| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2000: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1998: ¥1,500,000 (Direct Cost: ¥1,500,000)
|
|---|
| Research Abstract |
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
|
