| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1999: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
We had studied the basic research at the view points for prodrug gene therapy. Among the anti-cancer drugs for gynecologic tumors, now we have studied CPT-11 (as a pro-drug), carboxylesterase (as an activator for CPT-11), and the cancer cells' up-sensitibity to prodrug CPT-11 through the insersion of this enzyme's molecular gene.
By Potter et al. reported that enzyme activity of Rabbit carboxylhydrate was seven times superior to Human carboxylhydrate. Using the RT-PCR methods, we had picked up the gene clone of 1.7 KB Rabbit carboxylesterase c-DNA from Rabbit liver, and also done the gene clone of 1.7 KB Human carboxylesterase c-DNA from human leukemia cell HEL.There after, to compare the activities among these enzymes, we made three chimeras by some combining those cDNAs, and put those into the pCMV-tag2a mammalian expression vectors that had frag-tag sequence at the N-terminals. Five kinds of expression vectors were introduced into gynecological tumor cells. Transiently every vectors were introduced into 293T cell line. After forty hours, every carboxylesterase activities were determined. Introduced two cell lines showed higher enzyme activity.
At last, using Tet-off system, Rabbit carboxylesterase c-DNA inserted into expression vector, was introduced into ovarian cancer cell line SKOV3, and established two cell lines of hishly expression carboxylhydrate.
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