Copper alters the conformation and transcriptional activity of the tumor suppressor protein p53 in human Hep G2 cells.
The tumor suppressor protein p53 plays a role in the molecular response to DNA damage by acting as a DNA-binding transcription factor that regulates specific target genes to arrest the cell cycle, induce repair mechanisms, and initiate apoptotic cell death. To test the effect of copper on the transcriptional activity of p53, Hep G2 cells were transiently transfected with a luciferase reporter gene downstream from multiple p53 response elements. Co-transfection with the p53 gene resulted in a 6-fold increase in luciferase activity, showing that p53 acts as a transcription factor in this system. However, in the presence of copper, luciferase activity was significantly reduced. Oligonucleotide arrays representing 145 known p53-associated genes were hybridized with biotinylated cDNAs from mRNA extracted from control and copper-treated Hep G2 cells. Among the genes that were differentially regulated were fos, RB1, glutathione peroxidase, TGF-beta, and 15-lipoxygenase, a gene known to be activated by mutant p53. Although control Hep G2 cells synthesize wild-type p53, immunocytochemistry identified not only wild type, but also mutant p53 in the presence of copper and other agents that induce oxidative damage. Thus, this report not only identifies genes that may play a role in copper-mediated apoptosis, but also suggests that copper-induced oxidative processes result in the synthesis of mutant p53 with altered transcriptional properties.
Tassabehji
Nadine M
NM
Florida State University, 237 Biomedical Research Facility, Tallahassee, FL 32306-4340, USA.
VanLandingham
Jacob W
JW
Levenson
Cathy W
CW
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D014408:Biomarkers, Tumor / Q000378:metabolism; D019921:Biotinylation; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3; D061945:Caspase Inhibitors; D045744:Cell Line, Tumor; D003300:Copper / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010084:Oxidation-Reduction; D011487:Protein Conformation / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D017304:Annexin A5; D014408:Biomarkers, Tumor; D061945:Caspase Inhibitors; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D003300:Copper; D008156:Luciferases; C505407:CASP3 protein, human; D053148:Caspase 3