A new method for maturity-dependent fractionation of neutrophil progenitors applicable for the study of myelodysplastic syndromes

We applied our new method, maturity-dependent fractionation of bone marrow-derived neutrophil progenitors, to a study of gene expression profiles during granulopoiesis in myelodysplastic syndromes. CD34(+) cells with low density [F1], CD11b(-)/CD16(-) [F2], CD11b(+)/CD16(-) [F3] and CD11b(+)/CD16(low) [F4] with intermediate density, CD11b(+)/CD16(int) [F5] and CD11b(+)/CD16(high) [F6] with high density were isolated from six patients. Although AML1 and C/EBP-ϵ mRNA peaked at F1 and F4, respectively, in healthy individuals, C/EBP-ϵ was maximized at F2/F3 in all patients, two of whom showed simultaneous peaks of AML1 at F2. Thus, this fractionation is useful to detect mistimed induction of granulopoiesis-regulating genes in myelodysplastic syndromes

Membrane type 1-matrix metalloproteinase/Akt signaling axis modulates TNF-α-induced procoagulant activity and apoptosis in endothelial cells.

Membrane type 1-matrix metalloproteinase (MT1-MMP) functions as a signaling molecule in addition to a proteolytic enzyme. Our hypothesis was that MT1-MMP cooperates with protein kinase B (Akt) in tumor necrosis factor (TNF)-α-induced signaling pathways of vascular responses, including tissue factor (TF) procoagulant activity and endothelial apoptosis, in cultured human aortic endothelial cells (ECs). TNF-α (10 ng/mL) induced a decrease in Akt phosphorylation within 60 minutes in ECs. A chemical inhibitor of MMP, TIMP-2 and selective small interfering RNA (siRNA)-mediated suppression of MT1-MMP reversed TNF-α-triggered transient decrease of Akt phosphorylation within 60 minutes, suggesting that MT1-MMP may be a key regulator of Akt phosphorylation in TNF-α-stimulated ECs. In the downstream events, TNF-α increased TF antigen and activity, and suppressed the expression of thrombomodulin (TM) antigen. Inhibition of Akt markedly enhanced TNF-α-induced expression of TF antigen and activity, and further reduced the expression of TM antigen. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-α. Moreover, TNF-α induced apoptosis of ECs through Akt- and forkhead box protein O1 (FoxO1)-dependent signaling pathway and nuclear factor-kB (NF-kB) activation. Knockdown of MT1-MMP by siRNA reversed apoptosis of ECs by inhibiting TNF-α-induced Akt-dependent regulation of FoxO1 in TNF-α-stimulated ECs. Immunoprecipitation demonstrated that TNF-α induced the changes in the associations between the cytoplasmic fraction of MT1-MMP and Akt in ECs. In conclusion, we show new evidence that MT1-MMP/Akt signaling axis is a key modifier for TNF-α-induced signaling pathways for modulation of procoagulant activity and apoptosis of ECs

Phase II Study of Dasatinib safety and efficancy for CP-CML at Tohoku

第73回日本血液学会学術集

The role of HMGA2 in the proliferation and expansion of a hematopoietic cell in myeloproliferative neoplasms

Philadelphia chromosome-negative myeloproliferative neoplasms (MPN), which include polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by clonal proliferative hematopoiesis with increased blood cell count. Clonal expansion mechanisms in MPN and related disorders such as myelodysplastic syndromes (MDS) remain to be elucidated. Although mutations in the JAK2 gene lead to a proliferative hematopoiesis in majority of MPN and some MDS, the mutation alone does not cause a clonal expansion. In addition to JAK2 mutations, several genetic abnormalities, including TET2 and polycomb group genes involving epigenetic regulation have been reported in patients with MPN. Moreover, overexpression of HMGA2 due to removal of specific sites in its 3' untranslated region for regulatory let-7 micro RNAs may contribute to the proliferative hematopoiesis with conferring a growth advantage at the level of a hematopoietic stem cell in some cases with MPN

Impairment of FOS mRNA stabilization following translation arrest in granulocytes from myelodysplastic syndrome patients.

Although quantitative and qualitative granulocyte defects have been described in myelodysplastic syndromes (MDS), the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. We recently found that FOS mRNA elevation under translation-inhibiting stimuli was significantly smaller in granulocytes from MDS patients than in healthy individuals. The aim of this study is to clarify the cause of the impaired FOS induction in MDS. We first examined the mechanisms of FOS mRNA elevation using granulocytes from healthy donors cultured with the translation inhibitor emetine. Emetine increased both transcription and mRNA stability of FOS. p38 MAPK inhibition abolished the emetine-induced increase of FOS transcription but did not affect FOS mRNA stabilization. The binding of an AU-rich element (ARE)-binding protein HuR to FOS mRNA containing an ARE in 3'UTR was increased by emetine, and the knockdown of HuR reduced the FOS mRNA stabilizing effect of emetine. We next compared the emetine-induced transcription and mRNA stabilization of FOS between MDS patients and healthy controls. Increased rates of FOS transcription by emetine were similar in MDS and controls. In the absence of emetine, FOS mRNA decayed to nearly 17% of initial levels in 45 min in both groups. In the presence of emetine, however, 76.7±19.8% of FOS mRNA remained after 45 min in healthy controls, versus 37.9±25.5% in MDS (P<0.01). To our knowledge, this is the first report demonstrating attenuation of stress-induced FOS mRNA stabilization in MDS granulocytes

Schematic diagram describing the mechanisms of MT1-MMP/Akt signaling axis in TNF-α-dependent procoagulant activity and apoptosis of ECs.

<p>A crucial role of MT1-MMP in Akt-dependednt signaling pathways in TNF-α stimulation. MT1-MMP in the cytoplasm of ECs forms a complex with Akt in the intracellular signaling pathways in TNF-α-stimulated ECs. The interaction between MT1-MMP and Akt regulates TNF-α-induced changes in TF and TM expression in ECs and contributes to endothelial apoptosis through FoxO1 phosphorylation as well as caspase-3 activation.</p