Combined oligonucleotide and protein microarray temporal analysis of p53-mediated apoptosis
Black AP., Wee E., Lambe T., Bailey A., Jones L., Ogg GS.
Background: P53-mediated apoptosis involves a complex process induced largely by p53 acting as a transcription factor. We hypothesised that p53 expression would lead to transcriptional events that rapidly change during apoptosis and that protein array analysis would give a more comprehensive picture of p53-mediated apoptosis than mRNA alone. Materials and Methods: We over-expressed p53 in lymphoblastoid cell lines and performed temporal analysis of functional apoptosis, assessing mRNA levels by oligo microarray and protein levels by novel antibody microarray and Western blot. Results: mRNA levels varied over time. At least 10 genes that showed enhanced expression, such as APAF-1 and GADD45, contained the p53 target sequence confirming their nature as primary p53 targets. Changes in mRNA expression did not correlate directly with changes in protein expression. Conclusion: Array analysis of protein expression in addition to mRNA expression gave a more complete assessment of p53-mediated apoptosis; for example, enhanced levels of bcl-2 and E2F2 protein were detected which, along with APAF-1, are involved in the mitochondrial apoptotic process. These data extend existing p53 array findings to correlate with protein microarray data and functional apoptosis. Furthermor e they emphasize the importance of combined proteomic and genomic approaches to the investigation of p53-mediated apoptosis and other cellular processes.