Mariet T. Esselink

An inhibitor of PI3-K differentially affects proliferation and IL-6 protein secretion in normal and leukemic myeloid cells depending on the stage of differentiation

In this study, we examined the involvement of the phosphatidylinositol 3-kinase (PI3-K) and p70S6 kinase signal transduction pathway in the interleukin-1(IL-1)-mediated proliferation and cytokine production by normal and leukemic myeloid cells. Total AML blast populations, early progenitor (CD34(+)/CD36(-)) cells, and more differentiated (CD34(-)/CD36(+)) cells were treated with the PI3-K inhibitor Ly294002 and p70S6K inhibitor rapamycin. The effects on proliferation, IL-6 protein secretion, and intracellular signaling cascades were determined and compared with normal CD34(+) cells and monocytes. The function of the PI3-K pathway was dependent on the differentiation state of the AML cell population. In immature blasts, the IL-1-induced proliferation was strongly inhibited by Ly294002 and rapamycin, without a distinct effect on IL-6 protein production. In contrast, in mature monocytic blast cells inhibition of the PI3-K signaling route had a stimulatory effect on IL-6 protein secretion. Interestingly, these findings were not specifically linked to the malignant counterpart but were also observed with normal CD34(+) sorted cells vs mature monocytes. Evidence is provided that the Ly294002-induced increase in IL-6 protein secretion is linked to the cAMP dependent signaling pathway and not to changes in the phosphorylation of ERK or p38. However, although the enhanced IL-6 protein secretion is cAMP dependent, it was not found to be mediated by protein kinase A (PKA) or by the GTP-ase Rap1. This study indicates that inhibition of the PI3-K signaling pathway has an inhibitory effect on cell proliferation but a stimulatory effect on IL-6 expression mediated by a cAMP-dependent but PKA-independent route.

Stem cell factor synergistically enhances thrombopoietin-induced STAT5 signaling in megakaryocyte progenitors through JAK2 and Src kinase

Stem cell factor (SCF) has a potent synergistic effect during megakaryopoiesis when administered in combination with the major megakaryocytic cytokine, thrombopoietin (TPO). In this study we analyzed the underlying mechanisms with regard to STAT5 activity. TPO stimulation of MO7e cells resulted in STAT5 transactivation, which could be enhanced 1.6‐fold by costimulation with SCF, whereas SCF alone did not induce STAT5 transcriptional activity. This costimulatory effect of SCF was reflected in an increase in TPO‐induced STAT5 DNA binding and increased and prolonged STAT5 tyrosine phosphorylation in both MO7e cells and primary human megakaryocyte progenitors. In contrast, serine phosphorylation of STAT5 was constitutive and associated with an inhibitory effect on STAT5 transactivation. Signal transduction pathways that might synergize in TPO‐mediated STAT5 transactivation were analyzed using specific pharmacological inhibitors and indicated an essential role for Janus‐activated kinase 2 (JAK2) and a partial role for Src‐family kinases. Costimulation with SCF was found to increase and prolong tyrosine phosphorylation of JAK2 and the TPO receptor c‐mpl. In addition, the Src kinase inhibitor SU6656 partially downregulated the additional effect of SCF costimulation on STAT5 tyrosine phosphorylation. SCF‐induced enhancement of JAK2 phosphorylation was not affected by inhibition of Src kinase, suggesting that both JAK2 and Src kinase mediate STAT5 tyrosine phosphorylation. Synergistic activation of JAK2 and Src kinase may thus contribute to the enhanced STAT5 signaling in the presence of TPO and SCF.

Effect of ultrafilterable platinum concentration on cisplatin and carboplatin cytotoxicity in human tumor and bone marrow cells in vitro

The importance of the ultrafilterable platinum (fPt) fraction of cisplatin (CDDP) and carboplatin (CBDCA) for cytotoxicity and myelotoxicity was studied in vitro. By incubating CDDP or CBDCA with fetal calf serum (PCS) various fractions of fPt were prepared and determined by atomic absorption spectroscopy. A relation of % fPt fraction and incubation time (h) of 87e-1123t(r = −0.99) and 101e-o.oo87t (r = −0.99) were determined for CDDP and CBDCA, respectively. Cytotoxicity in the human small cell lung carcinoma cell line GLC4 and fPt fraction were closely related for CDDP (r = 0.99) and for CBDCA r = 0.97). However, at a similar fPt fraction the concentrations inhibiting cell survival by 50% (IC50) of CBDCA exceeded that of CDDP by a factor of 10-18 with 4 h exposure and a factor of 5 with continuous exposure. Tested in the range of peak concentrations in plasma of patients and at a clinically relevant fPt fraction of 10%, CDDP was not toxic for human bone marrow cells in the CFU-GM assay, whereas it was toxic at fPt fractions of 50% and 90%. However, CBDCA was myelotoxic at a (clinically relevant) fPt fraction of 50%, and also at 75% and 90%. The use of different fPt fractions, produced by the incubation method described in this study, permits the study of platinum drugs in vitro while approximating in vivo conditions might be used to evaluate myelotoxicity of new platinum drugs prospectively. For CDDP and CBDCA the fraction fPt determines cytotoxicity on tumor cells, and their different fPt fraction in patients account at least partly for their difference in myelotoxicity.

CELL CULTURE AND IMMUNE FUNCTION TESTS

Concerning the role of white blood cells in blood transfusions different aspects have to be considered: 1. Antibody formation against HLA and cell specific antigens by the patients immune system. 2. The influence of transfusion on the composition of different white blood cell populations in the patient. Several studies have demonstrated that supportive hemotherapy may influence the immune system: improvement of graft rejection in renal transplantation [1]; alterations in lymphocyte subpopulations in hemophilia patients [2]. 3. The properties and the function of the white blood cells of the donor, which have been collected for transfusion. For instance granulocytes used in transfusion for granulocytopenic patients. But most important are here perhaps the bone marrow grafts now that bone marrow transplantation has become an established procedure in the treatment of hematological disorders.