Zhang Lingzhi, Zambon Alexander C, Vranizan Karen, Pothula Kanishka, Conklin Bruce R, Insel Paul A
Department of Pharmacology, University of California San Diego, La Jolla, California 92093, USA.
J Biol Chem. 2008 Feb 15;283(7):4304-13. doi: 10.1074/jbc.M708673200. Epub 2007 Nov 29.
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.
第二信使环磷酸腺苷（cAMP）通过蛋白激酶A（PKA）发挥作用，但其诱导细胞凋亡的机制尚不清楚。在此，我们评估了线粒体的作用，并通过比较野生型（WT）S49淋巴瘤细胞和S49变体D(-)（cAMP抗性）分析了cAMP/PKA促进的细胞凋亡中的基因表达。D(-)缺乏cAMP促进的细胞凋亡，但具有野生型水平的PKA活性和cAMP促进的G(1)期生长停滞。用8-氯苯硫基-cAMP（8-(4-氯苯硫基)-腺苷-3':5'-环磷酸）处理WT S49细胞24小时可诱导线粒体膜电位丧失、细胞色素c和SMAC从线粒体释放，并增加caspase-3活性，而D(-) S49细胞则无此现象。基因表达分析（使用Affymetrix 430 2.0芯片）显示，用8-氯苯硫基-cAMP孵育的WT和D(-)细胞在2小时（约800个转录本）和6小时（约1000个转录本）时，cAMP调节的基因表达程度相似但不完全相同（|倍数|>2，p<0.06）；相比之下，在24小时时，WT和D(-)细胞中分别有大约2500个和约1100个转录本发生了变化。我们采用回归分析、聚类和功能注释相结合的方法来识别WT和D(-)细胞之间差异表达的转录本，发现cAMP介导的对参与转录抑制、细胞凋亡、细胞周期、RNA剪接、高尔基体和溶酶体的mRNA的调控存在差异。这两种细胞系在环磷酸腺苷反应元件结合蛋白（CREB）磷酸化、转录抑制剂ICER（诱导型cAMP早期阻遏物）的表达以及凋亡抑制因子（IAP）和Bcl家族基因的cAMP调节表达方面存在差异。这些发现表明，cAMP/PKA促进的淋巴细胞凋亡是通过线粒体介导的事件发生的，这意味着这种凋亡涉及多个生化途径中的基因网络。
