| Co-Investigator(Kenkyū-buntansha) |
KONDO Shinichi Kobe University, School of Medicine, Assistant Professor, 医学部・附属病院, 講師 (50153721)
RYO Ryukichi Kobe University, School of Medicine, Assistant Professor, 医学部・附属病院, 講師 (00159237)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1993: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1992: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Our present study was designed to clarify why the same megakaryocyte progenitor cells respond to various cytokines at different stages of magakaryocyte development. First, we analyzed the expressions of IL-3 receptor bata-subunit(IL-3R) mRNA and IL-6 receptor(IL-6R) mRNA during magakaryocyte differentiation. The molecular mechanism by which cytokine binding to its receptor exerts diverse biological responses remains unknown. To clarify the intracellular signal pathway in megakartocyte differentiation, we examined the role of ras p21 -like small GT P-binding proteins such as smg P21 and rho P21 in megakaryocyte development. In addition, we tried to establish the cell line with megakaryocyte lineage, which are unresponsive to cytokines such as leukemia inhibitory factor(LIF), IL-3, GM-CSF, and 11-6. We characterized that cell line to eluciade the mechanism by which the megakaryocytic leukemia cell proliferates and differentiates. Our results are as follows.
1. We found that IL-3 R mRNA wa
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s expressed constitutively in CMK cells and was transiently down-regulated by TPA and IL-6(megalaryocyte-differentiating agents), while the expression of IL-6 mRNA was increased by TPA in association with the differentiation of megakaryocytes. These results provides evidence that regulation of expression of cytokine receptor mRNA may be one the mechanisms for modulating the responsiveness of megakaryocyte progenitor cells to different cytokines during megakaryocyte development.
2. Both smg p21 mRNA and smg P21B mRNA were detected in CMK cells, and their levels were markedly elevated by TPA, which caused the differentiation of CMK cells into more mature megakaryocytes. The smg p21 protein molecules also increased during the TPA-induced diffrentiation of CMK cells. The mRNA level of glycoportein(GP)IIb was increased by TPA, but the time course of increase in the smg p21 mRNA levels was more rapid than that of the increase in the GP IIb mRNA level. These results imply that smg P21 may play an important role in regulating the megakaryocyte differentiation.
3. CMK cells constitutively expressed rho A and rho C mRNAs. Further, addition of C3 exoenzyme(C3) caused the inacitivation of rho protein in CMK cells and thereafter the megakaryocyte polyploidization. This result reveals that rho proteins may function as a regulator of the megakaryocyte polyploidization.
4. The new megakaryocytic leukemia cell line, designated KHI84, was established from the patient, who had the initial symptoms of swelling of the right cervical lymphnode. Surface marker analysis showed that KHI84 cells expressed GP Ib and GP IIb/IIIa antigens. However, Northern blot analysis revealed that platelet factor 4 mRNA, a mature megakaryocyte marker, was absent in KHI84 cells. In addition, neither cytokines such as LIF, IL-3, GM-CSF, and IL-6 nor TPA caused the proliferation and/or differentiation of KHI84 cells. These findings indicate that KHI85 is a useful cell line for investigating the mechanism of megakaryopoiesis. Less
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