The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells

The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca(2+)/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34(+) progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34(+) cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34(+) and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBPepsilon, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.     

Automated analysis of differentiation-induced leukemic cells during all-trans retinoic Acid therapy of acute promyelocytic leukemia

During differentiation-induction therapy of acute promyelocytic leukemia (APL) patients with all-trans retinoic acid (ATRA), a variety of APL-derived bizarre granulocytic cells appear in the peripheral blood. To evaluate the differentiation induction of leukemic cells, we have developed a new scattergram analyzing program with an automated hematology analyzer and compared the data with the flow cytometry measuring the expression of differentiation-associated cell surface antigens, CD11b and CD16. We used the fluorescence intensity and side scatter as parameters of granulocytic maturation in the analysis with the automated hematology analyzer. The analysis of 2 ATRA-treated APL patients and in vitro study using HL-60 cells demonstrated that the levels of fluorescence intensity and side scatter decreased as accompanied with granulocytic maturation, and these changes were parallel with the results of flow cytometry. Our automated scattergram analysis of cell differentiation will contribute to general, objective, and real-time evaluation of differentiation-induction therapy of APL with ATRA.     

New strategies for the treatment of acute myelogenous leukemia: differentiation induction--present use and future possibilities

A differentiation block and an accumulation of immature myeloid cells characterize acute myelogenous leukemia (AML). However, native AML cells usually show some morphological signs of differentiation that allow a classification into different subsets, and further differentiation may be induced by exposure to various soluble mediators, for example, all-trans retinoic acid (ATRA) and several cytokines. Combination therapy with ATRA and chemotherapy should now be regarded as the standard treatment of the acute promyelocytic leukemia (APL) variant of AML. Although several agents can also induce leukemic cell differentiation for other AML subgroups, in vitro studies as well as clinical data have demonstrated that these agents often have heterogeneous effects on the leukemic progenitors. This makes the clinical impact of differentiation induction therapy for individual patients difficult to predict. However, differentiation induction should be regarded as a promising therapeutic approach, especially as a part of immunotherapy or in combination with intensive chemotherapy to increase the susceptibility of AML blasts to drug-induced apoptosis. Although the morphology-based French-American-British classification was used to identify APL as an AML subset that required a special treatment, it seems unlikely that this classification alone can be used to identify new subsets of AML patients with special therapeutic requirements. Future studies on differentiation induction in AML should therefore focus on A) the identification of therapeutic agents with more predictable effects; B) the use of clinical and laboratory parameters to define new subsets of AML patients in which differentiation induction has a predictable and beneficial effect, and C) the characterization of how AML blast sensitivity to drug-induced apoptosis is altered by differentiation induction.     

Lithium Chloride Promotes Apoptosis in Human Leukemia NB4 Cells by Inhibiting Glycogen Synthase Kinase-3 Beta

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML). With the application of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), APL becomes one of best prognosis of leukemia. However, ATRA and ATO are not effective against all APLs. Therefore, a new strategy for APL treatment is necessary. Here, we investigated whether lithium chloride (LiCl), a drug used for the treatment of mental illness, could promote apoptosis in human leukemia NB4 cells. We observed that treatment with LiCl significantly accelerated apoptosis in NB4 cells and led to cell cycle arrest at G2/M phase. Moreover, LiCl significantly increased the level of Ser9-phosphorylated glycogen synthase kinase 3β(p-GSK-3β), and decreased the level of Akt1 protein in a dose-dependent manner. In addition, LiCl inhibition of c-Myc also enhanced cell death with a concomitant increase in β-catnin. Taken together, these findings demonstrated that LiCl promoted apoptosis in NB4 cells through the Akt signaling pathway and that G2/M phase arrest was induced by increase of p-GSK-3β(S9).     

NLS-RARα Inhibits the Effects of All-trans Retinoic Acid on NB4 Cells by Interacting with P38α MAPK

Nuclear localization signal retinoic acid receptor alpha(NLS-RARα), which forms from the cleavage of promyelocytic leukemia-retinoic acid receptor alpha(PML-RARα) protein by neutrophil elastase(NE), possesses an important role in the occurrence and development of acute promyelocytic leukemia(APL). However, the potential mechanism underlying the effects of NLS-RARα on APL is still not entirely clear. Here, we investigated the effects of NLS-RARα on APL NB4 cells and its mechanism. We found that all-trans retinoic acid(ATRA) could promote differentiation while inhibit proliferation of APL NB4 cells via upregulating the expression of phosphorylated p38α mitogen-activated protein kinase(p-p38α MAPK). We also found that NLS-RARα could inhibit differentiation while accelerate proliferation of NB4 cells via downregulating the expression of p-p38α protein in the presence of ATRA. Furthermore, immunofluorescence and co-immunoprecipitation assays confirmed NLS-RARα interacted with p38α protein directly. Finally, application of PD169316, an inhibitor of p38α protein, suggested that recruitment p38α-combinded NLS-RARα by ATRA eventually caused activation of p38α protein. In summary, our study demonstrated that ATRA cound promote differentiation while inhibit proliferation of APL NB4 cells via activating p38α protein after recruiting p38α-combinded NLS-RARα, while NLS-RARα could inhibit the effects of ATRA in the process.     

IL-1对维甲酸诱致高白细胞症的影响

使用生物活性检测法检测ATRA治疗前后APL患者血清中IL-1活性的动态变化,并分析IL-1活性与外周血白细胞数变化的相关性。观察经ATRA作用后不同时相NB4细胞、巨噬细胞和血管内皮细胞上清液中IL-1活性的动态变化,并以RT-PCR法检测NB4细胞中IL-1 mRNA表达的变化。结果表明,患者外周血白细胞数及粒细胞数与血清中IL-1活性的变化相关;ATRA作用后,NB4细胞、巨噬细胞和血管内皮细胞上清液中IL-1活性均呈时间依赖性增高;ATRA可上调NB4细胞中IL-1的表达。结论:ATRA治疗APL过程中,多种细胞来源的IL-1可能参与了高白细胞症的发生。     

Diagnostic challenges related to myeloid/natural killer cells, a variant of myeloblasts

The authors report herein two diagnostically challenging cases centered on the myeloid/natural killer (myeloid/NK) cells, a variant of myeloblasts, to illustrate the importance of advanced flow cytometric immunophenotyping and an updated understanding of surface markers in hematopoietic malignancies. Myeloid/NK cell acute leukemia is a very rare subtype of leukemia. Although its NK-cell nature is debatable, it represents a variant of leukemia with distinct morphological and immunophenotypical features. The first case is a de novo myeloid/NK-cell acute leukemia with a striking clinical, morphologic and immunophenotypic resemblance to acute promyelocytic leukemia (APL), but which could be distinguished by its CD11a, CD18, CD117 and CD9 expression. This case illustrates the importance of utilizing the APL surrogate surface phenotype of HLA-DR(low), CD11a(low) and CD18(low) by flow cytometric study to rule in/out APL immunophenotypically. In the second case, we show that myeloid/NK-cell blasts can present as a variant of blasts in a preleukemic disease as refractory anemia with excess blasts-1 (RAEB-1), where the blasts were negative for CD34, CD117 and HLA-DR. The recognition of such blast variant is important in appropriately classifying such preleukemic diseases by blast percentage.     

三氧化二锑诱导急性早幼粒细胞白血病细胞凋亡的研究

目的 研究锑剂三氧化二锑（Sb2O3)对早幼粒细胞白血病细胞株NB4凋亡的诱导作用，以寻求早幼粒细胞白血病治疗的新方法。方法 采用细胞生长曲线，形态学及硝基四氮唑蓝（NBT）还原试验，判定NB4细胞的生长，分化及功能。采用细胞周期分析和DNA电泳研究细胞凋亡。结果 Sb2O3能诱导早幼粒白血病细胞凋亡，且具有时间，剂量依赖性。结论 Sb2O3能有效地诱导早幼粒白血病细胞凋亡，提示锑剂诱导细胞半亡的疗法，有望成为临床治疗早幼粒细胞白血病的新方法。     

乌苯美司下调c-myc表达与其增强全反式维甲酸诱导NB4细胞

目的： 了解氨肽酶抑制剂乌苯美司（bestatin）能否增强全反式维甲酸（ATRA）对NB4细胞的诱导分化作用，及此过程中NB4细胞c-myc mRNA表达水平的改变。 方法： MTT法检测药物抑制细胞生长的作用。流式细胞仪测细胞表面分化抗原CD11b及四氮唑蓝（NBT）还原实验检测NB4细胞的分化。RT-PCR检测细胞c-myc mRNA表达水平。 结果： 50 mg/L、75 mg/L、100 mg/L乌苯美司与10 nmoL/L ATRA联合处理72 h，均能明显增强NB4细胞的NBT还原能力，与10 nmoL/L ATRA组差异显著（P<0.05，P<0.01）。从48 h到96 h，100 mg/L乌苯美司时间依赖性地增强10 nmoL/L ATRA诱导NB4细胞的NBT还原能力，与相应时点10 nmoL/L ATRA组差异明显（P<0.01）。100 mg/L乌苯美司与10 nmoL/L ATRA联合应用72 h，NB4细胞CD11b表达率明显高于10 nmoL/L ATRA组（P<0.01）。50 mg/L、75 mg/L、100 mg/L乌苯美司与10 nmoL/L ATRA联合处理4 h，NB4细胞c-myc mRNA表达水平明显低于单用ATRA组（P<0.05，P<0.01）；药物联合应用各组NB4细胞的c-myc mRNA表达水平与NBT还原能力之间呈负相关（r=-0.917,P<0.05）。 结论： 乌苯美司可能通过下调NB4细胞c-myc mRNA的表达，从而增强ATRA诱导NB4细胞分化的作用。     

Spontaneous migration of acute promyelocytic leukemia cells beneath cultured bone marrow adipocytes with matched expression of the major histocompatibility complex

Leptin, secreted by adipocytes in bone marrow stimulates proliferation of acute myeloid leukemia cells. PML-RARalpha-expressing acute promyelocytic leukemia (APL) cells express high levels of leptin receptor (OB-R). Adipocytes derived from mesenchymal stem cells (MSC) protect APL cells from drug-induced apoptosis partially through the interaction of the membrane-bound form of leptin and OB-R. We report that when NB4 APL cells migrated beneath a cultured MSC-derived adipocyte layer, apoptosis in most of the cells below the adipocytes was blocked. Major histocompatibility complex (MHC) expression of NB4 cells and MSCs matched (both were HLA-DR negative and HLA-ABC positive). This match in MHC expression may play a role in the intimate cell-to-cell interactions that support the anti-apoptotic effects of the membrane-bound leptin signaling pathway.     

NRP-1/CD304 expression in acute leukemia: a potential marker for minimal residual disease detection in precursor B-cell acute lymphoblastic leukemia

Neuropilin-1 (NRP-1)/CD304 is a marker for plasmacytoid dendritic cells. We determined the distribution of NRP-1/CD304 expression on normal hematopoietic cells and in 167 acute leukemias by flow cytometry. NRP-1/CD304 surface expression was frequent in precursor B-cell acute lymphoblastic leukemia (36/51 [71%]) and uncommon in acute myeloid leukemia (22.9%). In acute myeloid leukemia, expression was noted in all (4/4) acute myeloid leukemias with the M4eo subtype and in 50% of specimens (6/12) with complex cytogenetics. On hematopoietic cells, NRP-1/CD304 was expressed on normal erythroid progenitors, plasma cells, and B-cell progenitors, as well as plasmacytoid dendritic cells. Expression was not consistently detected on other hematopoietic cell types. Owing to this distribution of expression, the detection of NRP-1/CD304 alone on a hematopoietic cell cannot be used to determine plasmacytoid dendritic cell differentiation. Finally, we show that NRP-1/CD304 is overexpressed in 30% of precursor B-cell acute lymphoblastic leukemia samples compared with normal B-cell progenitors, allowing for its potential use as a marker for the detection of minimal residual disease.     

Podocalyxin: a marker of blasts in acute leukemia

Podocalyxin is a CD34 family member expressed by podocytes, vascular endothelium, mesothelium, and a subset of hematopoietic progenitors. Podocalyxin expression was not observed in the hematopoietic cells of normal adult bone marrow samples. However, podocalyxin was expressed by blasts in 30 (77%) of 39 cases of acute myeloid leukemia (AML), 22 (81%) of 27 cases of acute lymphoblastic leukemia (ALL), and 13 (87%) of 15 cases of cutaneous myeloid sarcoma. No correlation with CD34 expression by immunohistochemical analysis was seen. Wilms tumor 1 (WT1) expression was detected in blasts in 17 AML cases (44%) and 21 ALL cases (78%). There was no correlation between WT1 and podocalyxin expression. We conclude that podocalyxin is expressed commonly by blasts in ALL and AML. Analysis of the expression of CD34 and podocalyxin increases sensitivity for the immunophenotypic detection of leukemic blasts compared with the analysis of CD34 alone. Therefore, podocalyxin seems to complement CD34 as a useful hematopoietic blast marker. The physiologic role of podocalyxin in leukemic blasts remains unknown.     

The comparison of bone marrow kinetics between patients with acute myeloid leukemia and acute promyelocytic leukemia after induction chemotherapy

Abstract

Background and Aim: Recently, acute promyelocytic leukemia (APL) has shifted from the most hazardous to the best curable type of acute myeloid leukemia. Anthracyclines, all-trans retinoic acid (ATRA) and arsenic derivatives are the most important developments for the treatment of APL. ATRA promotes the terminal differentiation of malignant promyelocytes to mature neutrophils. We aimed to compare platelet and neutrophil recovery time after induction chemotherapy in patients with acute myeloid leukemia (AML) and APL.

Materials and Methods: Two hundred and fifteen patients with AML and APL, who were diagnosed and treated in our tertiary care center between the years of 2001 and 2018 were evaluated.

Results: One hundred and eighty one AML patients (84.2%) and 34 (15.8%) APL patients were included in this study. The time between neutrophil nadir after induction chemotherapy and neutrophil recovery was longer in APL patients than in AML patients [30.5 (4–52) vs. 20 (5–58), p?<?0.001]. The time between platelet nadir after induction chemotherapy and platelet recovery was longer in APL patients than in AML patients [21.5 (4–42) vs. 17 (4–45), p?=?0.02].

Conclusion: Neutrophil and platelet recovery times were longer in APL patients than in AML patients in our present study. In 60?days, mortality rate was higher in APL patients than AML patients. Non-relapse mortality (NRM) rate was similar between two groups. There was a significant difference between two groups in terms of NRM causes. Platelet and neutrophil recovery time is very important because infection is the most important cause of NRM.     

Construction of protein profile classification model and screening of proteomic signature of acute leukemia

The French-American-British (FAB) and WHO classifications provide important guidelines for the diagnosis, treatment, and prognostic prediction of acute leukemia, but are incapable of accurately differentiating all subtypes, and not well correlated with the clinical outcomes. In this study, we performed the protein profiling of the bone marrow mononuclear cells from the patients with acute leukemia and the health volunteers (control) by surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI_TOF_MS). The patients with acute leukemia were analyzed as unitary by the profiling that were grouped into acute promyelocytic leukemia (APL), acute myeloid leukemia-granulocytic (AML-Gran), acute myeloid leukemia-monocytic (AML-Mon) acute lymphocytic leukemia (ALL), and control. Based on 109 proteomic signatures, the classification models of acute leukemia were constructed to screen the predictors by the improvement of the proteomic signatures and to detect their expression characteristics. According to the improvement and the expression characteristics of the predictors, the proteomic signatures (M3829, M1593, M2121, M2536, M1016) characterized successively each group (CON, APL, AML-Gra, AML-Mon, ALL) were screened as target molecules for identification. Meanwhile, the proteomic-based class of determinant samples could be made by the classification models. The credibility of the proteomic-based classification passed the evaluation of Biomarker Patterns Software 5.0 (BPS 5.0) scoring and validated application in clinical practice. The results suggested that the proteomic signatures characterized by different blasts were potential for developing new treatment and monitoring approaches of leukemia blasts. Moreover, the classification model was potential in serving as new diagnose approach of leukemia.     

Antigen shared by human hemopoietic precursor cells and T and B lineage cells

In this study, we investigated antigens present at the surface of acute myeloblastic leukemia (AML) cells by using the monoclonal antibody (MAb) approach. The MAb AGF43 reacted with acute myeloid and lymphoid leukemia cells and chronic lymphocytic leukemia cells and was unreactive against chronic myeloid leukemia cells. A large proportion of AML blasts showing minimal or no differentiation (AML-M1) were intensely labeled by AGF43 in contrast to a smaller percentage of blasts showing partial differentiation (AML-M2 and acute myelomonocytic leukemia). The AGF43 antigen is expressed by bone marrow lymphoid (TdT+) and myeloid (CFU-GM) progenitor cells, 95% of B cells and 65% of T cells in the blood and absent from monocytes. Only 17% of normal myeloblasts were weakly stained by AGF43. Sections of tonsil and spleen were used to confirm that, unlike antibodies to MHC class II antigens, AGF43 stained a majority of T cells and macrophages were unreactive. In conclusion, the MAb AGF43 identifies a new precursor cell antigen. The distribution of this antigen during normal myelopoiesis and on AML cells support the suggestion that acute myeloid leukemias originate in pluripotent or closely related myeloid stem cells.     

Effects of vitamin D on the growth of normal and malignant B-cell progenitors.

As the effects of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) (VD, calcitriol) on the proliferation and differentiation potential of normal and leukaemic cells in vitro of myeloid lineage are known, we investigated the response to VD on the growth of both normal and malignant lymphoid progenitors. Effects of vitamin D on normal human lymphoid progenitors and B lineage acute lymphoblastic leukaemia (ALL) progenitors were assessed by using an in vitro cell colony assay specific for either B or T cell lineages. The expression of VDR on B untreated malignant progenitors at diagnosis was investigated by RT-PCR analysis. VD induced a significant inhibition of normal lymphoid cell progenitors growth of both T and B lineage. VD inhibited significantly also the growth of malignant B cell lineage lymphoid progenitors, without inducing cytotoxic effect. As it has been reported that VD effects on activated lymphocytes are mediated by 1,25-(OH)2-D3 nuclear receptor (VDR), we investigated VDR expression on malignant B cell progenitors. We did not detect VDR expression on these cells examined at diagnosis. We demonstrated that VD inhibited in vitro the clonogenic growth of both normal and malignant lymphoid B cell progenitors and that this inhibitory effect on malignant B cell progenitors was not related to VDR. Our work contributes to understanding of the mechanism of action of this hormone in promoting cellular inhibition of clonal growth of malignant lymphoid B cell progenitors, suggesting that the regulation of some critical growth and differentiation factor receptors could be a key physiological role of this hormone.     

Evidence for early hematopoietic progenitor cell involvement in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) represents a subtype of acute myeloid leukemia with characteristic morphologic, molecular, and immunophenotypic features. Previous immunophenotypic analyses have shown that leukemic cells in APL typically express the myeloid markers CD33 and CD13 but lack expression of the early hematopoietic progenitor cell antigens CD34 and HLA-DR. We analyzed selected immunophenotypic features of APL by flow cytometry and showed that 7 (41%) of 17 cases contained significant subsets of CD34+ leukemic cells: CD34+ myeloid cells predominated in 2 APL cases. By using a fluorescence-activated cell sorter-fluorescence in situ hybridization approach, we confirmed that the CD34+ cells harbored the t(15;17) translocation characteristic of APL. By using the same experimental approach, CD34+ populations were stratified into primitive CD34+ CD38- and committed CD34+ CD38+ progenitor cell subpopulations; cells in both subsets contained the t(15;17) translocation. The knowledge that APL may be partly or largely CD34+ is important for proper diagnosis. Furthermore, identification of the t(15;17) translocation in CD34+ CD38- blasts indicates that, in at least some cases, the leukemogenic mutation in APL occurs within primitive hematopoietic progenitor cells.     

Down-regulation of CD44 contributes to the differentiation of HL-60 cells induced by ATRA or HMBA

CD44 is highly expressed in human acute myeloid leukemia （AML） cells. Some experiments had shown that it was possible to reverse differentiation blockage in AML cells by CD44 ligation with specific antibodies, indicating that CD44 was closely related to the differentiation of leukemia cells. The differentiation of acute promyelocytic leukemia cell line HL-60 cells could be induced by all trans-retinoic acid （ATRA） and hexamethylene bisacetamide （HMBA）, but so far the mechanism was not demonstrated clearly. In the present study, we investigated whether ATRA or HMBA induced the growth arrest of HL-60 cells by down-regulating the expression of CD44. The results indicated that the proliferation of HL-60 cells was obviously inhibited and the differentiation was induced by both ATRA and HMBA. The decreased expression of CD44 and cyclin E mRNA, and the increased expression of p27 and p21 at mRNA levels were observed. Furthermore, there was a negative correlation between the expression of CD44 and p27. It was concluded that ATRA and HMBA played a role in the differentiation induction of HL-60 cells, which was mediated by the down-regulation of CD44, accompanied by down-regulation of cyclin E, and up-regulation of p27 and p21 mRNA. Cellular ＆ Molecular Immunology. 2007;4（1）：59-63.