G-CSF Stimulates Expression of Non-Phosphorylated Stathmin (Oncoprotein, Op18) in Hematopoietic Stem Cells.

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. In this study we investigated native hematopoietic CD34+ stem cells (HSCs) from BM in comparison to mobilized peripheral blood stem cells (mPBSCs) from G-CSF stimulated healthy donors. All the cell fractions were highly enriched (>99%). In comparative proteome analysis mPBSCs showed high levels of Stathmin compared to native HSCs from BM. We monitored Stathmin by fluorescent-microscopy of bone marrow smears and cytospins. We performed microarray-based gene expression profiles of the aforementioned cells focused on kinases regulating Stathmin’s activity. Western blotting and ion trap mass spectrometer (Bruker) were used to determine the differential phosphorylation. Mononuclear cells were isolated by a standard Ficoll 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.

Sample preparation: Total RNA was isolated from sorted 1 × 10e6 cells by standard methods using RNA isolation kit (Qiagen). For gene expression analysis topic-defined PIQOR™ stem cell microarrays (936 genes) were performed. Proteomics started with the determination of protein concentrations, 2D-gel-electrophoresis were described in Proteome Works System (BioRad). Sypro ruby and/or coomasie stained gels were used for protein identification after spot cutting by Q-TOF and ion trap mass spectrometer analyses.

1. The subcellular localization of the identified Stathmin and tubulin were performed by fluorescence and confocal microscopy of all cell fractions.

2. The microarray gene expression correlation shows different regulated genes e.g. kinases: PAK1, MAPK, ATM1, CDKN2A, cdk, PKA, PKCB, PKCD, PKCZ.

3. Stathmin (oncoprotein Op18) is expressed at very high levels in mPBSCs (Q-TOF), and remains non-phosphorylated (western blotting, ion trap mass spectrometry).

Our findings show that G-CSF stimulates Stathmin expression in CD34+ cells from BM and plays a key role in migration into peripheral blood. In microarray analyses we show that Stathmin phosphorylation will be regulated by different kinases in mPBSCs. But Stathmin is highly expressed in mPBSCs and is non-phosphorylated in its active form.