The response of cells of the murine megakaryocytic lineage to human interleukin 6 (IL-6) was asse in serum-depleted cultures using a variety of biological assays.IL-6 alone had no influence on megakaryocytic colony formation but augmented the numbers of these colonies induced by the multipotent colony-stimulating factor interleukin 3. However, in liquid marrow cultures, IL-6 alone promoted marked increments in megakaryocytic size and the activity of acetylcholinesterase, a marker enzyme ofthe lineage. Moreover, IL-6 induced a signicant shift toward higher ploidy classes when megakaryocytic DNA was quantitated by flow cytometry. To determine whether the influence of IL-6 on megakaryocytic maturation was direct, the factor was added to cultures of single megakaryocytes isolated from megakaryocytic colonies.Fifty-four percent ofthese cells increased in size compared with 19% of those grown without HL-6. The data show that human IL-6 is a potent direct-acting growth factor for murine megakaryocytes with activity promoting maturation of that lineage.Megakaryocytopoiesis is a process that encompasses proliferation of committed megakaryocytic progenitor cells (CFU-MK) and cellular maturation comprising nuclear endoreduplication (polyploidization), cytoplasmic enlargement, and accumulation of lineage markers (1-3). This process appears, at least in vitro, to be stimulated by a number of cytokines. The multipotent colony-stimulating factor interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin have been shown to promote the proliferation of megakaryocytic progenitors (4-6). Moreover, these growth factors can support not only proliferation, but also megakaryocytic maturation to some degree (7-9). These observations, nonetheless, do not exclude the idea that there exist growth factors that act predominantly to influence either proliferation or maturation. In this report the effects of recombinant human interleukin 6 (IL-6) on murine megakaryocytopoiesis are described. This 26,000 Mr glycoprotein with multiple biological activities has been purified to homogeneity from both murine and human sources and recently has been molecularly cloned (10-12). We now show that this cytokine significantly augments megakaryocytic maturation as assessed by size, acetylcholinesterase (AcChoEase) activity, and DNA content and that it synergizes with the megakaryocyte-growth-promoting activity of IL-3. MATERIALS AND METHODSMarrow Preparation. Marrow was flushed from the femurs of C57BL/6 mice with Iscove's modification of Dulbecco's medium (IMDM) supplemented with Nutridoma-SP (Boehringer Mannheim), a serum-free medium supplement. For culture studies, a single-cell suspension was made by repetitive expulsion through progressively smaller needles. For flow cytometry, a monocellular suspension was made by gentle filtration through 100-,um nylon mesh. In some experiments marrow cells were treated with 0.5 mM diisopropylfluorophosphate to inactivate endogenous AcChoEase (a marker enzyme of mega...

Bone marrow-derived stromal cells have engendered interest because of their therapeutic potential for promoting tissue vascularization and repair. When mononuclear cells isolated from mouse bone marrow were cultured in DMEM supplemented with 10% fetal bovine serum, cell populations arose that showed rapid proliferation and loss of contact inhibition. These cells formed invasive soft tissue sarcomas after i.m. injection into nude or scid mice. I.v. injection resulted in the formation of tumor foci in the lungs. The tumors were transplantable into syngeneic immunocompetent mice. Direct injection of cultured cells into immunocompetent mice also resulted in tumor formation. Karyotype analysis showed that increased chromosome number and multiple Robertsonian translocations occurred at passage 3 coincident with the loss of contact inhibition. The remarkably rapid malignant transformation of cultured mouse bone marrow cells may have important implications for ongoing clinical trials of cell therapy and for models of oncogenesis. (Cancer Res 2006; 66(22): 10849-54)

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The purpose of this trial was to evaluate the efficacy of 2-year consolidation therapy with nilotinib, at a dose of 300 mg twice daily, for achieving treatment-free remission in chronic myeloid leukemia patients with a deep molecular response (BCR-ABL1IS ≤0.0032%). Successful treatment-free remission was defined as no confirmed loss of deep molecular response. We recruited 96 Japanese patients, of whom 78 sustained a deep molecular response during the consolidation phase and were therefore eligible to discontinue nilotinib in the treatment-free remission phase; of these, 53 patients (67.9%; 95% confidence interval: 56.4–78.1%) remained free from molecular recurrence in the first 12 months. The estimated 3-year treatment-free survival was 62.8%. Nilotinib was readministered to all patients (n=29) who experienced a molecular recurrence during the treatment-free remission phase. After restarting treatment, rapid deep molecular response returned in 25 patients (86.2%), with 50% of patients achieving a deep molecular response within 3.5 months. Tyrosine kinase inhibitor withdrawal syndrome was reported in 11/78 patients during the early treatment-free remission phase. The treatment-free survival curve was significantly better in patients with undetectable molecular residual disease than in patients without (3-year treatment-free survival, 75.6 versus 48.6%, respectively; P=0.0126 by the log-rank test). There were no significant differences in treatment-free survival between subgroups based on tyrosine kinase inhibitor treatment before the nilotinib consolidation phase, tyrosine kinase inhibitor-withdrawal syndrome, or absolute number of natural killer cells. The results of this study indicate that it is safe and feasible to stop tyrosine kinase inhibitor therapy in patients with chronic myeloid leukemia who have achieved a sustained deep molecular response with 2 years of treatment with nilotinib. This study was registered with UMIN-CTR (UMIN000005904).

To determine the biologic activity of interleukin-6 (IL-6) on megakaryocytopoiesis and thrombocytopoiesis in vivo, the cytokine was administered intraperitoneally to mice every 12 hours at varying doses for five days or for varying time intervals, based on the kinetic analysis of IL-6 serum levels indicating the peak of 40 minutes following injection, with no detection at 150 minutes. A dose-response experiment showed that IL-6 increased platelet counts in a dose- dependent fashion at a plateau stimulation level of 5 micrograms. Administration of 5 micrograms of IL-6 reproducibly elevated platelet counts at five days by approximately 50% to 60% of increase. Moreover, a striking increase in megakaryocytic size in response to IL-6 was elicited by the treatment, but no change in megakaryocyte numbers; whereas IL-6 administration did not expand CFU-MK numbers. The in vivo studies in this manner had negligible effects on other hematologic parameters, with the minor exception of monocyte levels. These data show that IL-6 acts on maturational stages in megakaryocytopoiesis and promotes platelet production in vivo in mice, suggesting that IL-6 functions as thrombopoietin.