| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Shunji Dept. of Clinical Oncology, Cancer Chemo. Ctr., JAPANESE FOUNDATION FOR CANCER RESEARCH, Staff, 癌化学療法センター・臨床部, 研究員 (90221358)
ITO Yoshinori Dept. of Clinical Oncology, Cancer Chemo. Ctr., JAPANESE FOUNDATION FOR CANCER RESEARCH, Staff, 癌化学療法センター・臨床部, 研究員 (00261131)
HORIKOSHI Noboru Dept. of Clinical Oncology, Cancer Chemo. Ctr., JAPANESE FOUNDATION FOR CANCER RESEARCH, Director, 癌化学療法センター・臨床部, 部長 (00085648)
MIZUNUMA Nobuyuki Dept. of Clinical Oncology, Cancer Chemo. Ctr., JAPANESE FOUNDATION FOR CANCER RESEARCH, Staff, 癌化学療法センター・臨床部, 研究員 (40311229)
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| Research Abstract |
1)Binding of thymidylate synthase(TS) protein and TSmRNA
The repressive effect of TSmRNA translation is mediated by direct binding of TS itself to its own mRNA.We synthesized a completely degenerate, linear RNA pool of nucleotides sequences in vitro and isolated a high affinity RNA ligand that bind directly human TS protein through in vitro steps of selection and amplification. This single RNA molecule had binding affinity to TS protein having 20-fold greater than wild-type TS RNA sequences. Structure analysis of this RNA sequence revealed that it had a stem-loop structure having UGU loop element within the RNA sequence. Deletion and/or modification of this specific element within this RNA sequence seemed to be very critical in binding to TS protein.
2)Thymidylate synthase protein has a function as a RNA binding protein
In addition to TS's role in enzyme catalysis, there is evidence that TS also functions as an RNA binding protein. The translation of human TS mRNA is regulated by its own
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protein product via a negative autoregulatory mechanism. RNA gel shift assays revealed a specific interaction between TS protein and the protein-coding region of p53mRNA and in vitro translation studies demonstrated that this interacion resulted in the specific repression of p53mRNA translation. Western immunoblot analysis demonstrated that undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 cell line compared with that in the parent cell line. These findings suggest that TS regulates the expression of p53 at the translational level.
3)An improved schedule of 5-FU administration
TS is negatively autoregulated at the level of TSmRNA translation. To avoid the synthesis of new protein, we have developed a new improved administration schedule with 5-FU giving in a "on and off" manner. In vitro experiments revealed that every 12 hours "on and off" schedule over 96 hrs was nealy identical compared with 96 hrs contunuous exposure schedule. TS inhibition was maintained similarly up to 90% with both methods. Clinical pilot study using 5-FU 750-850mg/m^2 ON/OFF therapy revealed that one PR and seven NCs were obtained with decreased toxicity in patients with advanced gastrointestinal cancer resistant to prior 5-FU therapy. Less
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