MECHANISM OF CAMP-GROWTH REGULATORY FUNCTION

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Our long term goal is to elucidate the principles by which cAMP controls cell development, proliferation and diffrentiation. cAMP regulates a striking number of physiologic processes, including intermediary metabolism, cellular proliferation, and neuronal signaling by altering basic pattrens of gene expression via activation of cAMP response element (CRE)-directed transcription. The mechanism of the CRE-directed transcription in cell proliferation, however, is largely unexplored. To elucidate the role of the cAMP enhancer (CRE) in the control of cell proliferation, we used transcription factor-decoy oligonucleotide approach. Our studies revealed that a synthetic single-stranded oligonucleotide composed of the CRE sequence, which self-hybridizes to form a duplex/hairpin, can penetrate into cells, compete with CRE enhancers for binding transcription factors and specifically interfere with CRE- and Ap-1-directed transcription in vivo . This oligonucleotide restrained tumor cell proliferation, without affecting the growth of non-cancerous cells. These results suggest that the role of PKA in cancergenesis may involve its transcription of array of genes. Recent development of high throughput DNA microarray enables parallel analysis of expression profiles of thousands of genes in a single hybridization for complex biological systems. We examined by the microarray analysis the expression of global cellular genes that are involved in PKA-cAMP-pathways by overexpression/suppression of the wild type and mutant PKA R and C subunit genes that cause changes in the growth patterns and phenotype of cancer cells. We found that approximately 240 cDNAs, representing 10% of the total DNA elements on the array, were found to have significantly altered levels of expression after treatment with RIa antisense oligonucleotide (ODN)(48hr, 0.2 mM) which produced 50% growth inhibition. The cDNA expression for these candidate genes all exhibited greater than 2.5 fold alterations (either up- or downregulated). Importantly our results showed that in comparison to cells treated with antisense ODN, the antisense gene overexpressing cells exhibited striking overlaps in the expression profiles. The same DNA elements up- or downregulated in the antisense ODN treated cells were similarly up- or downregulated in the antisense gene overexpressing cells. On further analysis by Scatter plots, we found that less than 2% of the DNA elements which exhibited altered expression were selectively up- or down-regulated in either antisense ODN treated cells or antisense gene overexpressing cells. The microarray analysis of gene profiling is also underway in cells treated with CRE-decoy oligonucleotides. This approach will lead us to potentially survey all the genetic pathways and also to discover hitherto unrecognized novel genes that may be involved in tumor growth and tumorigenesis. Furthermore, the discovery of novel genes by this approach coupled with the genetic and biochemical analyses may unravel the mechanism of cAMP-deregulation underlying cancergenesis and offer new targets for drug development and novel treatment strategies for cancer.