Gene expression signatures of cAMP/protein kinase A (PKA)-promoted, mitochondrial-dependent apoptosis. Comparative analysis of wild-type and cAMP-deathless S49 lymphoma cells.

The second messenger cAMP acts via protein kinase A (PKA) to induce apoptosis by mechanisms that are poorly understood. Here, we assessed a role for mitochondria and analyzed gene expression in cAMP/PKA-promoted apoptosis by comparing wild-type (WT) S49 lymphoma cells and the S49 variant, D(-) (cAMP-deathless), which lacks cAMP-promoted apoptosis but has wild-type levels of PKA activity and cAMP-promoted G(1) growth arrest. Treatment of WT, but not D(-), S49 cells with 8-CPT-cAMP (8-(4-chlorophenylthio)-adenosine-3':5'-cyclic monophosphate) for 24 h induced loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and SMAC, and increase in caspase-3 activity. Gene expression analysis (using Affymetrix 430 2.0 arrays) revealed that WT and D(-) cells incubated with 8-CPT-cAMP have similar, but non-identical, extents of cAMP-regulated gene expression at 2 h (approximately 800 transcripts) and 6 h (approximately 1000 transcripts) (|Fold| > 2, p < 0.06); by contrast, at 24 h, approximately 2500 and approximately 1100 transcripts were changed in WT and D(-) cells, respectively. Using an approach that combined regression analysis, clustering, and functional annotation to identify transcripts that showed differential expression between WT and D(-) cells, we found differences in cAMP-mediated regulation of mRNAs involved in transcriptional repression, apoptosis, the cell cycle, RNA splicing, Golgi, and lysosomes. The two cell lines differed in cAMP-response element-binding protein (CREB) phosphorylation and expression of the transcriptional inhibitor ICER (inducible cAMP early repressor) and in cAMP-regulated expression of genes in the inhibitor of apoptosis (IAP) and Bcl families. The findings indicate that cAMP/PKA-promoted apoptosis of lymphoid cells occurs via mitochondrial-mediated events and imply that such apoptosis involves gene networks in multiple biochemical pathways.