Guy Laurent

Expression of β-catenin by acute myeloid leukemia cells predicts enhanced clonogenic capacities and poor prognosis

Activation of the Wnt/β-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 β-catenin was correlated to clonogenic capacity also in the acute myeloid leukemia (AML) setting. Eighty-two patients were retrospectively evaluated for β-catenin expression by Western blot. β-Catenin was expressed (although at various protein levels) in 61% of patients, and was undetectable in the remaining cases. In our cohort, β-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, β-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 β-catenin protein levels were dependent on post-transcriptional mechanisms involving the Wnt/β-catenin pathway only in leukemic cells. Indeed, β-catenin negative leukemic cells were found to increase β-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.

Potential Role for Ceramide in Mitogen-activated Protein Kinase Activation and Proliferation of Vascular Smooth Muscle Cells Induced by Oxidized Low Density Lipoprotein

Proliferation of vascular smooth muscle cells (SMC) is a hallmark in the pathogenesis of atherosclerotic lesions. Mildly oxidized low density lipoproteins (UV-oxLDL), which are mitogenic to cultured AG-08133A SMC, activate the sphingomyelin (SM)-ceramide pathway. We report here the following. (i) UV-oxLDL elicited a biphasic and sustained activation of MBP kinase activity, phosphorylation and nuclear translocation of p44/42 mitogen-activated protein kinase (MAPK), and [3H]thymidine incorporation, which were inhibited by PD-098059, a MAPK kinase inhibitor. (ii) The use of preconditioned media (from SMC pre-activated by UV-oxLDL) transferred to native SMC and blocking antibodies against growth factors suggest that UV-oxLDL-induced activation of MAPK and [3H]thymidine incorporation seem to be independent of any autocrine secretion of growth factors. (iii) UV-oxLDL-induced activation of a neutral sphingomyelinase, SM hydrolysis, ceramide production, and [3H]thymidine incorporation were inhibited by two serine-protease inhibitors (serpins), suggesting that a serpin-sensitive proteolytic pathway is involved in the activation of the SM-ceramide signaling pathway. (iv) UV-oxLDL-induced MAPK activation and [3H]thymidine incorporation were mimicked by ceramide generated in the plasma membrane by bacterial sphingomyelinase treatment or by addition of the permeant C2-ceramide. Serpins did not inhibit the MAPK activation and [3H]thymidine incorporation induced by C2-ceramide, indicating that activation of the MAPK and [3H]thymidine incorporation is subsequent to the stimulation of the SM-ceramide pathway. Taken together, these data suggest that mitogenic concentrations of UV-oxLDL are able to stimulate the SM-ceramide pathway through a protease-dependent mechanism and activate p44/42 MAPK, leading to proliferation of vascular SMC.

The phosphoinositide 3-kinase/Akt pathway is activated by daunorubicin in human acute myeloid leukemia cell lines

Daunorubicin induces apoptosis in myeloid leukemia cells by activation of neutral sphingomyelinase and ceramide generation occurring 4–10 min after daunorubicin addition. We show here that daunorubicin is able to increase the phosphoinositide 3‐kinase activity and enhance intracellular phosphoinositide 3‐kinase lipid products prior to ceramide generation. Daunorubicin activates Akt, a downstream phosphoinositide 3‐kinase effector. Interestingly, the phosphoinositide 3‐kinase inhibitors wortmannin and LY294002 accelerate daunorubicin‐induced apoptosis in U937 cells. The phosphoinositide 3‐kinase/Akt pathway has been involved in cell survival following serum deprivation, tumor necrosis factor α, anti‐Fas and UV radiations. Our results suggest that anti‐tumor agents such as daunorubicin may also activate anti‐apoptotic signals that could contribute to drug resistance.

Human telomerase is regulated by erythropoietin and transforming growth factor-|[beta]| in human erythroid progenitor cells

Telomerase catalytic subunit (hTERT) exerts important cellular functions including telomere homeostasis, genetic stability, cell survival and perhaps differentiation. However, the nature of external or internal signals, which regulate hTERT expression in tissues, remains poorly understood. Thus, whereas it has been described that hTERT gene is regulated along the differentiation of primitive myeloid progenitors, the effect of specific cytokines on telomerase expression in each myeloid lineage is currently unknown. Based on these considerations, we have investigated hTERT expression in erythroid cells treated with erythropoietin (EPO) and transforming growth factor β (TGFβ), as putative positive and negative regulators, respectively. We describe here that EPO activates hTERT gene transcription in in vitro-expanded primary erythroid precursors as well as in UT7 erythroleukemia cells. In UT7 cells, this study shows also that EPO acts through a JAK2/STAT5/c-myc axis. In contrast, TGFβ blocks EPO signaling downstream of c-myc induction through a Smad3-dependent mechanism. Finally, hTERT appears to be efficiently regulated by EPO and TGFβ in an opposite way in erythropoietic cells, arguing for a role of telomerase in red blood cell production.

The cell cycle regulator CDC25A is a target for JAK2V617F oncogene

The JAK2(V617F) mutation is present in the majority of patients with polycythemia vera and one-half of those with essential thrombocythemia and primary myelofibrosis. JAK2(V617F) is a gain-of-function mutation resulting in constitutive JAK2 signaling involved in the pathogenesis of these diseases. JAK2(V617F) has been shown to promote S-phase entry. Here, we demonstrate that the CDC25A phosphatase, a key regulator of the G1/S cell-cycle transition, is constitutively overexpressed in JAK2(V617F)-positive cell lines, JAK2-mutated patient CD36(+) progenitors, and in vitro-differentiated proerythroblasts. Accordingly, CDC25A is overexpressed in BM and spleen of Jak2(V617F) knock-in mice compared with wild-type littermates. By using murine FDC-P1-EPOR and human HEL and SET-2 cell lines, we found that JAK2(V617F)-induced CDC25A up-regulation was caused neither by increased CDC25A transcription or stability nor by the involvement of its upstream regulators Akt and MAPK. Instead, our results suggest that CDC25A is regulated at the translational level through STAT5 and the translational initiation factor eIF2α. CDC25A inhibition reduces the clonogenic and proliferative potential of JAK2(V617F)-expressing cell lines and erythroid progenitors while moderately affecting normal erythroid differentiation. These results suggest that CDC25A deregulation may be involved in hematopoietic cells expansion in JAK2(V617F) patients, making this protein an attracting potential therapeutic target.

Degadration of mismatch repair hMutSα heterodimer by the ubiquitin‐proteasome pathway

Mismatch repair plays a critical role in genome stability. This process requires several proteins including hMSH2/hMSH6 (hMutSα) heterodimer involved in the first stage of the process, the mispair recognition. We previously reported that in U937 and HL‐60 cell lines, hMSH2 and hMSH6 protein expression was much lower than that in HeLa and KG1a cells. Here, we showed that the decreased expression of hMutSα results from differences in the degradation rate of both proteins by the ubiquitin‐proteasome pathway. Our data suggest that in human cell lines, ubiquitin‐proteasome could play an important role in the regulation of hMutSα protein expression, thereby regulating mismatch repair activity.

Alteration of the daunorubicin-triggered sphingomyelin-ceramide pathway and apoptosis in MDR cells: Influence of drug transport abnormalities

We have previously shown that in myeloid leukemic cells, daunorubicin (DNR) induces apoptosis via the activation of the sphingomyelin‐ceramide pathway. We have now investigated sphingomyelin (SM) hydrolysis, ceramide generation, and apoptosis in vincristine‐selected multidrug resistant (MDR) HL‐60 cells (HL‐60/Vinc), compared with their parental counterparts. We show that DNR triggers the SM cycle (stimulation of neutral sphingomyelinase, SM hydrolysis, and ceramide generation) and apoptosis in both parental and MDR cells, when used at isotoxic doses (i.e., 1 and 100μM for HL‐60 and HL‐60/Vinc, respectively). However, in MDR cells treated with either 10μM DNR or 1μM DNR in association with the P‐glycoprotein (P‐gp) blocker verapamil (treatment conditions which yield an intracellular DNR concentration similar to that achieved with 1μM in the parental cells), we were unable to detect SM hydrolysis, ceramide generation and apoptosis. This implies that inhibition of the DNR‐induced SM cycle in MDR cells is not directly related to P‐gp. We have also investigated the influence of intracellular drug localization on the DNR‐induced SM‐cycle in HL‐60/Vinc cells. In these cells, DNR at 10μM is mainly localized in cytoplasmic vesicles, while the drug is diffusely distributed when used at 100μM. A diffuse distribution pattern was also observed when MDR cells were treated with 1μM DNR in association with the cyclosporine derivative PSC‐833, but not with verapamil. In parallel, PSC‐833, but not verapamil, restored the induction of the SM cycle and the apoptotic potential of DNR, and markedly increased drug cytotoxicity in MDR cells. Our results suggest that altered intracellular drug transport plays an important role in limiting ceramide generation and cell death in MDR cells. Int. J. Cancer 81:580–587, 1999.

TNFα 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 TNFα, 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 TNFα, leukemic cells may exert cytotoxicity against myeloid progenitors. This finding may have important clinical implications in the context of diseases characterized by TNFα accumulation, such as AML or myelodisplasic syndromes.