1998 Fiscal Year Final Research Report Summary
Basic study of a novel anti-leukemic agent, G-CSF-Pseudomonas exotoxin fusion protein
| Project/Area Number |
09557082
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Hematology
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| Research Institution | The University of Tokyo |
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Principal Investigator |
TOJO Arinobu The University of Tokyo, Institute of Medical Science, Lecturer, 医科学研究所, 講師 (00211681)
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| Co-Investigator(Kenkyū-buntansha) |
ASANO Shigetaka The University of Tokyo, Institute of Medical Science, Professor, 医科学研究所, 教授 (50134614)
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| Project Period (FY) |
1997 – 1998
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| Keywords | G-CSF / Pseudomonas exotoxin / leukemia |
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| Research Abstract |
A chimeric toxin in which the cell surface binding domain of Pseudomonas exotoxin was replaced with mature human granulocyte colony-stimulating factor(G-CSF)was produced in. Escherichia coli, partially purified and tested for its biological activity on a G-CSF-dependent murine myeloid leukemia cell line, NFS60. This fusion protein, termed as G-CSF-PE40, can displace[^<125>I]G-CSF binding to its receptor. After 48 hrs' incubation in the presence of G-CSF, G-CSF-PE40 inhibited protein synthesis and revealed cytotoxicity in NFS60 cells in a concentration dependent manner. The half maximal dose of G-CSF-PE40 for protein synthesis inhibition(ID_<50>)in NFS60 cells was estimated at around 100 pM.Additionally, relatively low concentration of G-CSF-PE40 stimulated DNA synthesis in NFS60 cells after 16 hrs' incubation in the absence of G-CSF, suggesting that G-CSF-PE40 also can transduce a transient mitogenic signal. Next, we placed the signal sequences derived from bacterial alkaline phosphata
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se[phoA]in front of G-CSF, resulting in marked improvement in production of the chimeric toxin, which was secreted into the periplasmic fraction and could be purified without denaturation-refolding steps. Purified G-CSF-toxin was applied to test its activity in normal and leukemic mice. C57/BL6 mice were intraperitoneally injected with about 100 ng of G-CSF-toxin for three consecutive days. These mice showed profound decrease in the number of peripheral neutrophils by the last injection without significant influences on other types of blood cells, but they recovered from neutropenia within 1 week after the last injection. Next, we challenged young-adult female SJLJ mice which were intravenously injected with 10^6 L103 syngeneic myeloid leukemia cells and would die of leukemia within 3 weeks(median : Day 15, n=4). When mice inoculated with L103 cells were simulataneously treated with a single injection of G-CSF-toxin, the survival periods of mice were markedly prolonged(median : Day 30, n=5). These results have encouraged us to apply G-CSF-PE40 to the future treatment of myeloid leukemias. Less
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