Analysis of the DNA-binding activity of p53 mutants using functional protein microarrays and its relationship to transcriptional activation.
Sequence-specific DNA binding is the key function through which tumor suppressor p53 exerts transactivation of the downstream target genes, often being impaired in cancer cells by mutations in the TP53 gene. Functional protein microarray technology enables a high-throughput parallel analysis of protein properties within one experiment under the same conditions. Using an array approach, we analyzed the DNA binding activity of wild type p53 protein and of 49 variants. Our results show significant differences in the binding properties between the p53 mutants. The C-terminal mutant R337C displayed the highest DNA binding activity on the array. However, the same mutant showed only a partial activation in the reporter gene assay and almost no activation of downstream target genes after transfection of expression vector into cells lacking endogenous p53. These observations demonstrate that DNA binding itself is not sufficient for activating the p53 target genes in at least some of the p53 mutants and, therefore, in vitro studies might not always reflect in vivo conditions.
Center of Molecular Biology and Gene Therapy, Department of Internal Medicine - Hematooncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Cernopolni 9, CZ-625 00 Brno, Czech Republic.
Jitka
J
Malcikova
Center of Molecular Biology and Gene Therapy, Department of Internal Medicine - Hematooncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Cernopolni 9, CZ-625 00 Brno, Czech Republic.
Boris
B
Tichy
Loschmidt Laboratories, Institute of Experimental Biology and National Center for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5/A4, CZ-625 00 Brno, Czech Republic.
Jiri
J
Damborsky
Center of Molecular Biology and Gene Therapy, Department of Internal Medicine - Hematooncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Cernopolni 9, CZ-625 00 Brno, Czech Republic.
Jitka
J
Kabathova
Center of Molecular Biology and Gene Therapy, Department of Internal Medicine - Hematooncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Cernopolni 9, CZ-625 00 Brno, Czech Republic.
Martin
M
Trbusek
Center of Molecular Biology and Gene Therapy, Department of Internal Medicine - Hematooncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Cernopolni 9, CZ-625 00 Brno, Czech Republic.
Jiri
J
Mayer
Center of Molecular Biology and Gene Therapy, Department of Internal Medicine - Hematooncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Cernopolni 9, CZ-625 00 Brno, Czech Republic.
Sarka
S
Pospisilova
D001665:Binding Sites; D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism*; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D040081:Protein Array Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism*
D050505:Mutant Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA