Soon-Nam Lee

Mobilization Kinetics of CD34+ Cells in Association with Modulation of CD44 and CD31 Expression during Continuous Intravenous Administration of G‐CSF in Normal Donors

The aim of the present study is to evaluate the kinetics of CD34+ cells and investigate the potential modulation of CD44 and CD31 expression on CD34+ cells during continuous i.v. administration of G‐CSF, thus to elucidate the possible mechanism of peripheral blood progenitor cell (PBPC) mobilization. Fifteen healthy donors were enrolled in this study. G‐CSF (10 μg/kg/day) was administered for four consecutive days through continuous 24‐h i.v. infusion. For measurement of complete blood counts, CD34+ cell levels and their expression of CD44 and CD31, PB sampling was performed immediately before the administration of G‐CSF (steady‐state) and after 4, 8, 24, 48, 72, 96, and 120 h of G‐CSF administration. The percentage and absolute number of CD34+ cells significantly increased at day 3 (0.55 ± 0.09%, 51.12 ± 24.83 × 103/ml) and day 4 (0.47 ± 0.09%, 46.66 ± 24.93 × 103/ml), compared to the steady‐state level (0.06 ± 0.09%, 2.03 ± 5.69 × 103/ml). At day 3 to day 5 following the onset of G‐CSF administration, a strong decrease of CD44 and CD31 expression was observed on mobilized CD34+ cells compared to controls: the relative fluorescence intensity of CD44 and CD31 was, respectively, 50%‐70% and 40%‐90% lower than that of controls. We conclude that continuous i.v. administration of G‐CSF apparently results in more rapid mobilization of CD34+ cells, and downregulation of CD44 and CD31 on CD34+ cells is likely to be involved in the mobilization of PBPC after treatment with G‐CSF.

Distinct patterns of apoptosis in association with modulation of CD44 induced by thrombopoietin and granulocyte‐colony stimulating factor during ex vivo expansion of human cord blood CD34+ cells

The insufficient number of haemopoietic stem cells (HSCs) in cord blood (CB) is the major potential limitation to widespread use of CB for marrow replacement. Cytokine‐mediated ex vivo expansion has been proposed as a means of increasing the number of CB HSCs for transplantation. However, the biology of CB HSCs during cytokine‐mediated ex vivo expansion, such as apoptosis or expression of adhesion molecules, has not yet been elucidated. We have investigated the patterns of apoptosis and CD44 expression on human CB CD34+ cells during ex vivo expansion. CD34+ cells isolated from human CB were cultured in a stroma‐free liquid culture system with thrombopoietin (TPO), flt3‐ligand (FL), stem cell factor (SCF), and/or granulocyte‐colony stimulating factor (G‐CSF). During the culture, for up to 5 weeks, apoptosis was measured by staining with 7‐amino‐actinomycin D (7‐AAD) along with concurrent immunophenotyping of CD34 and CD44 with three‐colour flow cytometry. In the cultures with TPO, an apoptotic fraction with down‐regulated CD44 appeared from the fourth day up to the second week. G‐CSF also induced apoptosis but in a different manner; the apoptotic fraction without down‐regulation of CD44 appeared unremittingly for up to 5 weeks. FL did not induce apoptosis or down‐regulation of CD44. These findings show that apoptosis is indeed involved in the regulation of CB CD34+ cells in ex vivo expansion and the patterns of apoptosis are dependent on the type of cytokines used. The distinct patterns of apoptosis suggest different mechanisms of TPO and G‐CSF in inducing apoptosis, which still remains to be elucidated.

Regulation of megakaryocytic differentiation of K562 cells by FosB, a member of the Fos family of AP-1 transcription factors

Abstract.The regulation of megakaryocytic differentiation is poorly understood. Using K562 cells, which can partly recapitulate the process in response to phorbol 12-myristate 13-acetate (PMA), we performed microarray-based gene expression profiling to identify genes that play significant roles in megakaryopoiesis. Here, we describe the function of FosB, an AP-1 transcription factor. FosB is induced in PMA treated K562 cells in a sustained manner and forms an active AP-1 protein-DNA complex. Down-regulation of FosB with specific shRNAs inhibited the induction of CD41, a specific cell surface marker of megakaryocytes. We also show that activation of the PKC-MEK-ERK signaling pathway is required for induction of FosB and CD41. Finally, we cross-examined the microarray data in conjunction with gene function annotation data to identify additional target genes of FosB. We define 3 genes, INHBA, CD9, and ITGA2B as regulatory targets of FosB and show that CD9, in particular, is a direct target of FosB.

Rac1 regulates heat shock responses by reorganization of vimentin filaments: identification using MALDI-TOF MS.

Rac1 has been implicated in a wide variety of biological processes, including actin remodeling and various signaling cascades. Here we have examined whether Rac1 might be involved in heat shock-induced cell signaling. We found that Rat2 stable cells expressing a dominant negative Rac1 mutant, RacN17 (Rat2-RacN17), were significantly more tolerant to heat shock than control Rat2 cells, and simultaneously inhibited the activation of SAPK/JNK by heat shock compared to control Rat2 cells. However, no discernible effect was observed in typical heat shock responses including total protein synthesis and heat shock protein synthesis. To identify the proteins involved in this difference, we separated the proteins of both Rat2 and Rat2-RacN17 cell lines after heat shock using two-dimensional gel electrophoresis and identified the differentially expressed proteins by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) after in-gel trypsin digestion. Differentially expressed proteins between two cell lines were identified as vimentin. Rat2-RacN17 cells showed significant changes in vimentin as well as marked changes in vimentin reorganization by heat shock. The vimentin changes were identified as N-terminal head domain cleavage. These results suggest that Rac1 plays a pivotal role in the heat shock-induced signaling cascade by modifying intermediate vimentin filaments. Cell Death and Differentiation (2001) 8, 1093–1102