MECHANISM OF CAMP-GROWTH REGULATORY FUNCTION

我们的长期目标是阐明cAMP控制细胞发育、增殖和分化的原理。CAMP通过激活cAMP反应元件(CAMP)诱导的转录改变基因表达的基本模式，调节大量的生理过程，包括中间代谢、细胞增殖和神经元信号转导。然而，Cre引导的转录在细胞增殖中的机制在很大程度上尚不清楚。为了阐明cAMP增强子(Cre)在控制细胞增殖中的作用，我们采用了转录因子-诱骗寡核苷酸的方法。我们的研究表明，由Cre序列组成的人工合成的单链寡核苷酸可以穿透细胞，与Cre增强剂竞争结合转录因子，并在体内特异性干扰Cre和AP-1的转录。这种寡核苷酸抑制肿瘤细胞的增殖，而不影响非肿瘤细胞的生长。这些结果提示，PKA在肿瘤发生中的作用可能与其转录一系列基因有关。最近发展的高通量DNA微阵列使复杂生物系统的单个杂交中数千个基因的表达谱得以并行分析。我们通过微阵列分析，通过过度表达/抑制野生型和突变型PKA R和C亚单位基因来检测参与PKA-cAMP途径的全局细胞基因的表达，这些基因导致癌细胞的生长模式和表型发生变化。我们发现，在RIA反义寡核苷酸(ODN)(48小时，0.2 mM)处理后，大约240个cDNA，占阵列上总DNA元件的10%，显著改变了表达水平，产生了50%的生长抑制。这些候选基因的cdna表达均表现出2.5倍以上的变化(或上调或下调)。重要的是，我们的结果表明，与反义ODN处理的细胞相比，过表达反义基因的细胞在表达谱上显示出显著的重叠。在反义ODN处理的细胞中上调或下调的相同DNA元件在反义基因过表达的细胞中也同样上调或下调。进一步的散点图分析发现，在反义ODN处理的细胞和反义基因过表达的细胞中，只有不到2%的DNA元件表达发生了选择性的上调或下调。基因图谱的微阵列分析也在用CRE-圈套寡核苷酸处理的细胞中进行。这种方法将使我们有可能调查所有的遗传途径，并发现到目前为止尚未认识到的可能与肿瘤生长和肿瘤发生有关的新基因。此外，通过这种方法发现新的基因，结合遗传和生化分析，可能会解开cAMP-去调控癌症发生的机制，并为癌症的药物开发和新的治疗策略提供新的靶点。

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.