The Role of HTm4 in Haematopoietic Cell Differentiation

• Main Authors: Xiu-Wen Huang, 黃琇雯
• Other Authors: Jon, J.L. Ko
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/63890882355520980850

碩士 === 國立陽明大學 === 生物藥學研究所 === 97 === HTm4 is a hematopoietic-specific cell-cycle regulator. Its expression in hematopoietic cells is highly regulated. In the cascade of hematopoietic differentiation, HTm4 is detected consistently in B cell lineages with CD19+ phenotype, in some subpopulations but not all of myelocytic lineages with CD11b+ phenotype, and none in cells with CD3+ marker, which is considered T cell lineage specific. To study the expression and influences of HTm4 in the differentiation of hematopoietic cells, we set out to establish murine embryonic stem (ES) cells with either HTm4+/EGFP or HTm4Floxed/EGFP phenotype. The former will aid us in the detection of HTm4, as signified by the co-expression of EGFP, expression in all hematopoietic cells derived from ES cells under induction of differentiation. In this study, we established haploid ES cell (HTm4+/EGFP), at a frequency of 1/300 ES clones analyzed. The ES cell (HTm4+/EGFP) had been induced to differentiate along the B cell lineage, and the expression of HTm4 examined simultaneously with that of CD19. So far, we found that the expression of HTm4 may not in all CD19+ B cell population, in contrast to our previous observation derived from the differentiation of human hematopoistic stem cells. Work also in progress is the procurement of ES (HTm4Floxed/EGFP) cells. It is established that the overexpression or reduction of HTm4 would inhibit the cell cycle progression, though the point of this blockade is still unknown. To ascertain the stated point of blockade, we analyzed HTm4-knockdown K562 cells synchronized by either serum starvation or thymidine treatment. Cells synchronized by serum starvation, G0 phase blockade, failed to show any effect of HTm4 knockdown, while that with thymidine, G1-S phase blockade, showed prominent cell-cycle delay in HTm4 knockdown cells. This delay lasted for about 24 hours, in comparision to that of the control and parental line. It seems that HTm4 is functioning at G1/S transition, and probably, after the point of blockade by thymidine. Since p27Kip1 interacts with cdk2 and cyclin E that is also functioning in the G1/S transit, it likely coordinates with HTm4 in the regulation of cell cycle progression at G1/S transition. The reduction of HTm4 might enhance the interaction between p27Kip1 and CyclinE-CDK2 complexes, and subsequently delay cell-cycle progression. We have tried to acertain if the amount of p27Kip1 in the cyclinE-CDK 2 is higher in HTm4 knockdown cell than that of the control and parental line through fractionation and immunoprecipitation. Thus far, the data are inconsistent. Our alternative approach is to evaluate HTm4 knockdown effect in p27Kip1 knockdown cells. Cell-cycle arrest is known to affect erythrocytic differentiation. We set out to evaluate if the cell-cycle retardation caused by HTm4 knockdown also pertinent to erythrocytic differentiation. Employing Ara-C (1 μM) induction system, we showed that at 48 to 96 hr post induction HTm4 knockdown cells gave rise to more erythrocytes in late to terminal differentiation stage than that of the control and partental line, although the overall efficacy for the induction of erythroid differentiation remained the same for all groups. Our data suggested that under normal condition, HTm4 might participate in the temporal regulation of erythroid differentiation.