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。利用DNA微阵列，我们对暴露于反义基因的细胞中的基因表达进行了系统的表征，无论是外源性的还是内源性的。我们发现，以序列特异性的方式，针对蛋白激酶A的反义RIA改变了在细胞生长、分化和激活的特定阶段协调表达的基因簇的表达。定义增殖转化特征的基因下调，而定义分化逆转转化特征的基因在反义治疗的癌细胞和肿瘤中上调，但在宿主肝脏中不上调。在这种分化特征中，显示出最高诱导性的基因包括G蛋白Rap1和CDc42的基因。外源反义寡核苷酸诱导的表达特征与内源性反义基因过表达诱导的表达特征显著重叠。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. Using DNA microarrays, we have conducted a systematic charaterization of gene expression in cells exposed to antisense, either exogenously or endogenously. We have found that in a sequence-specific manner, antisense targeted to protein kinase A RIa alters expression of the clusters of coordinately expressed genes at a specific stage of cell growth, differentiation, and activation. The genes that define the proliferation-transformation signature are down-regulated, whereas those that define the differentiation-reverse transformation signature are up-regulated in antisense-treated cancer cells and tumors, but not in host-livers. In this differentiation signature, the genes showing the highest induction include genes for the G proteins Rap1 and Cdc42. The expression signature induced by the exogenously supplied antisense oligodeoxynucleotide overlaps strikingly with that induced by endogenous antisense gene overexpression. 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.