Molecular analysis of modulation for expression of pro-gastrin releasing peptide and gastrin releasing peptide receptor genes in lung epithelial cells

前列腺素释放肽(ProGRP)及其受体(GRPR)是调节正常和疾病状态下呼吸道上皮细胞分化和增殖的关键系统之一。由于尼古丁可上调或下调多种基因在呼吸道上皮细胞中的表达水平，我们首先探讨了尼古丁对肺上皮细胞proGRP和GRPR基因表达的分子影响。1 mM尼古丁可上调产生GRP的H128小细胞肺癌(SCLC)细胞的ProGRP基因表达。为了进一步研究其调控机制，将proGRP基因的5‘侧翼区与荧光素酶报告基因连接，并将其导入SBC-5小细胞肺癌细胞。当含有上游SCLC特异性增强子元件的质粒被导入细胞并被尼古丁刺激时，观察到更高的启动子活性。尼古丁还上调了HS-24肺鳞状细胞…中grpr基因的表达水平更多的癌细胞、H128细胞或A549腺癌细胞呈剂量依赖关系。此外，尼古丁暴露还导致GRPR基因5‘侧翼区启动子活性增加。接下来，在获得知情同意后，评估小细胞肺癌患者的血清proGRP(31-98)水平，肿瘤组织中proGRP和GRPR mRNA的表达，可选择性剪接的proGRP转录本的比例，以及免疫组织化学染色检测肿瘤中GRP蛋白的产生。只有在血清proGRP水平高的个体恢复的肿瘤组织中才能检测到proGRP mRNA。各亚型的比例基本相同，以I型和III型为主，II型较少。GRP蛋白的产生仅在血清前GRP水平高的个体的肿瘤中被证实。相反，GRPR转录本只在五个表达GRP原的肿瘤中的两个肿瘤细胞中检测到。最后，由于小细胞肺癌是一种预后极差的肺癌，需要探索新的治疗方法。在此背景下，使用腺病毒(Ad)介导的Cre/loxP系统，通过使用proGRP基因启动子在产生proGRP的SCLC细胞中获得足够的细胞类型特异性的转基因表达。将带有proGRP启动子的表达Cre的Ad载体(AdGRPCre)和含有CAG启动子的LacZ表达载体(AdGRPCre)共感染SBC-5细胞，并用一对loxP序列隔开，获得了SCLC细胞特异性LacZ基因的表达。此外，在体外用另一个Ad载体(AxCALNLhBax-α)和AdGRPCre一起转移的Bax基因完成了对SCLC细胞的特异性诱导凋亡。接种SBC-5细胞和AdGRPCre和AxCALNLhBax-α共感染裸鼠的肿瘤生长抑制实验也证实了抗肿瘤作用。这些观察结果表明，proGRP和GRPR之间存在重要的分子相互作用，并且转基因的细胞特异性表达对人小细胞肺癌具有潜在的治疗优势。较少

Progastrin-releasing peptide (proGRP) and GRP receptor (GRPR) are one of the key systems regulating respiratory epithelial cell differentiation and proliferation in the normal and disease conditions. Because nicotine is known to up- or downregulate the levels of expression of multiple genes in airway epithelial cells, we first explored the molecular effects of nicotine on expression of genes for proGRP and GRPR in the lung epithelium. ProGRP gene expression was upregulated by exposure to 1 mM nicotine in GRP-producing H128 small cell lung cancer (SCLC) cells. To further characterize the modulating mechanisms, the 5' flanking regions of the proGRP gene were linked to the luciferase reporter gene and transfected into SBC-5 SCLC cells. Higher promoter activity was observed when cells were transfected with the plasmid containing the upstream SCLC-specific enhancer element and stimulated with nicotine. Nicotine also upregulated the levels of GRPR gene expression in HS-24 squamous cell lung … More cancer cells, H128 cells, or A549 adenocarcinoma cells in a dose-dependent manner. In addition, nicotine exposure caused increase in promoter activity of the 5'-flanking region of the GRPR gene as well. Next, individuals with SCLC were evaluated, after obtaining informed consent, for the level of serum proGRP (31-98) measured by ELISA, expression of both proGRP and GRPR mRNA in SCLC tumor tissues, the proportions of alternatively spliced proGRP transcripts, and production of GRP protein in tumors by using immunohistochemical staining. ProGRP mRNA could be detected only in tumor tissues recovered from individuals with high serum proGRP levels. The proportions of mRNA subtypes in each case were nearly the same, revealing type I and type III to be predominant, whereas type II being scarce. GRP protein production was demonstrated in tumors exclusively from individuals exhibiting high serum proGRP levels. In contrast, GRPR transcripts were detectable only in cancer cells from two of five proGRP-expressing tumors. Finally, because SCLC is lung carcinoma with extremely poor prognosis, novel therapeutic approaches need to be explored. In this context, an adenovirus (Ad)-mediated Cre/loxP system was used to achieve sufficient cell type-specific expression of transgenes in proGRP-producing SCLC cells by using the proGRP gene promoter. SCLC cell-specific lacZ gene expression was achieved in proGRP-expressing SBC-5 cells by coinfection of a Cre-expressing Ad vector with proGRP promoter (AdGRPCre) and a lacZ-expressing vector with CAG promoter separated by a pair of loxP sequences. Further, SCLC cell-specific induction of apoptosis was accomplished by using the Bax gene transferred by another Ad vector (AxCALNLhBax-alpha) along with AdGRPCre in vitro. Antitumor effect was also confirmed in vivo by suppression of tumor growth in nude mice inoculated with SBC-5 cells and coinfected with AdGRPCre and AxCALNLhBax-alpha. These observations suggest the important molecular interaction between proGRP and GRPR, and potential therapeutic advantage of the cell-specific expression of the transgene for human SCLC tumors. Less