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

原胃泌素释放肽（Progastrin-releasing peptide, proGRP）和GRP受体（GRPR）是调节呼吸道上皮细胞在正常和疾病状态下分化和增殖的关键系统之一。由于已知尼古丁可上调或下调气道上皮细胞中多个基因的表达水平，我们首先探索了尼古丁对肺上皮中proGRP和GRPR基因表达的分子效应。暴露于1mm尼古丁可上调产grp的H128小细胞肺癌（SCLC）细胞的ProGRP基因表达。为了进一步表征调节机制，将proGRP基因的5'侧区与荧光素酶报告基因连接，并转染到SBC-5 SCLC细胞中。当细胞转染含有上游sclc特异性增强子元件的质粒并受到尼古丁刺激时，观察到更高的启动子活性。尼古丁在HS-24鳞状细胞肺癌细胞、H128细胞和A549腺癌细胞中也呈剂量依赖性上调GRPR基因表达水平。此外，尼古丁暴露也引起GRPR基因5'侧区域启动子活性的增加。接下来，在获得知情同意后，通过免疫组织化学染色评估SCLC患者血清proGRP（31-98）水平，SCLC肿瘤组织中proGRP和GRPR mRNA的表达，proGRP转录物的选择性剪接比例以及肿瘤中GRP蛋白的产生。ProGRP mRNA仅在血清ProGRP水平高的个体恢复的肿瘤组织中检测到。每种病例中mRNA亚型的比例几乎相同，显示I型和III型占主导地位，而II型较少。GRP蛋白的产生仅在血清中显示高proGRP水平的个体的肿瘤中得到证实。相比之下，GRPR转录物仅在5个表达progrp的肿瘤中的2个肿瘤细胞中检测到。最后，由于SCLC是一种预后极差的肺癌，需要探索新的治疗方法。在这种情况下，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细胞中实现了充分的细胞类型特异性转基因表达。通过将含有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（由一对loxP序列分隔）共同感染，在表达proGRP的SBC-5细胞中实现了SCLC细胞特异性lacZ基因的表达。此外，通过另一种Ad载体（AxCALNLhBax-alpha）与AdGRPCre一起转移Bax基因，在体外完成了SCLC细胞特异性诱导凋亡。在体内，通过接种SBC-5细胞并同时感染AdGRPCre和AxCALNLhBax-alpha，抑制裸鼠肿瘤生长，也证实了抗肿瘤作用。这些观察结果提示了proGRP和GRPR之间重要的分子相互作用，以及转基因在人类SCLC肿瘤中细胞特异性表达的潜在治疗优势。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