| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
Megakaryocytes are unique cells, developing a polyploid DNA content regularly during the normal life cycle of the cell. Understanding the true nature of megakaryocytic polyploidization and the mechanisms that control polyploidization in megakaryocytes have been hampered partly because we have not had a suitable model system for resolving these issues. To investigate these issues, we have established a novel factor-dependent human leukemia cell line YMP91.The proliferation of YMP91 depends on both signals from c-kit and gp130.Four independent subclones of YMP91, designated YMP91-A, -B, -C, and -D have been established by limiting dilution method in the presence of SCF and IL-6/sIL-6R. The cells in the cytospin smear of YMP91-C are heterogeneous in size, and large polyploid cells can be observed in 10 - 20 %, whereas the cells of YMP91-A are homogeneous in size and there are no multinucleated cells. Platelet peroxidase activity examined by electron microscopic analysis of YMP91-A is negative, but that of YMP91-C is positive along with abundant cytoplasmic fine structures, suggesting these sublines show distinct phenotypes in respect of megakaryocytic maturation. YMP91 and its sublines may thus be expected to serve a model system for clarifying the mechanisms regulating the megakaryopoiesis. We have hypothesized that the difference of phenotypes in terms of megakaryocytic maturation in each subline should be caused from the changes in gene expression. To identify the changes in gene expression in these sublines, we have utilized a CDNA microarray system to analyze the expression of over 2,304 genes. Comparison of YMP91-A and - C revealed 53 genes that showed significant difference in the expression level, and that of YMP91-A and -D did 23 genes. These candidates that might play an important role in megakaryocytopoiesis are under investigation with Northern blot analysis for quantitative evaluation and some of them are also being functionally evaluated.
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