|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1998 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1997 : ¥2,400,000 (Direct Cost : ¥2,400,000)
To select the suitable cells for promoter assay, expression of RNA arid protein of squamous cell carcinoma antigen (SCCA) 2 was studied in 40 human cell lines including cervical, skin and other squamous cell carcinoma cell lines, and endometrial, ovarian and other adenocarcinoma cell lines, and normal keratiocytes. ELIZA was used to detect SCCA protein expression in supernatant of each cell. This ELIZA did not differentiate SCCA1 and SCCA2 because of high homology of these 2 proteins. RT-PCR was used to detect SCCA2 RNA expression in each cell. Northern blot analysis was not used because of high homology (98%) of DNAs between SCCA1 and SCCA2. SCCA protein was detested in the supernatant of each squamous cell carcinoma cell line ; 18 ng/ml in SKG llla cells, 1 ng/ml in normal human keratinocyte, NHK, and <0.01 ng/ml in all adenocarcinoma cells. DNA-PCR analysis showed the clear bands of cDNAs of SCCA 1 and SCCA2 with high specificity from 1O^2 to 10^9 copies. RT-PCR analysis demonstrated the results which was well correlated with those of the ELIZA and adenocarcinoma cells did not show any bands. Transcription start site was 9bp upstream from the first codon, which was determined by SMART system. Deletion assay demonstrated the high promoter activity in the 250-500 bp upstream from the first codon. This promoter activity is the most in the SKG llla cells, 10% of that of SKGllla in the NHK, and absent in the SKOV3 cells. Promoter activity of SCCA2 is highly specific in the squamous cell carcinoma and promissing for the tissue specific gene therapy for squamous cell carcinoma.
deletion assayによりプロモーター活性は250-500bpのところに最も高いことが認められ、この活性はELIZA、RT-PCRとほぼ同様にSKG IIIaに最も高く、NHKではその10%程度であり、SKOV3には認められなかった。以上により、SCCA2のプロモーター活性は転写開始領域より上流250-500bpに最も高く扁平上皮癌に組織特異的であることが判明した。