Nucleophosmin status may influence the therapeutic decision in de novo acute myeloid leukemia with normal karyotype.
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Acute Disease↗
Biomedical subjects
C Demur
Publications and source records attributed to C Demur.
A crosstalk between the Wnt and the adhesion-dependent signaling pathways governs the chemosensitivity of acute myeloid leukemia.
Relapses following chemotherapy are a major hindrance to patients' survival in acute myeloid leukemia (AML). To investigate the role of the hematopoietic niche in the chemoresistance of leukemic cells, we examined two pathways: one mediated by adhesion molecules/integrins, and the other by soluble factors of the morphogen Wnt pathway. In our study, both the adhesion of leukemic blasts to fibronectin and the addition of Wnt antagonists induced, independently, resistance of AML cells to daunorubicin in a cell survival assay. Using pharmacological inhibitors and siRNA, we showed that both resistance pathways required the activity of the glycogen synthase kinase 3beta (GSK3beta). Moreover, the AML cell protection downstream of GSK3beta was mediated by NF-kappaB. A link between the adhesion and the Wnt pathway was found, as adhesion of U937 on human osteoblasts, a component of the hematopoietic niche, triggered the secretion of the Wnt antagonist sFRP-1 and supported resistance to daunorubicin. The osteoblast-conditioned medium could also confer chemoresistance to U937 cells cultured in suspension, and this cell protective effect was abrogated after depletion of sFRP-1. In the context of this potential double in vivo resistance, modulators of the common signal GSK3beta and of its target NF-kappaB could represent important novel therapeutic tools.
Antibiotics, Antineoplastic↗
Expression of beta-catenin by acute myeloid leukemia cells predicts enhanced clonogenic capacities and poor prognosis.
Activation of the Wnt/beta-catenin pathway has recently been shown to be crucial to the establishment of leukemic stem cells in chronic myeloid leukemia. We sought to determine whether beta-catenin was correlated to clonogenic capacity also in the acute myeloid leukemia (AML) setting. Eighty-two patients were retrospectively evaluated for beta-catenin expression by Western blot. beta-Catenin was expressed (although at various protein levels) in 61% of patients, and was undetectable in the remaining cases. In our cohort, beta-catenin expression was correlated with the clonogenic proliferation of AML-colony forming cells (AML-CFC or CFU-L) in methylcellulose in the presence of 5637-conditioned medium, and more strikingly with self-renewing of leukemic cells, as assessed in vitro by a re-plating assay. In survival analyses, beta-catenin appeared as a new independent prognostic factor predicting poor event-free survival and shortened overall survival (both with P<0.05). Furthermore, variations in beta-catenin protein levels were dependent on post-transcriptional mechanisms involving the Wnt/beta-catenin pathway only in leukemic cells. Indeed, beta-catenin negative leukemic cells were found to increase beta-catenin in response to Wnt3a agonist in contrast to normal counterparts. Altogether, our data pave the way to the evaluation of Wnt pathway inhibition as a new rationale for eradicating the clonogenic pool of AML cells.
Cell Line, Tumor↗
TNFalpha stimulates NKG2D-mediated lytic activity of acute myeloid leukemic cells.
The mechanism by which leukemic cells interfere with normal hematopoiesis remains unclear. We show here that, whereas the leukemic KG1a cells are naturally devoid from cellular cytotoxicity, once activated by TNFalpha, they display cytolytic activity toward various cellular targets including CFU-GM. This mechanism is dependent on stimulation of the granzyme B/perforin system. In addition, KG1a cells expressed the NKG2D receptor and its signal-transducing adaptator DAP 10, which were functional as confirmed by redirected lysis experiments. Interestingly, flow cytometry analysis of 20 samples of patients with acute myeloid leukemia (AML) (FAB M0-M5) revealed the expression of NKG2D (40%) and other natural cytotoxicity receptors (40% for NKp30, 74% for NKp44, 39% for NKp46) by a pool >15% of leukemic cells. Furthermore, CD34+ hematopoietic progenitors undergoing granulomonocytic differentiation expressed NKG2D ligands. Altogether, we propose a model in which, upon stimulation by TNFalpha, leukemic cells may exert cytotoxicity against myeloid progenitors. This finding may have important clinical implications in the context of diseases characterized by TNFalpha accumulation, such as AML or myelodisplasic syndromes.
Acute Disease↗
[Hemophagocytic syndrome associated with neutropenia after chemotherapy].
MATERIAL AND METHODS: This retrospective study reports 15 cases of hemophagocytic syndrome in children treated in our department during a eight-year period. RESULTS: Underlying diseases were acute lymphoblastic leukemia (n = 8) acute myeloblastic leukemia (n = 6) and Burkitt lymphoma (n = 1). Hemophagocytic syndrome was suspected after chemotherapy, in case of an unusual prolonged febrile neutropenia (n = 14) or isolated thrombocytopenia (n = 1). That fever was associated with cutaneous, pulmonary, hematologic, digestive and cardiac signs. Biological disorders included hypoprotidemia, hyponatremia, increased liver enzymes and fibrinopenia. Thrombocytopenia was observed in all patients and was associated with neutropenia for 14 of them. Diagnosis of hemophagocytic syndrome was always confirmed by bone marrow aspiration (infiltration with activated macrophages). Infection was documented in eight children. The treatment of hemophagocytic syndrome relied on steroids and resolution of symptoms occurred within three days of therapy. No recurrence of hemophagocytic syndrome was observed with a median follow up of two years and a half. CONCLUSION: Such complication should be suspected in cases of prolonged febrile neutropenia and/or thrombocytopenia, and confirmed by bone marrow aspiration. Indeed, steroid therapy is effective and chemotherapy can be then pursued.
Adrenal Cortex Hormones↗
Genotypical instability in undifferentiated cells: precursors for environmental adaptability?
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Adaptation, Biological↗
Lethal host-versus-graft disease and hypereosinophilia in the absence of MHC I-T-cell interactions.
Neonatal injection of semiallogeneic spleen cells in BALB/c mice induces a self-limited state of chimerism that promotes the differentiation of donor-specific CD4 T cells toward the Th2 phenotype. Here we show that injection of spleen cells from beta2-microglobulin-deficient (BALB/c x C57BL/6) F1 mice into BALB/c newborns with a disrupted beta2-microglobulin (beta2m) gene results in a lethal lymphoproliferative disorder associated with uncontrolled Th2 response, long-term persistence of donor B cells, and sustained blood eosinophilia. Autoimmune manifestations are also enhanced and characterized by a severe autoantibody-mediated glomerulonephritis. Histological examination of the spleen shows a hyperplasia of periarteriolar lymphoid sheaths, with accumulation of eosinophils and basophils, and variable degree of fibrosis. Perivascular lymphoid infiltrates with eosinophils are also found in the lung and are correlated with disease severity. Such abnormalities are almost absent using beta2m-sufficient mice. These data demonstrate that induction of lymphoid chimerism in the absence of MHC class I-T-cell interactions results in a lethal form of host-versus-graft disease that represents a unique model of Th2-dependent chronic inflammatory disease associated with an hypereosinophilic syndrome in mice.
Animals↗
Diagnosis of acute basophilic leukemia.
De novo acute basophilic leukemia (ABL) is a rare form of acute leukemia. Most frequently, the blast cells are morphologically undifferentiated, and the recognition of the presence of coarse basophilic granules may be the first step in diagnosis of this rare disorder. These granules are metachromatic and MPO negative. Immunophenotyping shows myeloid markers and some more specifically associated antigens such as CD9 or CD25 which are strongly expressed. Lymphoid, erythroid or megakaryocytic markers are not significantly expressed. In the absence of basophilic granules, some cases are classified as AML M0 if they express myeloid markers, or undifferentiated leukemia if no markers are present. If specific immature basophilic or theta granules are present, only an electron microscopic study will enable the diagnosis of a basophilic lineage assignment. Some cases may be misdiagnosed if all these steps are not followed. After all these investigations, two types of ABL may be defined: 1-A pure ABL, monophenotypic with basophilic lineage involvement alone, which should be classified as AML M8 . Genetic studies in these cases are very important for understanding the leukemic process and in a few cases, we can suspect c-MYB oncogene involvement but further investigations are still necessary. 2- More frequently, acute leukemia can be a mixture of blasts from different lineages with an important but variable participation of mature or immature basophilic cells. These cases must be classified as AML/Baso or multiphenotypic acute leukemias and often present Phl -chromosomal abnormality.
Biomarkers↗
Plasmodium falciparum infection following allogeneic bone-marrow transplantation.
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Bone Marrow Transplantation↗
Lack of ceramide generation in TF-1 human myeloid leukemic cells resistant to ionizing radiation.
The mechanism(s) by which ionizing radiation (IR) induces cell death is of fundamental importance in understanding cell sensitivity and resistance. Here we evaluated the response to IR of two subclones of the autonomous human erythromyeloblastic cell line TF-1: TF-1-34 (which expresses CD34) and TF-1-33 (which lacks CD34). In clonogenic assays, TF-1-34 cells were found to be relatively less sensitive to IR compared to TF-1-33 cells based on the D0 determination (3.01 vs 1.56 Gy). Furthermore, after IR at 12 Gy, TF-1-33 cell viability decreased by approximately 50% within 24 h, whereas TF-1-34 cell growth was unaffected during this time. Gradual loss of TF-1-34 cell viability was observed only after 48 h. Morphological and molecular analysis revealed that TF-1-33 cells died of apoptosis, and TF-1-34 cells of delayed reproductive cell death. While IR produced comparable amounts of DNA double strand breaks (DSB) in both cell lines, TF-1-34 retained DSB much longer than TF-1-33 suggesting that radioresistance and the defective apoptotic response of TF-1-34 cells was not related to a higher DNA repair capacity. However, the lack of an apoptotic response in TF-1-34 was correlated to the absence of a sphingomyelin (SM)-ceramide (CER) signaling pathway. Indeed, IR triggered in TF-1-33 cells but not in TF-1-34, SM hydrolysis (25% at 12 Gy) and CER generation (>50%) through the activation of neutral but not acid sphingomyelinase. Synthetic cell permeate CER induced apoptosis in both TF-1-33 and TF-1-34 cells. This study indicates that alterations of the SM-CER signaling pathway can significantly influence the cell death process as well as the survival of acute myeloid leukemia cells after IR exposure.
Antimetabolites↗
Acute myeloid leukemia cells with low P-glycoprotein expression and high rhodamine 123 efflux capacity display high clonogenicity.
This study was designed to correlate the clonogenic capacity of acute myeloid leukemia (AML) cells with P-glycoprotein (P-gp) expression level and P-gp-mediated efflux capacity. Fifty AML cell samples were tested for P-gp expression using MRK16 monoclonal antibody and flow cytometry. Among them, 12 samples were selected for sorting experiments according to the following two criteria: their clonogenic capacity in methylcellulose in the presence of 5637 conditioned medium, and the heterogeneity of P-gp distribution in leukemic cells. For each of these 12 samples, leukemic cells which displayed the highest P-gp expression level (P-gp++) and P-gp- leukemic cells were sorted after MRK16 staining and seeded into methylcellulose for primary clonogenic assay. In each case, the number of CFU-L in the P-gp fraction was significantly higher than that of the P-gp++ fraction (P < 0.01); the median number of CFU-L for 10(5) seeded cells being 147 (range 3-1855) and 495 (range 60-4100) for P-gp++ and P-gp- populations, respectively. Furthermore, in order to correlate clonogenic capacity and P-gp function, AML cells were stained with rhodamine 123 (Rh 123), washed and then sorted after 4 h incubation at 37 degrees C in Rh 123-free media on the basis of their residual fluorescence intensity before plating. For each of six samples, we found that the number of CFU-L in the AML cell fraction which displayed the most efficient Rh 123 efflux capacity (Rh 123dull) was significantly higher compared to that of the AML cell fraction which displayed high residual fluorescence signal (Rh 123bright) (P = 0.05); the median number of CFU-L for 10(5) seeded cells being 1025 (range 250-2240) and 296 (range 11-838) for Rh 123dull and Rh 123bright populations, respectively. Altogether this study suggests that, for an individual AML cell population, the clonogenic fraction is preferentially recruited in AML cells which display low P-gp expression and high P-gp-mediated efflux capacity.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Establishment and characterization of a human T-lymphocyte cell line immortalized by SV40 and with abnormal expression of TCR/CD3.
Human lymphocytes derived from the peripheral blood of a healthy woman were transfected with a plasmid carrying the simian virus 40 (SV40) large T antigen. The successfully transformed cells contained SV40 large T DNA and were negative for Epstein-Barr virus (EBV) and human T-cell leukaemia virus (HTLV)-1 genomes. The immortalized cell line was assigned to the T-lymphocyte lineage on the basis of morphological, immunological and cytochemical criteria. While the cells expressed CD1a and CD4 at the cell surface, the CD3 complex was solely intracytoplasmic. Immunoprecipitation studies indicated that these cells lacked T-cell receptor (TCR) alpha-chains but not beta-chains. They were negative for activation markers such as CD25, CD69 and major histocompatibility (MHC) class II molecules. In addition, the transformed cells exhibited a complete growth independency towards interleukin-2 (IL-2). However, after phorbol ester stimulation, CD25 and CD69 markers were expressed and IL-2 was secreted. This new human immortalized T-lymphocytic cell line, which is cell-surface TCR/CD3-negative, may be useful as an in vitro model for studying TCR/CD3 assembly, expression and signal transduction.
Antigens, Polyomavirus Transforming↗
Altered intracellular distribution of daunorubicin in immature acute myeloid leukemia cells.
We have used laser-assisted confocal microscopy to evaluate the intracellular distribution of daunorubicin (DNR) in acute myeloid leukemia (AML) cell lines and fresh AML cells according to their differentiation phenotype. In KG1a, KG1, TF-1 and HEL cells, which express the early differentiation marker CD34, DNR was distributed in perinuclear vesicles which could be associated with the Golgi apparatus, as suggested by the distribution of fluorescent probes specific for intracellular organelles. In contrast, U937 and HL-60 cells, which display a more mature phenotype, exhibited nuclear and diffuse cytoplasmic DNR fluorescence. DNR sequestration was not correlated with P-glycoprotein (P-gp) or multidrug resistance protein expression. Furthermore, PSC833, a potent P-gp blocker, had little effect on drug sequestration in CD34+ AML cells. We also tested the effect of metabolic inhibitors, cytoskeleton inhibitors and carboxy-ionophores on DNR distribution in both CD34- and CD34+ AML cells. However, only non-specific metabolic inhibitors restored nucleic/cytoplasmic distribution in CD34+ cells. In these cells, the intracellular distribution of doxorubicin and idarubicin was very similar to that of DNR, while the distribution of methoxymorpholinyl-doxorubicin was nuclear and diffusely cytoplasmic. In fresh AML cells, DNR was also concentrated in the perinuclear region in CD34+ but not in CD34- cells. However, DNR sequestration was not observed in normal CD34+ cells. Finally, our results show that DNR is sequestered in organelles in CD34+ AML cells via an active mechanism which appears to be different from P-gp-mediated transport. Abnormal DNR distribution may account for the natural resistance of immature AML cells to anthracyclines.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Multicentric evaluation of the MDR phenotype in leukemia. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.
The wide discrepancies in the frequency of 'positive' samples for multidrug resistance (MDR) phenotype within the same type of tumor observed in the literature justified the need for the definition of consensus recommendations. To define standard techniques of MDR phenotype measurement, we ran a large multicentric evaluation of the different methods available. Thirty-six French centers participated in the study, and 742 samples of 2-10 x 10(6) viable cells were sent by overnight express mail between December 1993 and February 1996. The same batches of MRK16, 4E3 and UIC2 were used. Nineteen samples of leukemia (12 AML, 1 ALL, 6 lymphoproliferative syndromes) and six leukemic cell lines with different levels of MDR expression were tested. Five meetings reached agreement concerning the guidelines for each technique, except immunocytochemistry. The 19 fresh samples were tested by each center using one to four techniques among cytofluorometry, immunocytochemistry, functional tests and RT-PCR. Five samples were diagnosed as 'negative' according to local criteria, with few discordant results (0 to 16% of 'positive' results). For all the 14 remaining samples, large discrepancies were observed from center to center, and from one technique to another. No correlations could be found between techniques. Flow cytometric analysis of cells already exposed to MRK16 or control IgG2A, fixed in paraformaldehyde and sent to centers did not reduce the discrepancies between centers in two of the four samples with moderate expression, emphasizing the role of histogram interpretation. The use of alternative monoclonal antibodies (4E3 and UIC2) did not reduce the discrepancies observed. In a second step, the K562 parental cell line, a low resistant subline (K562/HHT100, x7 resistance index to DNR) and a high resistant subline (K562/HHT300, x125 resistance index to DNR) were sent blindly three times, with an increasing level of recommendations for flow cytometry. Dramatic improvements were observed in cytometric results when the result was expressed as the ratio of arithmetic mean of fluorescence of antibody (10 microg of MRK16)/arithmetic mean of fluorescence of control (10 microg IgG2A): the proportion of expected results increased from 61 to 100% for K562, and from 37 to 85% for K562/HHT100. For uptake and drug efflux measurements, the use of 1 h uptake of 0.1 microM of rhodamine, followed by 1 h efflux +/-10 microM of verapamil, permitted an increased reproducibility of the technique from 71 to 100% for K562 and K562/HHT100. Whatever the technique used, concordant results were obtained for K562/HHT300. The immunocytochemistry, using several antibodies (MRK16, JSB1 and C219) gave many non-interpretable results (44%), due to a frequent high background and discordant results between antibodies in the same centers, and discordant conclusions between centers. The group does not recommend this technique for circulating tumoral cells.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Acute basophilic leukaemia and translocation t(X;6)(p11;q23).
We report two infants with acute basophilic leukaemia associated with a t(X;6)(p11;q23) as the sole abnormality. Morphologic evidence of basophilic lineage was provided by light and electron microscopy. Both patients also had a similar presentation on diagnosis, characterized by clinical signs consistent with a hyperhistaminaemia syndrome, i.e. urticarian rashes and gastro-intestinal disorders evocative of peptic ulcer. Immunophenotypes differed in the two patients, one expressing CD24, CD13 and CD33, whereas only CD117 was found in the other. Basophilic acute leukaemia, a rare group among acute leukaemias, might be nonrandomly associated with a specific chromosomal abnormality, t(X;6)(p11;q23). This new entity might also be identifiable by an uncommon clinical presentation and occurrence in infancy.
Antineoplastic Combined Chemotherapy Protocols↗
Vincristine degradation by serum from leukemic patients: role of myeloperoxidase.
Myeloperoxidase (MPO) has been shown to catalyze the in vitro degradation of vincristine (VCR). Given that MPO is a lysosomal enzyme that can be released into the circulation by both normal activated and leukemic myeloid cells, we investigated the possibility that sera from patients with acute myeloblastic leukemia (AML) might exhibit an increased capacity to degrade VCR. 31 serum samples (23 from patients with acute myeloblastic leukemia and 8 from patients with other conditions) were analyzed after incubation with ((3)H)VCR by using HPLC. Sera from patients with AML demonstrated an increased ability to breakdown VCR when compared to either normal sera or to sera from patients with lymphoid leukemias. VCR degradation was significantly increased by adding hydrogen peroxide, an electron donor for MPO, to the sera and was almost completely inhibited by adding 1 mM acetaminophen, an inhibitor of MPO. VCR peroxidation in the presence of hydrogen peroxide correlated both with the number of leukemic blasts in the circulation at the time the sera were obtained and with serum MPO concentrations determined by an immunoassay. These data suggest that the inactivity of VCR in AML may be due in part to its rapid peroxidation to inactive species by the MPO of leukemic myeloblasts.
Acetaminophen↗
Minimally differentiated erythroleukaemia (AML M6 'variant'): a rare subset of AML distinct from AML M6. Groupe Français d'Hématologie Cellulaire.
We describe eight cases of erythroleukaemia distinct from FAB-AML M6, which demonstrate minimal erythroid differentiation not associated with a myeloblastic component. Three infants (including a Down's syndrome) and two adults presented with a de novo leukaemia. One case was preceded by an untreated refractory anaemia with excess of blasts and one by polycythaemia vera. One case presented with an inaugural blast crisis of chronic myeloid leukaemia. In four patients the leukaemic cells showed a proerythroblast-like morphology. The four other were initially classified as undifferentiated AL (two cases) or AML MO (two cases) because of the immature aspect of the cells, their lack of myeloperoxidase activity and the absence of B, T lymphoid and myeloid (My) marker expressions apart from the CD33 antigen. Immunophenotyping in three cases showed an immature erythroblast profile (glycophorins A and B+, spectrin+). In the five others the erythroid nature was recognized by the expression of ABH blood group system on fresh cells (four cases) and glycophorin A on cells after 3 d in vitro culture with erythropoietin (EPO) + IL3 (two cases). Moreover, an erythroid colony growth of leukaemic origin was observed in three patients. In conclusion, the study of erythroid marker expression is of particular importance when immunophenotyping leukaemic cells with a proerythroblast-like morphology or an undifferentiated aspect and a HLA DR-, CD36++, B-, T-, My- (CD33 +/-) phenotype. We propose the term AML M6 'variant' for this rare type of AML.
Aged↗
Lack of correlation between expression and function of P-glycoprotein in acute myeloid leukemia cell lines.
In a panel of acute myeloblastic leukemia (AML) cell lines, representative of distinct differentiation stages, we investigated the possible correlation between drug-resistance and both expression and function of the multidrug resistance (MDR)-related P-glycoprotein (P-gp). The AML cell lines were KG1a, KG1, TF1, HEL, ML1, and two non drug-selected P-gp positive subclones originating from HL-60 (HL-60JD) and U937 (U937AQ). All these cells overexpressed the mdr1 gene (analyzed by RT-PCR) and displayed variable levels of P-gp expression. Flow cytometric semi-quantitative evaluation of P-gp with two P-gp specific monoclonal antibodies (MRK16 and UIC2) showed the following P-gp expression hierarchy: TF1 < KG1a < HEL < KG1 < HL-60JD < ML1 < U937AQ; the latter expressing 13 times more P-gp than TF1. When P-gp function was assessed by Rhodamine 123 (Rh123) efflux kinetics, we found that only KG1a and KG1 cells, which have an early (immature) CD34+ CD33- CD38- phenotype, and to a lesser extent TF1, with an intermediate (CD34+ CD33+ CD38+) phenotype, displayed significant P-gp activity which could be inhibited by both verapamil and SDZ PSC 833. In contrast, the other more mature CD33+ CD34- AML cell lines presented no Rh123 efflux capacity although they expressed higher P-gp levels. Daunorubicin (DNR) accumulation studies showed that inhibitors of P-gp increased DNR accumulation only in the immature AML cells whereas they had no impact on the mature AML cell lines. MTT drug cytotoxicity assay confirmed that the immature AML cells were 10-15-fold more resistant to DNR than the mature AML cells. Although P-gp inhibitors were able to increase the cytotoxicity of DNR in AML cells which displayed functional P-gp, they could not increase DNR cytotoxicity to levels comparable to that of the CD34- CD33+ cells, suggesting that DNR resistance of immature AML cells may not solely be related to P-gp. With drug-selection, AML subclones displayed higher levels of P-gp expression and higher extruding capacities, and therefore chemoresistance, and this independently of their initial differentiation phenotype. Finally, this study provides evidence for a lack of correlation between expression and function of P-gp in AML cells; this relationship being dependent upon leukemic cell differentiation in unselected myeloid leukemic cells.
ATP Binding Cassette Transporter, Subfamily B, Mem↗