oxidative stress and longevity, we isolated three novel paraquat-resistant mutants, mev-5, mev-6, and mev-7, from the nematode Caenorhabditis elegans. They all showed the Dyf (defective in dye filling) phenotype, but not always resistance to heat or UV. Life-span extension was observed only in the mev-5 mutant at 26 degrees C. These results indicate that longevity is uncoupled with the phenotype of paraquat resistance.