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The study was designed to evaluate the implication of apoptosis in myeloid leukemic cell death induced by daunorubicin (DNR) and to identify the possible factors which may influence this process. DNR-induced apoptosis was characterized by morphology and DNA fragmentation in six leukemic myeloid cell lines which expressed different differentiation phenotypes. In phenotypically mature HL-60 and U937 cells, DNR induced typical apoptosis with characteristic morphological changes and intense internucleosomal DNA fragmentation within a narrow concentration range (0.5-2 microM). When these cells were treated with higher doses of DNR, large DNA fragments (100 kbp), but not internucleosomal fragments, were identified. DNR-induced DNA fragmentation in HL-60 and U937 was inhibited by antioxidants such as N-acetylcysteine (N-ac) or pyrrolidine-dithiocarbamate (PDTC). In the phenotypically immature KG1a, KG1, HEL and ML1 cell lines DNR induced no characteristic apoptotic morphological features as well as very low levels of internucleosomal DNA fragmentation, whereas large DNA fragments (200 kbp) were observed in KG1a treated with 7 microM DNR. Since the latter expressed P-glycoprotein (P-gp), the role of P-gp in the lack of apoptotic response to DNR was investigated. One P-gp inhibitor (verapamil) slightly improved DNR-induced DNA fragmentation in KG1a cells whereas the combination of verapamil and buthionine-sulfoximine (BSO), which depletes glutathion store, further increased internucleosomal DNA fragmentation. In conclusion, DNR induced internucleosomal DNA fragmentation in some but not all AML cells; the magnitude of this process being influenced by both intracellular drug concentration and oxidative balance.

Basic fibroblast growth factor (bFGF or FGF-2) is an angiogenic and pleiotropic factor involved in the proliferation and differentiation of numerous cell types. It is expressed mostly in tissues of mesoderm and neuroectoderm origin, and plays an important role in the mesoderm induction, together with transforming growth factor-beta (TGF-beta). Although hematopoietic cells derive from the mesoderm, relatively few studies have addressed the role of bFGF in the hematopoietic system until recently. It appears that bFGF is expressed and produced by bone marrow stromal cells, as well as by cells from several mature peripheral blood lineages. It is released and stored in the bone marrow extra-cellular matrix. FGF-receptors (FGF-Rs) are expressed on nearly every cell of hematopoietic origin tested so far. Growing evidence shows that bFGF can positively regulate hematopoiesis, by acting on various cellular targets: stromal cells, early and committed hematopoietic progenitors, and possibly some mature blood cells. It synergizes with hematopoietic cytokines, or antagonizes the negative regulatory effects of another factor, TGF-beta, thus potentially playing a central role in hematopoiesis.

Recent reports have suggested that basic fibroblast growth factor (bFGF) could play a permissive role in hematopoiesis, in combination with specific colony-stimulating factors. We investigated the expression of bFGF and FGF-receptors (FGF-Rs) in leukemic cell lines of various hematopoietic lineages. Three protein isoforms of bFGF of approximately 18, 22 and 24 kDa were detected in the myeloid cell line K562, but not in myelomonocytic or lymphoid (T or B) cell lines. In vitro-induced differentiation of K562 cells did not change the pattern of expression of the different bFGF isoforms. Accordingly, the mRNA of bFGF was found expressed in K562, but not in other leukemic lines tested, as assayed by reverse transcript amplification (RT-PCR). Using the same technique, we searched for the presence of high affinity FGF-Rs on these cells: in eight out of ten cell lines tested, mRNA for at least one FGF-R among FGF-R1, FGF-R3 or FGF-R4 was expressed, whereas FGF-R2 was never detected. We found that two cell lines were responsive to bFGF in different biological assays: (i) in K562 myeloid cells induced to differentiate by hemin, preincubation with bFGF and heparin increased cell viability and decreased hemin-induced DNA fragmentation, without affecting erythroid differentiation; and (ii) in U937 monocytic cells, the production of plasminogen activator was increased by bFGF or aFGF in combination with heparin. Binding experiments with 125I-bFGF (up to 200 pM) in the presence of heparin revealed high affinity receptors on the K562 and U937 cell lines (1177 +/- 440 and 392 +/- 184 sites/cell, Kd = 61.7 +/- 8.6 and 43.1 +/- 13.5 pM, respectively). Thus our results strongly suggest that cells of hematopoietic origin could express functional FGF-receptors.

In order to determine the role of interleukin 2 (IL2) on the proliferation of leukemic cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) we studied the production of IL2, the function of IL2 receptors (IL2R) expressed on T-ALL cells and their IL-2-dependent in vitro proliferation. Leukemic cells from six out of 17 T-ALL/T-cell non-Hodgkin's lymphoma patients with a prothymocyte (stage I) or a mature thymocyte (stage III), but not with a common thymocyte (stage II) phenotype, could proliferate, in a dose-dependent manner, in response to recombinant IL2 (rIL2) and anti-Tac and TU27 moAbs as well as polyclonal anti-IL2 purified immunoglobulin G could inhibit this IL2-induced cell proliferation. Both crude or/and Amicon-concentrated media conditioned by T-ALL cells from 10 out of 13 tested patients contained IL2 activity as assessed by colorimetric biological and immunoenzymatic assays; this biologic activity was due to a 14.5 kDa molecule adsorbed by anti-IL2 antibodies in an immunoaffinity assay. Although less than 10% of fresh leukemic cells expressed IL2R alpha (Tac) chain, a 24 h cell incubation in the absence of any mitogenic stimulation induced IL2R alpha chain expression in five out of 13 patients (11-83% Tac+ cells). Morever, Tac mRNA transcripts could be detected in fresh cells from all 10 patients tested. Staining of fresh leukemic cells with an IL-2R beta-chain-specific monoclonal antibody and flow cytometry analysis revealed that 4-13% of leukemic cells were positive. Binding experiments with 125I-rIL2 showed a small number of high affinity IL2R on fresh cells from three T-ALL patients (114-200 sites/cell, dissociation constant = 101-181 pm). Finally, antibodies against IL2R alpha, IL2R beta and IL2 could inhibit both IL2 driven and spontaneous cell proliferation of most patients' T-ALL cells, although in some cases an heterogenous pattern of inhibition was observed. Taken together, these findings strongly suggest that an IL2/IL2R-dependent mechanism could be involved in the proliferation of some T-ALL cells.

The effect of phorbol myristate acetate (PMA) on the expression of interleukin 2 receptors (IL-2R), production of IL-2 and IL-2-dependent proliferation of acute lymphoblastic leukemia T cells (T-ALL cells) from 10 patients was studied. First, the effect of PMA on the expression of cell surface markers was assessed: a decrease of CD3 and CD4, and an enhanced expression of CD8 molecule were observed on T-ALL cells. Moreover, PMA exhibited an heterogenous effect on various activation-associated molecules such as a decreased expression of transferrin receptor and T10 molecule and an induced expression of 4F2 and CD9 molecules. It is known that functional high-affinity IL-2R are composed of at least two IL2 binding molecules, the alpha (p55) and beta (p70) chains. We found that PMA induced the expression of both IL-2R alpha and IL-2R beta chains, as well as IL-2 production by T-ALL cells. These effects were time- and dose-dependent. Cross-linking experiments with 125I-labelled recombinant IL-2 (125I-rIL-2) revealed both p55 (IL-R alpha) and p70 (IL-2R beta) IL-2-binding polypeptides, whereas binding equilibrium assays on PMA-treated cells demonstrated the presence of a low number (31-413) of high-affinity binding sites/cell in five out of six cases analysed, as well as intermediate affinity IL-2R (1234-3919 sites/cell) in four out of six cases, according to the time of incubation with PMA. In two cases tested high-affinity IL-2R on PMA-treated T-ALL cells could internalize 125I-rIL-2 at 37 degrees C. PMA alone enhanced the spontaneous proliferation of T-ALL cells in three cases, whereas a clear synergy between IL-2 and PMA could be detected in three patients' cells. Moreover, exogenous rIL-2 enhanced cell proliferation of PMA-preincubated T-ALL cells in four cases studied. Taken together, these observations indicate that a short-term incubation of T-ALL cells with PMA can activate the IL-2/IL-2R system on these cells without inducing strong modifications of their differentiation status. These results thus suggest that this system may be involved in the proliferation process of some activated immature T cells.

The pre-B Reh-6 leukemic cells do not express membrane interleukin-2 (IL-2)-R alpha (Tac or p55) chain; however, their incubation with PMA induces the expression of both high and low affinity IL-2-R. Northern analysis of nonstimulated Reh-6 as well as leukemic cells from patients with acute B cell precursor lymphoblastic leukemia displayed a constitutive expression of p55 mRNA transcripts, which could be enhanced by PMA. Both actinomycin-D and cycloheximide could abrogate PMA-induced p55 membrane expression, suggesting the need for de novo mRNA and protein synthesis. The increased PMA-induced p55 mRNA accumulation was an early event (4 hr) and could be enhanced, specifically, by rIL-2 because anti-Tac moAb inhibited this rIL-2-mediated effect. Immunofluorescence and cross-linking studies using 125I-rIL-2 failed to reveal membrane-associated p55 protein on both Reh-6 and patients' leukemic cells. Conversely, immunogold staining and electron microscopy studies, revealed p55 immunoreactive molecules in the cytoplasm but not in the nucleus of all Reh-6 cells. Using a sensitive EIA, p55 molecules could be detected in cell lysates but not the culture supernatants of Reh-6 cells, suggesting that p55 was not released into the culture medium. These results indicate that constitutively expressed p55 mRNA on pre-B leukemic cells is translated into a relative immunoreactive protein that cannot be expressed on cell surface for unknown, yet, reasons.