Mechanism of cAMP-growth regulatory function

cAMP-生长调节功能的机制

• 批准号: 6761999
• 负责人: Y S CHO-CHUNG
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6761999
• 关键词: antineoplastics; biological signal transduction; cAMP response element binding protein; cell cycle; cell differentiation; cell growth regulation; cell proliferation; cellular oncology; cyclic AMP; cyclic AMP receptors; gene expression; genetic regulatory element; intracellular transport; microarray technology; molecular site; protein kinase A; tissue /cell culture

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.

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应答元件（CRE）介导的转录改变基因表达的基本模式，调节大量生理过程，包括中间代谢、细胞增殖和神经元信号传导。然而，在细胞增殖中的CRE指导的转录的机制在很大程度上是未探索的。为了阐明cAMP增强子（CRE）在细胞增殖调控中的作用，我们采用转录因子-诱饵寡核苷酸方法。我们的研究表明，由CRE序列组成的合成单链寡核苷酸，其自身杂交形成双链体/发夹，可以渗透到细胞中，与CRE增强子竞争结合转录因子，并在体内特异性干扰CRE和Ap-1定向转录。这种寡核苷酸抑制肿瘤细胞增殖，而不影响非癌细胞的生长。这些结果提示PKA在肿瘤发生中的作用可能与其对一系列基因的转录有关。高通量DNA微阵列技术的发展使得在复杂的生物系统中，可以在一次杂交中并行分析数千个基因的表达谱。利用DNA微阵列，我们进行了一个系统的charaterization基因表达的细胞暴露于反义，无论是外源性或内源性。我们已经发现，以序列特异性方式，针对蛋白激酶A RIa的反义改变了在细胞生长、分化和活化的特定阶段协同表达基因簇的表达。定义增殖-转化特征的基因被下调，而定义分化-逆转转化特征的基因在反义处理的癌细胞和肿瘤中被上调，但在宿主肝脏中没有。在这种分化特征中，显示出最高诱导的基因包括G蛋白Rap1和Cdc42的基因。外源性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明显重叠。基因谱的微阵列分析也在用CRE-decoy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，并发现迄今尚未认识到的新基因，可能参与肿瘤生长和肿瘤发生。此外，通过这种方法发现新的基因，结合遗传和生化分析，可能会揭示癌症发生的cAMP失调机制，并为药物开发和癌症治疗提供新的靶点。