| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 96 === HTm4 is a hematopoietic cell cycle regulator that is expressed mainly in the hematopoietic tissues. Our previous data showed that the expression of HTm4 is controlled in a cell differentiation stage- and cell type-dependent fashion. The ectopic expression of HTm4 in U937 entailed a prominent cell cycle arrest at G0/G1. This blockade was attributed likely to the enhanced dephosphorylation of CDK2 by KAP (CDK associated phosphatase) upon the binding of HTm4 to the latter. Additionally, in vitro induction experiments by others have determined that the blockade of cell cycle progress at G1 phase is mandatory for a hematopoietic progenitor cell undertaking the differentiation pathway. Hence, we set out to study if HTm4 participates in the differentiation of hematopoietic progenitor cells.
We first established HTm4 knockdown K562 clones as the target model for our study. Two clones (siHTm4 #5 and #9, with knockdown efficiency at 80 % and 88 %, respectively) were used, together with a siLuc #9 and a parental K562 as the controls. The proliferation rate assay using trypan blue staining method and cell cycle synchronization using double thymidine blockade method illustrated that the knockdown of HTm4 protracted the progression of cell cycle in the targeted K562. Upon the induction of erythrocytic differentiation employing Ara-C (1 μM), the knockdown clones showed more differentiated cells than that of the control group.
Megakaryocytic differentiation induced by PMA treatment for 96 hours showed that some HTm4 knockdown cells with the DNA content rising up to 16N ploidy level, whereas that of control cells did not increase beyond 16N. Pertinent to the ploidy studies, the HTm4 knockdown clones were determined to be more mature cells than that of the control group in the cascade of megakaryocytic differentiation determined after Wright’s stain treatment.
Regarding to differentiation, the retardation of cell cycle progress manifested by HTm4-knockdown seemed to have thrust the progression of terminal differentiation in K562 cells. This observation is in parallel with the consensus notion that the ability of Ara-C or PMA in the induction of terminal differentiation in hematopoietic progenitor cells is partly due to its inhibitory effect on cell cycle progression in the target cells.
Here, we demonstrated that HTm4-knockdown protracted cell cycle progression, while previous publications showed this effect could also be achieved by the ectopic expression of HTm4. It is likely that the reduction of HTm4 protein might have allowed more binding of p27, a more potent inhibitor, to CDK2, and hence the enhanced retardation of cell cycle progression, while the same effect incurred by over-expression of HTm4 was through its enhanced interaction with KAP and consequently the dephosphorylation of CDK2.
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