Mobilisation strategies for normal and malignant cells.

This review evaluates the latest information on the mobilisation of haemopoietic stem cells for transplantation, with the focus on what is the current best practice and how new understanding of the bone marrow stem cell niche provides new insights into optimising mobilisation regimens. The review then looks at the mobilisation of mesenchymal stromal cells, immune cells as well as malignant cells and what clinical implications there are.

Early myeloid cells expressing c-KIT isoforms differ in signal transduction, survival and chemotactic responses to Stem Cell Factor.

Isoforms of the receptor tyrosine kinase, c-KIT, differ in the presence or absence of a GNNK tetrapeptide in the extracellular juxtamembrane region. When expressed in murine NIH3T3 cells, these isoforms of c-KIT showed differential activation of signaling pathways and proliferation in response to Stem Cell Factor (SCF). However, c-KIT is not normally expressed by fibroblasts, but plays a key role in hematopoiesis. Because signaling pathways and cellular responses mediated by c-KIT differ in different cell types, we studied the effects of SCF stimulation on factor-dependent murine early myeloid cells expressing human GNNK+ or GNNK- c-KIT. As in fibroblasts, SCF activation of the GNNK- isoform resulted in stronger, more rapid receptor phosphorylation, and activation of Src kinases, while only a minor effect on the phosphatidylinositol 3-kinase pathway was observed. Similarly, more rapid Src kinase-dependent internalisation of the GNNK- isoform occurred in response to SCF. In contrast to fibroblasts, only minor differences in ERK activation were seen indicating that early hematopoietic cells, unlike fibroblasts, are not dependent on Src kinases for activation of this pathway in response to SCF. Enhanced SCF-dependent growth was observed in GNNK- c-KIT expressing cells due to lower cell attrition. The rate of cell division was similar. Importantly, cells expressing the GNNK- isoform showed a greater chemotactic response to SCF.

OCT-1-mediated influx is a key determinant of the intracellular uptake of imatinib but not nilotinib (AMN107): reduced OCT-1 activity is the cause of low in vitro sensitivity to imatinib.

Intrinsic sensitivity of newly diagnosed chronic myeloid leukemia (CML) patients to imatinib (IC50(imatinib)) correlates with molecular response. IC50(imatinib) is defined as the in vitro concentration of drug required to reduce phosphorylation of the adaptor protein Crkl by 50%. We now show that interpatient variability in IC50(imatinib) is mainly due to differences in the efficiency of imatinib intracellular uptake and retention (IUR). In 25 untreated CML patients, the IC50(imatinib) strongly correlated (R (2) = -0.484, P = .014 at 2 muM imatinib) with the IUR of [(14)C]imatinib. The addition of prazosin, a potent inhibitor of OCT-1 cellular transporter, reduced the IUR and eliminated interpatient variability. IC50 values for the more potent BCR-ABL inhibitor nilotinib (AMN107) did not correlate with IC50(imatinib) (R(2) =-0.0561, P > .05). There was also no correlation between IC50(nilotinib) and the IUR for [(14)C]nilotinib (R (2) = 0.457, P > .05). Prazosin had no effect on nilotinib IUR, suggesting that influx of nilotinib is not mediated by OCT-1. In conclusion, whereas OCT-1-mediated influx may be a key determinant of molecular response to imatinib, it is unlikely to impact on cellular uptake and patient response to nilotinib. Determining interpatient and interdrug differences in cellular uptake and retention could allow individual optimization of kinase inhibitor therapy.

Role of c-KIT expression level and phosphatidylinositol 3-kinase activation in survival and proliferative responses of early myeloid cells.

The receptor tyrosine kinase c-KIT and its ligand Stem Cell Factor (SCF) are critical in haemopoiesis but pathways linking receptor activation to specific responses in progenitor cells are still unclear. We have investigated the role of c-KIT expression level and the phosphatidylinositol 3-kinase (PI3-K) pathway in survival and cell division of early myeloid cells in response to SCF. Two factor-dependent murine early myeloid cell lines, FDC-P1 and Myb-immortalised haemopoietic cells (MIHC), were transduced to express wild-type c-KIT or a mutant form of the receptor (Y721F) that lacks the major recruitment site for the p85 regulatory subunit of PI3-K. Several clones expressing different receptor levels were analysed in each case. Growth of cells expressing either the wild-type or Y721F mutant KIT was strongly dependent on receptor level within the physiological range. Using an assay that allows quantitative measurement of the contributions of cell survival and cell division, diminished cell growth in response to SCF under limiting conditions of receptor copy number or PI3-K recruitment was shown to be almost entirely due to decreased cell survival. Further studies with the PI3-K inhibitor LY294002 indicated that PI3-K activation was also required for cell division. Alternate binding and/or indirect activation of PI3-K could support cell division mediated by Y721F mutant KIT, but was insufficient for the survival response.

Macrophage colony-stimulating factor receptor c-fms is a novel target of imatinib.

Imatinib is a tyrosine kinase inhibitor that suppresses the growth of bcr-abl-expressing chronic myeloid leukemia (CML) progenitor cells by blockade of the adenosine triphosphate (ATP)-binding site of the kinase domain of bcr-abl. Imatinib also inhibits the c-abl, platelet-derived growth factor (PDGF) receptor, abl-related gene (ARG) and stem-cell factor (SCF) receptor tyrosine kinases, and has been used clinically to inhibit the growth of malignant cells in patients with CML and gastrointestinal stromal tumors (GISTs). Although initially considered to have minimal effects of normal hematopoiesis, recent studies show that imatinib also inhibits the growth of some nonmalignant hematopoietic cells, including monocyte/macrophages. This inhibition could not be attributed to the known activity profile of imatinib. Here, we demonstrate for the first time that imatinib targets the macrophage colony-stimulating factor (M-CSF) receptor c-fms. Phosphorylation of c-fms was inhibited by therapeutic concentrations of imatinib, and this was not due to down-regulation in c-fms expression. Imatinib was also found to inhibit M-CSF-induced proliferation of a cytokine-dependent cell line, further supporting the hypothesis that imatinib affects the growth and development of monocyte and/or macrophages through inhibition of c-fms signaling. Importantly, these results identify an additional biologic target to those already defined for imatinib. Imatinib should now be assessed for activity in diseases where c-fms activation is implicated, including breast and ovarian cancer and inflammatory conditions.