ROLE OF CAMP-DEPENDENT PROTEIN KINASE IN GROWTH CONTROL

营依赖性蛋白激酶在生长控制中的作用

• 批准号: 6435167
• 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/6435167
• 关键词: analog; antineoplastics; carcinoma; cell differentiation; cell growth regulation; chemical carcinogenesis; cyclic AMP; cytotoxicity; enzyme structure; growth inhibitors; isozymes; leukemia; multidrug resistance; neoplasm /cancer pharmacology; neoplastic cell; pharmacokinetics; protein kinase A; sarcoma; tissue /cell culture; transfection /expression vector; viral carcinogenesis

Our long term goal is to understand the regulation of cell growth by cAMP-dependent protein kinase (PKA) by an in-depth investigation of the structure, function and expression of the regulatory (R) and catalytic (C) subunits of PKA. Two isoforms of PKA, PKA-I and PKA-II, share a common catalytic (C) subunit but contain distinct regulatory (R) subunits, RI and RII, respectively. It is known that expression of PKA-I and its regulatory subunit, RIa is increased in human cancer cell lines, in primary tumors, and in cells after transformation. Our studies revealed the following: (1) Use of antisense strategy: RIa antisense oligodeoxynucleotide or antisense RIa gene overexpression is used to demonstrate the sequence-specific inhibition of RIa gene expression and in vivo tumor growth inhibition. The loss of RIa by the antisense resulted in rapid increase in RIIb and PKA-IIb holoenzyme. Pulse-chase experiments demonstrated that RIIb protein increased its half-life 3-6 fold in antisense treated cells. Thus, RIIb in the holoenzyme complex is stabilized, exhibiting an increased half-life. Through this biochemical adaptation, in the antisense-treated cancer cells, the ratio of PKA-I to PKA-II changes to that similar to that of normal cells.; (2) Recombinant gene technology: Retroviral-vector mediated overexpression of wild type and point-mutated RIa, RIIa, RIIb and C subunits of PKA to demonstrate distinctive roles of the R subunit isoforms in cell growth, differentiation, and reverse transformation; and (3) Discovery of extracellular PKA (ECPKA):the free catalytic (C) subunit of PKA is excreted in conditioned medium of various cancer cells, and in serum of cancer patients; the ECPKA expression was upregulated 10-fold as compared with normal serum. Importantly, this study provided the means of modulating the ECPKA expression by changing expression of the intracellular PKA-I and PKA-II. Namely, (1) overexpression of RIa in expression vector, which upregulates intracellular PKA-I, can markedly upregulate ECPKA expression; (2) overexpression of RIIb, which downregulates PKA-I in the cell and reverts the transformed phenotype, downregulates ECPKA; and (3) A mutation in the Ca gene that prevents myristylation allows the intracellular PKA upregulation but blocks the ECPKA increase, suggesting that the N-terminal myristyl group of Ca is required for ECPKA expression. It was further shown that ECPKA upregulation is reduced in cancer cells maintaining hormone-dependency (a normal cell property), such as hormone-dependent breast cancer as compared to the hormone-independent breast cancer. These observations indicate that this phenomenon of ECPKA expression can provide innovative approach to cancer diagnosis, prognosis, and hormone-dependence detection (breast cancer).

我们的长期目标是通过对PKA的调节（R）和催化（C）亚基的结构，功能和表达进行深入研究，了解CAMP依赖性蛋白激酶（PKA）对细胞生长的调节。 PKA，PKA-I和PKA-II的两个同工型共享一个共同的催化（C）亚基，但分别包含不同的调节（R）亚基，RI和RII。 众所周知，PKA-I及其调节亚基的表达在人类癌细胞系，原发性肿瘤和转化后细胞中增加。 我们的研究揭示了以下内容：（1）使用反义策略：RIA反义寡脱氧核苷酸或反义RIA基因过表达用于证明对RIA基因表达和体内肿瘤生长抑制的序列特异性抑制作用。反义因RIA的损失导致RIIB和PKA-IIB Holoenzyme迅速增加。 脉搏练习实验表明，RIIB蛋白在反义处理的细胞中增加了其半衰期3-6倍。 因此，全酶复合物中的RIIB稳定，表现出增加的半衰期。 通过这种生化适应，在反义治疗的癌细胞中，PKA-I与PKA-II的比率变化与正常细胞相似的比率。 （2）重组基因技术：逆转录病毒载体介导的野生型和点突变的RIA，RIIA，RIIB和C亚基的过表达，以表现出R亚亚元素在细胞生长，分化和反向转化中的独特作用； （3）发现细胞外PKA（ECPKA）：PKA的游离催化（C）亚基在各种癌细胞的条件培养基和癌症患者的血清中排泄； 与正常血清相比，ECPKA表达上调了10倍。 重要的是，这项研究提供了通过改变细胞内PKA-I和PKA-II的表达来调节ECPKA表达的手段。 也就是说，（1）上调细胞内PKA-I的表达载体中RIA的过表达可以显着上调ECPKA的表达； （2）RIIB的过表达，它下调了细胞中的PKA-I并恢复转化的表型，下调ECPKA； （3）CA基因中防止肉豆蔻酰化的突变允许细胞内PKA上调，但阻止了ECPKA的增加，这表明Ca的N末端肉豆蔻基是ECPKA表达所必需的。 进一步表明，与非激素依赖性乳腺癌相比，维持激素依赖性（正常细胞特性）的癌细胞中ECPKA上调降低了，例如激素依赖性乳腺癌。 这些观察结果表明，这种ECPKA表达现象可以为癌症诊断，预后和激素依赖性检测提供创新的方法（乳腺癌）。