Different kinases acting on stathmin (oncoprotein, Op18) in human healthy and malignant hematopoietic stem cells

17527 Background: Stathmin/Op18 is a cytosolic phosphoprotein which regulates the dynamics of microtubules. This regulation is important in mitosis during cell division and in the migration of cells in modification of the cytoskeleton. The process of tumor proliferation and metastasis is characterized by high rates of mitosis and migration into distant tissues. Stathmin itself is regulated by kinases through phosphorylation of mainly 4 different serin sides. In this study, we investigated stathmin- and its kinases expression in native hematopoietic CD34+ stem cells (HSCs) from bone marrow (BM) in comparison to mobilized peripheral blood stem cells (mPBSCs) from G-CSF stimulated donors and leukemic CD34+ cells from patients with AML. Methods: Mononuclear cells were isolated by a standard Ficoll-Hypaque gradient separation method from the different blood sources. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (>99%) CD34+ cells fractions. In comparative proteome analysis, we detected the protein expression of stathmin in mPBSCs, AML CD34+ cells, and in native HSCs from BM. We performed microarray-based gene expression profiles of these cells and focused on kinases regulating stathmin’s activity. Furthermore, we monitored stathmin and its relevant kinases by FACS analyses of the enriched cell fractions and by fluorescence microscopy of bone marrow smears and cytospins. Results: In this study, we have shown in comparative proteome analysis (Q-TOF-MS/MS) that stathmin is expressed in G-CSF mobilized hematopoietic stem cells for the first time and in AML cells. In microarray analysis we indentified up- and down-regulated kinases: MAPK, PAK1, PKC beta/zeta, MEKK3 and CDKs. Accordingly, we demonstrated in FACS analyses and in immunofluorescence microscopy the high intracellular expression of PKCzeta in AML cells and MEKK3 as well PAK1 in mPBSCs. Conclusions: Our findings show that G-CSF stimulates Stathmin expression in mPBSCs and plays a key role in migration into peripheral blood. Furthermore, we show the different expression of kinases acting on stathmin in mPBSCs and AML cells. Consequently, stathmin and its relevant kinases promise to become a future target in therapies of malignant processes. No significant financial relationships to disclose.