Patrice Dumain

Study of the apoptosis induced in vitro by antitumoral drugs on leukaemic cells

A flow cytometric method for simultaneous apoptotic cell detection and cell cycle analysis was applied on the U937 cell line. Four antitumoral drugs currently used in the treatment of acute myeloid leukaemia were studied in vitro: DNR, IDR, MITO and Ara-C. Our results show a dissociation between the cytostatic effect (the block in the cell cycle observed for low drug concentrations) and the cytotoxic effect (the induction of apoptosis induced by higher concentrations) for all the tested molecules. Low concentrations of Ara-C induced a block in the S phase while higher concentrations (>10(-7) M) induced apoptosis at the G1-S boundary. Low concentrations of anthracyclines (<40 nM DNR and <20nM IDR) induced a block in G2 without apoptosis. Apoptosis was induced in G1 and/or early S phases by higher concentrations of anthracyclines. The concentration inducing 50% apoptosis (IC50) was found to be, respectively, 200 and 40 nM for DNR and IDR. Analysis of MITO-treated cells showed a parallel increase in the percentages of S phase and apoptotic cells. However, the bivariate analysis showed that apoptosis did occur in a population with G1 DNA content. For two other drugs (CAM and COLC), apoptosis occurred for the same concentrations and in the same phase as the block (in S and G2M, respectively). The IC50 of MITO was found to be 100 nM. Cotreatment of the cells with colchicin and either Ara-C or IDR showed that the passage through mitosis was not necessary for the completion of apoptosis at the G1-S boundary. Short incubations of U937 cells with high concentrations of anthracyclines were found to be efficient in inducing further apoptosis. We conclude that, for all the assayed molecules, the cytotoxic and/or cytostatic effects of the antitumoral drugs tested greatly depend on the concentrations used and that, depending on their in vivo pharmacokinetics, the induction of apoptosis could be an important mechanism of action for some of these drugs.

Effect of Pentoxifylline on Apoptosis of Cultured Cells

Apoptosis or programmed cell death (PCD) was measured in two human cell models by flow cytometric analysis. Blood neutrophils underwent spontaneous apoptosis in short-term culture. Pentoxifylline (PTX) inhibited spontaneous neutrophil PCD. We confirmed that granulocyte/macrophage colony-stimulating factor (GM-CSF) inhibited apoptosis of polymorphonuclear neutrophils. Treatment with both GM-CSF and PTX did not increase the inhibition of PCD by either GM-CSF or PTX alone. Because apoptosis could be due to the accumulation of H2O2 in the culture medium, and because PTX has been described to reduce peroxide production, we studied the effect of adding catalase to the medium. Catalase reduced the neutrophil apoptosis and this effect was cumulative with the effect of PTX. Camptothecin, an inhibitor of topoisomerase I, induces a block in the S-phase of the cell cycle followed by apoptosis of the U937 cell line. This drug-induced apoptosis was partially inhibited by PTX, whereas the S-phase cell block was not affected. In conclusion, PTX was found to inhibit apoptosis in two different human cell types. In neutrophils, this effect appears to occur regardless of the inhibition of phosphodiesterase activity and inhibition of H2O2 release.

Bcr-abl translocation can occur during the induction of multidrug resistance and confers apoptosis resistance on myeloid leukemic cell lines.

Apoptosis was studied in parental and mdr-1 expressing U937, HL60 and K562 myeloid leukemic cell lines using mdr unrelated inducers of apoptosis such as Ara-C, cycloheximide, serum deprivation, ceramide, monensin and UV irradiation. Apoptosis was efficiently induced by all these treatments in U937 and HL60 cells while K562 cells exhibited an apoptosis-resistant phenotype except with UV and monensin. The pattern of apoptosis resistance in mdr-1 expressing U937 (U937-DR) and HL60 (HL60-DR100) was similar to that presented by K562. This apoptosis-resistant phenotype of mdr cells was not overcome by concentrations of verapamil inhibiting the P-gp 170 pump. The acquisition of this phenotype was posterior to the mdr-1 expressing phenotype since a HL60-DR5 variant, selected at the beginning of the induction of resistance, presented a low level of mdr-1 expression without resistance to apoptosis. The variations observed in the Fas (CD95) expression between sensitive and resistant cells were not sufficient to account for apoptosis resistance. However, a high expression in Abl antigen was found in all the apoptosis-resistant cells. RT – PCR and Western blot analysis showed that this increase in Abl antigen content was accompanied by the expression in U937-DR and HL60-DR100 cells of a hybrid bcr/abl mRNA and a 210 kD Bcr/Abl protein which was constitutive in K562. This expression was due to the translocation of abl and the amplification of the bcr-abl translocated gene. These results are in agreement with the role of Bcr/Abl tyrosine protein kinase as an inhibitor of apoptosis independently of the mdr-1 expression. They also suggest that translocation of the abl gene in the bcr region is a highly probable rearrangement in the mdr-1 expressing myeloid cells and that Bcr/Abl tyrosine kinase effect on apoptosis needs the regulation of intracellular pH and is inactive against UV-induced apoptosis.

Flow cytometric study of idarubicin and daunorubicin accumulation and the effect of verapamil in leukemic cell lines and fresh cells from patients with acute non-lymphoblastic leukemia

By using flow cytometry, the intracellular accumulation (Acc) of idarubicin (IDA) and daunorubicin (DNR) and the effect of verapamil (VRP) on both anthracycline accumulation (VRP index) were studied in leukemic cell lines (K562 and HL60 and their two DNR-resistant subclones) and fresh leukemic cells. IDA accumulated more than DNR in both parental (K562: p < 0.03 and HL60: 0.09) and resistant cell lines (p < 0.01 for both cell lines) irrespective of whether or not they were treated with VRP. VRP index was higher for DNR than for IDA (p < 0.05). Similar results were observed in fresh leukemic blasts from 25 patients with ANLL (IDA Acc superior to DNR Acc: p < 0.0001; higher VRP index for DNR than for IDA: p < 0.01). The higher Acc of IDA than DNR seen in fresh leukemic cells could explain the better clinical efficacy of IDA reported in patients with ANLL.

SYNTHESIS OF BCL-2 IN RESPONSE TO ANTHRACYCLINE TREATMENT MAY CONTRIBUTE TO AN APOPTOSIS-RESISTANT PHENOTYPE IN LEUKEMIC CELL LINES

BACKGROUND Some forms of chemoresistance in leukemia may start from failure of tumour cells to successfully undergo apoptosis and Bcl-2 may play a role in this defect. Therefore, we evaluated the Bcl-2 content and synthesis in relation with the apoptotic potential in leukemic cell lines after anthracycline treatment. METHODS U937, HL60, and K562 cells and their drug resistant (DR) variants were treated with varying concentrations of Idarubicin (IDA). Apoptosis was evaluated by fluorescence microscopy after acridine orange staining. Bcl-2 and Bax content were evaluated either by flow cytometry after indirect immunolabelling or by Western blot. RESULTS High Bcl-2 contents were not related to a poor ability to undergo apoptosis in U937, HL60, K562 and their DR variants. IDA induced a concentration-dependent increase in Bcl-2 content in all cell lines as long as they do not perform apoptosis. Enhanced Bcl-2 expression was inhibited by cycloheximide, actinomycin D, or antisense oligonucleotide directed against bcl-2 mRNA. Bcl-2 expression was also increased in the resistant U937 variant after serum deprivation or C2-ceramide treatment. The synthesis of Bcl-2 led to an increased Bcl-2/Bax ratio solely in the cells with an apoptosis-resistance phenotype. CONCLUSIONS These data suggest that exposure to IDA induces Bcl-2 expression in leukemic cell lines, and that this mechanism could contribute to apoptosis resistance and participate in the acquisition of chemoresistance. They also confirm that the evolution of the Bcl-2/Bax ratio reflects apoptotic ability better than the steady state level of Bcl-2 expression.

Membrane thrombomodulin levels are decreased during hypoxia and restored by cAMP and IBMX

Thrombomodulin is a membrane glycoprotein expressed by endothelium and is a receptor for thrombin. The thrombin-thrombomodulin complex inactivates the procoagulant activity of thrombin and catalyzes activation of protein C(1). The decrease in thrombomodulin expression at the surface of the vascular endothelium may contribute to the development of thrombosis. In vitro studies have shown that cytokines (TNF, IL-1) or endotoxins down regulate thrombomodulin expression (2, 3, 4) whereas histamine (5), retinoic acid (6) and cAMP analogs (7, 8) increase its expression. Hypoxia is frequently related to disorders of the vascular system such as atheromatous arterial disease and venous insufficiency (9). Clinically, hypoxia induces an increase in vascular permeability (10) and a prothrombotic tendency (12). In vitro, it has been demonstrated that hypoxia is able to modify endothelial properties. In effect, hypoxia has been shown to down regulate thrombornodulin expression on bovine arterial endothelial cells (BAEC) and microvascular endothelial cells (11, 13, 14). Moreover, it has been demonstrated that hypoxia decreases endothelial cell barrier function by lowering cAMP levels (15). cAMP is the major up regulator of thrombomodulin expression, so we investigated the effect of agents able to increase cAMP levels on membrane thrombomodulin expression in hypoxic conditions on HUVEC

Fluorescent in situ hybridization on flow‐sorted cells as a tool for evaluating minimal residual disease or chimerism after allogeneic bone marrow transplantation

We studied the feasibility and the sensitivity of fluorescent in situ hybridization (FISH) using leukemic or host/donor-specific probes on flow-sorted cells to assess minimal residual disease (MRD) or chimerism in transplanted patients in complete remission. We first performed experimental models of MRD and chimerism by mixing HL60 cells and normal lymphocytes in different proportions. Over 80% HL60 cells were obtained from mixtures of 5% HL60 cells in peripheral blood mononuclear cells (PBMC). We then evaluated MRD and mixed chimerism in a chronic myelogenous leukemia patient in relapse after allogeneic sex-mismatched bone marrow transplantation (BMT), who had received a donor lymphocyte infusion (DLI). Three months after DLI, mixed chimerism was observed in each bone marrow (BM)-sorted lineage (CD13+, CD14+, CD20+, and CD3+), with the highest level of recipient cells in the granulocytic lineage (CD13+). Five months after DLI, host cells were at a low level but remained detectable in the granulocytic lineage. In the same sample, the bcr-abl gene was detected in the granulocytic lineage and not in the lymphocytes. We also studied chimerism in an aplastic anemia sex-mismatched transplanted female patient. We determined the proportion of recipient total lymphocytes, CD4+ and CD8+ lymphocytes, and CD14+ monocytes under cyclosporin A therapy on five peripheral blood samples and one BM sample over 5 months. Results showed a regular decrease in recipient total lymphocytes (26.6% to 10.6%) and monocytes (20.7% to 8%). CD8(+)-recipient cells decreased rapidly, while CD4+ remained stable (17%). This work demonstrates the feasibility of FISH after cell sorting, combining the sensitivities of both flow cytometry and FISH and the specificities of both immunophenotyping and genotype analysis.

Influence Of Hypoxia And Hypoxia-Reoxygenation On Endothelial P-Selectin Expression

P-selectin is an endothelial adhesion molecule involved in the initial step of the neutrophil recruitment. We investigated the effect of hypoxia (95% N2, 5% CO2) and of hypoxia-reoxygenation (95% air, 5% CO2) on the expression of P-selectin by human umbilical vein endothelial cells (HUVEC). P-selectin expression was detected by immunolabelling and quantified by flow cytometric analysis. Our data indicate that hypoxia induces an increase in P-selectin expression with a maximum reached after 90 minutes. A hypoxic exposure of 90 minutes results in a highly significant increase compared to normoxia (p < 0.001, n = 13). Furthermore, when a reoxygenation period follows 90 minutes of hypoxia, the initially elevated levels of P-selectin are dramatically enhanced with a maximum obtained after 60 minutes of reoxygenation.

Flow cytometric study of the activation of polymorphonuclear cells.

The activation of human polymorphonuclear cells (PMN) by the chemotactic peptide, N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), or the protein kinase C activator, phorbol myristate acetate (PMA), was studied using flow cytometry. Two probes were used to evaluate PMN activation: 1) a monoclonal antibody (MoF11) directed against an antigen (Ag) expressed on the membrane of monocytes and of activated PMN; 2) rhodamine phalloidin was used at the cytoplasmic level to measure the F‐actin content. The expression of MoF11 antigen was found to be 3 to 5 times greater on the membrane of PMN activated by either FMLP or PMN as compared with membrane expression of the same Ag on resting PMN. This increase was found to be dose dependent for the two activators. Kinetic studies showed that a maximum response was observed in 1 to 2 min at 37° when FMLP was used, whilst a similar response required 10 min when PMA was used. The same discrepancy with activators was observed when actin polymerization was measured by labelling with rhodamine phalloidin. However, pretreatment of PMN with cytochalasin B inhibited actin polymerization whilst MoF11 antigen expression was increased, suggesting that the MoF11 antigen could be stored in granules of resting PMN. The study of actin polymerization and of MoF11 antigen expression, separately or in combination, could be a useful tool for the detection of activated PMN in biological samples.

Flow cytometric evaluation of fas expression in relation to response and resistance to anthracyclines in leukemic cells.

BACKGROUND Cell chemosensitivity to cytotoxic drugs has been attributed to their ability to trigger apoptosis. The emergence of resistance in drug-exposed cells is often characterized by the appearance of drug efflux mechanisms including P-gp transport. Nevertheless, mdr1 expression may coexist with other resistance features, in particular those interfering with apoptotic signaling pathways. METHODS Leukemic cell lines cultured in a progressively toxic environment were analyzed for Fas and P-gp expression by immunostaining and flow cytometry. Their mdr1 mRNA expression level was determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and their apoptotic response was microscopically evaluated. Activation of the Fas pathway was obtained by cross-linking the Fas receptor with the 7C11 anti-Fas agonist. RESULTS We demonstrate a dose-dependent Fas overexpression after short-term (18 h) incubation with daunorubicin. The subsequent sensitization to Fas activators led to a significant increase in the apoptotic response induced by 7C11. After long-term exposure to daunorubicin and acquisition of drug resistance, expression of P-gp was accompanied by a decrease in the number of Fas sites at the cell surface with a correlated desensitization to Fas-induced apoptosis. Additional alterations in the Fas signaling pathway can also be hypothesized in the most resistant Jurkat cell line. CONCLUSIONS The induction of Fas expression could be one of the mechanisms of action of chemotoxic drugs and thus might enhance the cell susceptibility to Fas-mediated apoptosis. On the contrary, the emergence of the multidrug resistance phenotype is associated with a down-regulation of Fas expression and possible defects in the Fas signaling pathway.