Determination of transcription start-site of CXCL14/BRAK in squamous cell carcinoma

Objectives: CXCL14/BRAK, a non-ELR motif chemokine, is highly expressed in all normal cells, but is not expressed or expressed negligible level in the most of head and neck squamous cell carcinomas (HNSCC) examined. We have reported that tumor suppressing chemokine BRAK was significantly down regulated by epidermal growth factor receptor (EGFR) signaling (Ozawa et al., Biochem. Biophys. Res. Commun. 348: 406-412, 2006). However, the mechanisms by which the gene is regulated are still unclear. So, for the first step to elucidate the mechanisms regulating BRAK gene expression, we first determined the transcriptional start site and promoter region of the gene.

Methods: HSC-3 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum. In order to determine promoter region of the gene, we constructed vectors containing presumptive promoter regions for the BRAK gene connected to the firefly luciferase gene and introduced them into HSC-3 cells. Promoter activities were determined by Dual-GloTM Luciferase Assay System (Promega). For determination of the transcriptional start-site, the 5'RACE method was employed by use of 5'-RACE CORE SET (TAKARA).

Results: We could not find any promoter activity in the 3,000 base pairs of the region upstream of the previously reported transcriptional start-site of the gene. By further screening for the promoter region, we did find the promoter activity in the exon-1 region of the afore-mentioned gene. We also identified the transcriptional start-site in this exon-1 region and found it to be located in the downstream of the promoter region.

Conclusions: We identified the new promoter region of the tumor suppressing BRAK gene to be located in the previously reported exon 1 and the transcription start site to be located downstream of this newly identified promoter.