| Project/Area Number |
11671624
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Ehime University |
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Principal Investigator |
HAMADA Katsuyuki Ehime University Faculty of Medicine, University Hosupital Assistant Professor, 医学部・附属病院, 講師 (90172973)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | ovarian cancer / promoter / gene therapy / tissue specificity / CA125 / transcription regulation / IAI3B / tumor marker / 転写因子 / 腺癌 / エンハンサー |
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| Research Abstract |
Genomic DNA was cloned from EMBL SP6/T7 phage library using 500 bp of 5' end of CA125 gene and sequenced 5 kb upstream of exon 1 of CA125 genomic DNA.Transcriptional start site was determined by SMART PCR cDNA Synthesis kit. Exon 1 is devided into exon 1A and exon 1B.It is reported that transcriptional start site of normal lymphocite is exon 1A.However, this study demonstrated that transcriptional start site of ovarian cancer cell line was exon 1B.Deletion mutant was constructed by using PCR, in which antisense primer was contructed including transcriptional start site of exon 1B.Sense primer was setted in every 250-500 bp size 5' upstream of promoter region. Luciferase assay was determined by dual luciferase assay kit (Promega). Significant transcriptional activity was demonstrated in 500 bp upstream of transctiprional start site. Enhancer activity was shown in 1825 bp uptream of transctiprional start site. Ovarian carcinoma HEY cell line, cervical carcinoma SKGIIIa cell lilne and normal human keratinocyte K42 cell line were used to determine the tissue specificity in the transcriptional activity of CA125 gene. The highest transcriptional activity of CA125 gene was shown in 1825 bp upstream of transcriptional start site. The transcriptional activity of CA125 gene was 7 times that of SV40 gene in HEY cells, 1.9 times that of SV40 gene in SKGIIIa and 1.8 times that of SV40 gene in K42 cells. These results shows the tissue specific activity of CA125 gene in ovarian carcinoma cells. Then we inserted the 1875 bp of CA125 gene into E1A promoter region of adenovirus to make a conditioned replicative adenovirus. This vector showed the high tissue specific anti-proliferative activity in ovarican carcinoma cells, because IC50 of this vector was 0.01 MOI in HEY cells and 100 MOI in SKGIIIa cells. From these results, gene therapy by using CA125 gene promoter may be tissue specific for ovarian carcinoma and promissing in the clinical trial of gene therapy of ovarian cancer.
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