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J. Biol. Chem., Vol. 283, Issue 7, 4304-4313, February 15, 2008

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Gene Expression Signatures of cAMP/Protein Kinase A (PKA)-promoted, Mitochondrial-dependent Apoptosis

COMPARATIVE ANALYSIS OF WILD-TYPE AND cAMP-DEATHLESS S49 LYMPHOMA CELLS^*

Lingzhi Zhang¹, Alexander C. Zambon¹, Karen Vranizan, Kanishka Pothula, Bruce R. Conklin^¶, and Paul A. Insel^||2

From the Departments of Pharmacology and ^||Medicine, University of California San Diego, La Jolla, California 92093, the Functional Genomics Laboratory, University of California Berkeley, California, 94720, and the ^¶Gladstone Institute of Cardiovascular Disease and the Departments of Medicine and Molecular and Cellular Pharmacology, University of California, San Francisco, California 94141

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 G₁ 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 (800 transcripts) and 6 h (1000 transcripts) (|Fold| > 2, p < 0.06); by contrast, at 24 h, 2500 and 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.

Received for publication, October 19, 2007 , and in revised form, November 26, 2007.

^* This work was supported by Grants from the Leukemia and Lymphoma Society, National Institutes of Health (GM61774), and National Institutes of Health sponsored Institutional Research and Academic Career Development Award (IRACDA) fellowship (GM06852). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains three supplemental tables.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Pharmacology, 9500 Gilman Dr., BSB 3073 UCSD 0636, La Jolla, CA 92093-0636. E-mail: pinsel{at}ucsd.edu.

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