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. Further more they emphasize the importance of combined proteomic and genomic approaches to the investigation of p53-mediated apoptosis and other cellular processes.