Differentiation induction of blast cells in two cases of childhood acute megakaryoblastic leukemia in vitro by interleukin-3 and interleukin-6: an ultrastructural cytochemical study.

Although hematopoietic growth factors influence renewal and differentiation of blast progenitors in acute myelogenous leukemia (AML), morphological maturation of leukemic blasts is thought a rare event, even when cultured in the presence of appropriate growth stimulants. However, light microscopic observation may not be sufficient to clarify precisely the effects of hematopoietic growth factors on the morphological differentiation of leukemic blasts. In this study, using cell culture techniques and electron microscopic cytochemistry for platelet peroxidase (PPO), we studied the effects of interleukin-3 (IL-3) and interleukin-6 (IL-6), both of which are considered to play an important role in normal megakaryocytopoiesis, on the growth and differentiation of blast cells from two patients with childhood acute megakaryoblastic leukemia (AMKL). In both of the two cases, IL-3 stimulated leukemic colony formation in methylcellulose culture, whereas IL-6 showed little such activity. However, in suspension culture, IL-6 was active in promoting megakaryocytic differentiation, although incomplete, as detected by increase in the number of PPO-positive cells, some having demarcation membrane-like structure. This effect was evident in culture with IL-6 alone in one patient, but it was detectable only when IL-6 was used in combination with IL-3 in the other patient. In contrast, IL-3 alone stimulated differentiation towards myeloid but not megakaryocytic lineage. These results indicate that IL-3 and IL-6 have a distinct role in leukemic megakaryocytopoiesis (IL-3 stimulates growth, whereas IL-6 promotes morphological differentiation) and that cooperation between these two cytokines functions most effectively for megakaryocytic differentiation of AMKL cells in a fashion similar to that for normal megakaryocytopoiesis.     

Expression of the CSF-1 gene in the blast cells of acute myeloblastic leukemia: association with reduced growth capacity

Myelopoietic growth factors are known to influence the growth in culture of malignant blast cells from human Acute Myeloblastic Leukemia (AML). We have used cDNA clones for the factor CSF-1 and its receptor fms to study DNA and RNA from the blasts of 25 AML patients. The CSF-1 gene was always in the germline configuration. CSF-1 mRNA was found in about half the blast populations. The cells were also studied for their growth properties in culture. A highly significant association was found between CSF-1 expression and poor growth in suspension culture. Most blast populations expressed fms; the number of fms expression negative samples was to small to permit the detection of any association between fms expression and growth or any interaction between the effects of the expression of the growth factor and its receptor. We propose that CSF-1 may be an important part of the mechanism determining the balance between self-renewal and determination in AML blast clones.     

Hydrocortisone in culture protects the blast cells in acute myeloblastic leukemia from the lethal effects of cytosine arabinoside

The blast cells in acute myeloblastic leukemia (AML) respond to many of the same regulatory mechanisms that control normal hemopoiesis. These include the growth factors that bind to membrane receptors and steroid hormones or vitamins that have intracellular receptors. We report the effects in culture of the steroid glucocorticoid hydrocortisone on freshly explanted AML blasts from patients and on two continuous AML cell lines. Only small changes in clonogenic cell numbers in suspension cultures were seen in the presence of hydrocortisone. The most striking effect of the hormone was on the sensitivity of blasts cells to cytosine arabinoside (ara-C). In contrast to the response of AML blast cells to retinoic acid, a ligand for intracellular steroid receptors that sensitizes some blast populations to ara-C, hydrocortisone reduced the toxic effects of the drug. The protective action of hydrocortisone was not mediated through the cell cycle since exposure of blasts to hydrocortisone did not affect the percentage of cells in DNA synthesis as measured with the tritiated thymidine (3HTdR) "suicide" technique. The hydrocortisone effect could be demonstrated using a pulse (20 min) exposure protocol. Blasts pulsed with increasing specific activities of 3HTdR showed the usual response pattern with an initial loss in plating efficiency to about 50% of control, followed by a plateau, regardless of whether the cells had been exposed to hydrocortisone. Control blasts exposed to increasing ara-C concentrations gave very similar dose-response curves; in striking contrast, blast cells cultured in hydrocortisone, then pulsed with ara-C did not lose colony-forming ability even though the same population was sensitive to 3HTdR. The hydrocortisone effect was dose and time related; protection from ara-C increased from 10(-8) to 10(-5) M and was seen after 4 hr exposure but required 8 hr to reach a maximum. We conclude that hydrocortisone can protect blasts from the lethal effects of ara-C even while the cells are in active DNA synthesis.     

Lytic activity against primary AML cells is stimulated in vitro by an autologous whole cell vaccine expressing IL-2 and CD80

Despite being of the myeloid lineage, acute myeloid leukaemia (AML) blasts are of low immunogenicity, probably because they lack the costimulatory molecule CD80 and secrete immunosuppressive factors. We have previously shown that in vitro stimulation of autologous peripheral blood mononuclear cells (PBMCs) with primary AML cells modified to express CD80 and IL-2 promotes proliferation, secretion of Th1 cytokines and expansion of activated CD8⁺ T cells. In this study, we show that allogeneic effector cells (from a healthy donor or AML patients) when stimulated with IL-2/CD80 modified AML blasts were able to induce the lysis of unmodified AML blasts. Effector cells stimulated with IL-2/CD80AML blasts had higher lytic activity than cells stimulated with AML cells expressing CD80 or IL-2 alone. Similarly, AML patient PBMCs primed with autologous IL-2/CD80 AML cells had a higher frequency of IFN-γ secreting cells and show cytotoxicity against autologous, unmodified blasts. Crucially, the response appears to be leukaemia specific, since stimulated patient PBMCs show higher frequencies of IFN-γ secreting effector cells in response to AML blasts than to remission bone marrow cells from the same patients. Although studied in a small number of heterogeneous patient samples, the data are encouraging and support the continuing development of vaccination for poor prognosis AML patients with autologous cells genetically modified to express IL-2/CD80.     

Phenotypically Dormant and Immature Leukaemia Cells Display Increased Ribosomal Protein S6 Phosphorylation

Mechanistic/mammalian target of rapamycin (mTOR) activity drives a number of key metabolic processes including growth and protein synthesis. Inhibition of the mTOR pathway promotes cellular dormancy. Since cells from patients with acute myeloid leukaemia (AML) can be phenotypically dormant (quiescent), we examined biomarkers of their mTOR pathway activity concurrently with Ki-67 and CD71 (indicators of cycling cells) by quantitative flow cytometry. Using antibodies to phosphorylated epitopes of mTOR (S2448) and its downstream targets ribosomal protein S6 (rpS6, S235/236) and 4E-BP1 (T36/45), we documented that these phosphorylations were negligible in lymphocytes, but evident in dormant as well as proliferating subsets of both mobilised normal stem cell harvest CD34+ cells and AML blasts. Although mTOR phosphorylation in AML blasts was lower than that of the normal CD34+ cells, p-4E-BP1 was 2.6-fold higher and p-rpS6 was 22-fold higher. Moreover, in contrast to 4E-BP1, rpS6 phosphorylation was higher in dormant than proliferating AML blasts, and was also higher in the immature CD34+CD38- blast subset. Data from the Cancer Genome Atlas show that rpS6 expression is associated with that of respiratory chain enzymes in AML. We conclude that phenotypic quiescence markers do not necessarily predict metabolic dormancy and that elevated rpS6 ser235/236 phosphorylation is characteristic of AML.     

Distribution of the cytoskeletal protein,Nestin, in acute leukemia

Abstract

Nestin is a neuroepithelial stem cell marker that is expressed in some types of tumor cells. Recent reports suggest that Nestin may be closely related to malignant cell proliferation and migration. Acute leukemia (AL) is characterized by a lack of differentiation, which results in uncontrolled proliferation in the bone marrow and accumulation of immature cells. The expression and function of Nestin in AL is unclear. We investigated Nestin immunohistochemical patterns of 87 patients that included 47 cases of acute myeloid leukemia (AML) and 40 cases of acute lymphoblastic leukemia (ALL), and 20 patients in complete remission (CR) from AML or ALL. We also investigated the clinico-pathological features of 87 cases of AL and their CR and overall survival (OS). Nestin was expressed in leukemic blasts and mature granulocytic cells in most cases (39/47) of AML. Conversely, Nestin was expressed in mature granulocytic cells in fewer cases (6/40) of ALL, but not in blasts. Nestin expression appeared in leukemic blasts of AML, but not ALL. Nestin expression in AML blast cells was not associated with CR or OS. We provide evidence that Nestin is expressed in AL and might be a useful immunohistochemical marker for identifying AML and ALL.     

Acute myelogenous leukemia blasts as accessory cells during in vitro T lymphocyte activation

The ability of acute myelogenous leukemia (AML) blasts to mediate costimulatory signals during T lymphocyte activation was investigated in an experimental model where monoclonal T cell populations were stimulated with standardized activation signals (anti-CD3, anti-CD2, and anti-CD28 monoclonal antibodies and phytohemagglutinin). Proliferative T cell responses were detected for all AML patients (n = 16) when irradiated leukemia blasts were used as accessory cells during activation. T cell cytokine release was also observed for all patients when nonirradiated AML accessory cells were used, and for most patients a broad cytokine response (interleukin (IL) 2, IL4, IL10, IL13, and interferon-gamma) was detected. However, both T cell proliferation and cytokine release showed a wide variation among AML patients, and T cell responsiveness was in addition dependent both on the nature of the activation signal and on differences between individual T cell clones. The accessory cell function of AML blasts showed no correlation with the release of any single immunomodulatory soluble mediator (IL1beta, IL6, TNF-alpha, soluble IL2 receptors) or the expression of any particular adhesion/costimulatory membrane molecule (CD54, CD58, CD80, and CD86) by the blasts. However, blocking studies with anti-CD58 and anti-CD80/86 monoclonal antibodies demonstrated that both pathways can be involved when AML blasts are used as accessory cells, but the relative importance and the final effects of signaling through these pathways differ between AML populations. Although there is a wide interpatient variation, we conclude that for a majority of patients the native AML blasts can mediate adequate costimulatory signals needed for accessory cell-dependent T cell activation.     

CD80 (B7-1) expression on human acute myeloid leukaemic cells cultured with GM-CSF, IL-3 and IL-6

Acute myeloid leukaemia (AML) blasts rarely express the B7 family of co-stimulatory molecules and do not elicit a clinically significant autologous T-lymphocyte anti-tumour response. The aim of this study was the in vitro modification of AML blasts to an antigen-presenting cell phenotype characterised by upregulated expression of the co-stimulatory molecule CD80 (B7-1). Circulating AML cells were induced to undergo partial differentiation in culture with the cytokines IL-3, IL-6 and GM-CSF; they exhibited variable upregulation of CD80 and continued to express MHC class I and II. These cells remained viable to day 20, in contrast with normal peripheral blood mononuclear cells (PBMNC), which did not survive under the culture conditions. In contrast to unmanipulated blasts, cultured leukaemic cells expressed B7-1. Where initial cytogenetic abnormalities were present, they were also seen in flow-sorted CD80-expressing cells after culture in cytokines, indicating their malignant origin. The immunogenic potential of these cultured cells was highlighted by allogeneic and autologous mixed lymphocyte reactions, in which both differentiated, but not unmanipulated, blasts produced expansion of T-lymphocyte numbers. Autologous cytotoxic T-lymphocyte (CTL) assays indicated specific killing of B7-1+ leukaemic cells, which was greatly enhanced after priming of the T-lymphocytes by B7-1+ blasts prior to the CTL assay, then enabling the CTL to lyse both unmanipulated and differentiated leukaemic cells.     

Vorinostat induces reactive oxygen species and DNA damage in acute myeloid leukemia cells

Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents.     

Characterization of a cell-type-restricted negative regulatory activity of the human granulocyte-macrophage colony-stimulating factor gene.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the proliferation and maturation of normal myeloid progenitor cells and can also stimulate the growth of acute myelogenous leukemia (AML) blasts. GM-CSF is not normally produced by resting cells but is expressed by a variety of activated cells including T lymphocytes, macrophages, and certain cytokine-stimulated fibroblasts and endothelial cells. Production of GM-CSF by cultured AML cells has been demonstrated, and GM-CSF expression by normal myeloid progenitors has been postulated to play a role in myelopoiesis. We have investigated the regulation of expression of GM-CSF in AML cell lines, and our results demonstrate the presence of a strong constitutive promoter element contained within 53 bp upstream of the cap site. We have also identified a negative regulatory element located immediately upstream of the positive regulatory element (within 69 bp of the cap site) that is active in AML cell lines but not T cells or K562 CML cells. Competition transfection and mobility shift studies demonstrate that this activity correlates with binding of a 45-kDa protein.     

The effects of recombinant CSF-1 on the blast cells of acute myeloblastic leukemia in suspension culture

Recombinant hemopoietic colony-stimulating factors (CSFs), including GM-CSF, G-CSF and IL-3, have been shown to be effective stimulators of both self-renewal and terminal differentiation of blast stem cells in acute myeloblastic leukemia (AML). We have examined the activity of a fourth growth factor, recombinant CSF-1 (or M-CSF), on the growth of leukemic blasts in culture. CSF-1 was found to be active on some, but not all, blast populations. In sensitive cells, CSF-1 often stimulated the production of adherent blast cells incapable of division. This observation leads us to suggest that CSF-1 may be useful in the treatment of selected cases of AML.     

Erlotinib antagonizes constitutive activation of SRC family kinases and mTOR in acute myeloid leukemia

Tyrosine kinases such as SRC family kinases (SFKs) as well as the mammalian target of rapamycin (mTOR) serine/threonine kinase are often constitutively activated in acute myeloid leukemia (AML) and hence constitute potential therapeutic targets. Here we demonstrate that the epidermal growth factor receptor (EGFR) inhibitor erlotinib, which has previously been shown to mediate antiproliferative/cytotoxic off-target effects in myelodysplastic syndrome (MDS) and AML blasts, reduces SFK overactivation. Erlotinib induced an arrest in the G₁ phase of the cell cycle that, in cells with constitutive SFK activation, could be recapitulated by chemical inhibition of SFKs with 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-α]pyrimidin-4-amine (PP2). Moreover, erlotinib inhibited the phosphorylation of mTOR targets like p70^SK6, stimulated the maturation of the autophagic marker LC3 and promoted the formation of autophagosomes. Notably, PP2 and the mTOR inhibitor rapamycin had a similar cell cycle-arresting activity to erlotinib, but neither of these compounds alone induced significant levels of cell death. Altogether, these results suggest that the therapeutic off-target effect of erlotinib may be linked to, yet cannot be entirely explained by, the inhibition of oncogenic signaling via SFKs and mTOR. Thus, combination therapies with erlotinib and rapamycin might be beneficial for MDS and AML patients.     

The simultaneous antagonistic effects of a T cell hybridoma product on the growth and the maturation of activated lymphocytes

Lymphocyte maturation and growth are two antagonistic events; therefore, successful antibody synthesis accompanied by clonal expansion, as seen in most humoral immune responses, must be the result of a delicate quantitative balance between maturation and growth factors. On the basis of this hypothesis, it should be feasible to search for a T cell product that alters this equilibrium and favors either B cell growth or maturation. In an attempt to isolate T cell hybridomas producing these activities, we fused BW5147 thymoma cells with Con A-activated spleen cells. One of the hybridomas obtained constitutively produces a product (or products) that selectively inhibits mitogen-induced replication but not maturation of normal but not transformed B lymphocytes. More importantly, the same product(s) supports Ig synthesis but not growth of LPS blasts, which suggests that we are dealing with a B cell maturation factor. The effect of this supernatant can be completely abrogated by the B cell mitogen DxS. In addition, the proliferative response of B cells to this ligand is unaffected by the hybridoma product. The implications of our results for understanding the mechanism of B cell triggering are discussed.     

Effects of transforming growth factor beta, tumor necrosis factor alpha, interferon gamma and LIF-HILDA on the proliferation of acute myeloid leukemia cells

A group of polypeptide factors that regulate cell growth and differentiation has been tested for their biological activities on the growth and differentiation of leukemic cells isolated from patients with Acute Myeloid Leukemias (AML). The effects of Transforming Growth Factor beta 1 (TGF beta), Tumor Necrosis Factor alpha (TNF alpha), Interferon gamma (IFN gamma) and LIF-HILDA were compared on leukemic cells cultured in vitro for seven days. Spontaneously growing leukemic cells were selected in order to study either inhibition or enhancement of proliferation induced by these factors. Only TGF beta 1 was found to induce a clear inhibition of leukemic proliferation in all cases tested. Recombinant TNF alpha and IFN gamma were found to induce either inhibition or enhancement of the proliferation on separate specimens. Under the conditions of culture, it was not possible to document any effect of LIF-HILDA. Cell differentiation and cell maturation were documented studying the modulation of cell surface antigens. TGF beta did not modify antigen expression on the cells surviving after 3 days in culture. Both TNF alpha and IFN gamma were found to enhance the expression of adhesion molecules and to a lesser extent, the expression of some lineage associated antigens. No effect of LIF-HILDA on antigen modulation was documented in the cases tested. These data confirm that TGF beta is by itself a potent inhibitor of the myeloid leukemia cells proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of cryopreservation on human leukaemic clonogenic cells (CFU-L).

The biological and clinical significance of growth characteristics of leukaemic clonogenic cells (CFU-L) cultured from patients has been the subject of many studies. While some investigators collect leukaemic cells in large numbers from blood of untreated patients and store them in a frozen state before use in experiment, others study fresh cells. Since cryopreservation may alter the proliferation and differentiation of CFU-L, we have followed its influence and that of DMSO, used as protective agent and known to be an inducer of differentiation in leukaemic blasts, on the clonogenicity of peripheral blast cells from patients with AML and CML in blast crisis. Our data show that a short incubation with 7.5% DMSO (with or without cryopreservation) induced increase in the clonogenicity and proliferation rate of CFU-L (without morphological changes). The possible causes of these effects as well as the question of aggressivity of leukaemic blasts after the short incubation with 7.5% DMSO are discussed.     

Prognostic significance of some clinical, morphological and cytogenetic findings in refractory anaemia (RA) and RA with sideroblasts

In 27 patients initially diagnosed as refractory anaemia (RA) or RA with sideroblasts (RA-S) according to the FAB-classification a number of clinical, morphological and cytogenetic parameters were correlated for prognostic significance. From these correlations it emerged that severe cytopenia is centrally positioned with regard to clinical course in RA and RA-S. Positive correlations were found to initial diagnosis, clonal cytogenetic abnormalities, progression to RA with an excess of blasts (RAEB) or acute myeloid leukaemia (AML), the percentage of bone marrow blast cells and prolonged half life for radioactively labeled iron. The degree of peripheral blood granulocytopenia, alone, was correlated to bone marrow hypoplasia. Moreover, the frequency of abnormal karyotypes was inversely correlated to bone marrow cellularity and proportional to the frequency of bone marrow blast cells. From these relationships it may be proposed that chromosome abnormalities are associated with prolonged blast cell generation times and inhibition of blast cell maturation resulting in reduced marrow cellularity and blast cell accumulation, and, in the peripheral blood, falling percentages of neutrophil granulocytes. With the blast cell accumulation the bone marrow cellularity again becomes hyperplastic and the preleukaemic condition is transformed into RAEB or AML.     

Tumor-selective action of HDAC inhibitors involves TRAIL induction in acute myeloid leukemia cells

Chromatin is a dynamic macromolecular structure epigenetically modified to regulate specific gene expression. Altered chromatin function can lead to aberrant expression of growth regulators and may, ultimately, cause cancer. That many human diseases have epigenetic etiology has stimulated the development of 'epigenetic' therapies. Inhibitors of histone deacetylases (HDACIs) induce proliferation arrest, maturation and apoptosis of cancer cells, but not normal cells, in vitro and in vivo, and are currently being tested in clinical trials. We investigated the mechanism(s) underlying this tumor selectivity. We report that HDACIs induce, in addition to p21, expression of TRAIL (Apo2L, TNFSF10) by directly activating the TNFSF10 promoter, thereby triggering tumor-selective death signaling in acute myeloid leukemia (AML) cells and the blasts of individuals with AML. RNA interference revealed that the induction of p21, TRAIL and differentiation are separable activities of HDACIs. HDACIs induced proliferation arrest, TRAIL-mediated apoptosis and suppression of AML blast clonogenicity irrespective of French-American-British (FAB) classification status, karyotype and immunophenotype. No apoptosis was seen in normal CD34(+) progenitor cells. Our results identify TRAIL as a mediator of the anticancer action of HDACIs.     

Decreased expression of DNAM-1 on NK cells from acute myeloid leukemia patients

This study tested the hypothesis that the expression of CD112 and CD155 (DNAM-1 ligands) on leukemic blasts induces a decreased expression of the activating receptor DNAM-1 on natural killer (NK) cells from acute myeloid leukemia (AML) patients. DNAM-1 is a co-receptor involved in the activation of NK cell cytotoxicity after its interaction with its ligands CD112 and CD155 on target cells. Here we study the expression of DNAM-1 on NK cells and DNAM-1 ligands on blasts from AML patients stratified by age. The results demonstrate that NK cells from AML patients younger than 65 years have a reduced expression of DNAM-1 compared with age-matched controls. The analysis of DNAM-1 ligands showed a high expression of CD112 and CD155 on leukemic blasts. An inverse correlation between CD112 expression on leukemic blasts and DNAM-1 expression on NK cells was found. Furthermore, downregulation of DNAM-1 was induced on healthy donors' NK cells after in vitro culture with leukemic blasts expressing DNAM-1 ligands. In conclusion, these results support the hypothesis that receptor-ligand crosslinking downregulates DNAM-1 expression on NK cells from patients <65 years of age. Considering the relevance of DNAM-1 in NK recognition and killing of leukemic cells, the reduced expression of this receptor on NK cells from AML patients can represent an additional mechanism of tumor escape.