Induction of Differentiation of Mouse Myeloid Leukemia M1 Cells by Serum of Patients with Chronic Myeloid Leukemia

We examined the capacities of sera from patients with myeloid leukemia to induce differentiation in mouse myeloid leukemic M1 cells. Higher differentiation-inducing activity (D-activity) was detected in sera of patients with chronic myelomonocytic leukemia or chronic myeloid leukemia (CML) than in sera of patients with acute myeloid leukemia and normal volunteers. The D-activity in the sera was lost on heating the sera at 56° for 30 min. The major peak of D-activity on Sephadex G-200 gel filtration had an apparent molecular weight of 160,000. The origin of the D-activity in sera of patients with CML was studied by culturing fractions of peripheral blood cells of patients with D-activity for 3 days and then measuring the ability of the conditioned medium (CM) to induce differentiation of M1 cells. The cells in the myeloblast and promyelocyte fraction differentiated spontaneously into macrophage-like cells during culture for 3 days and the cells in the late granulopoietic cell fraction differentiated into neutrophil-like cells. Higher D-activity was present in CM of cells in the myeloblast and promyelocyte fraction than in CMs of late granulopoietic cell fractions. These results suggest that human leukemic cells produce D-activity for M1 cells during their differentiation into macrophage-like cells.     

氨基甾体 Z1对 MEG-01白血病细胞的抑制增殖及诱导分化作用

目的：观察氨基甾体Z1对人慢性粒细胞白血病MEG-01细胞的抑制增殖和诱导分化作用。方法采用液体培养实验、集落培养实验、MTT实验观察氨基甾体Z1对MEG-01细胞的抑制增殖作用;采用Wright染色、AS-D NAE染色、细胞膜CD41表面标记物检测氨基甾体Z1对MEG-01细胞的诱导分化作用。结果10－8～10－4 mol／L的氨基甾体Z1连续作用后,细胞和集落计数明显减少,处理组MTT值显著降低,且呈浓度依赖;Wright染色显示处理组细胞有分化表现,AS-D NAE染色A值升高（P＜0．01）,流式细胞术检测处理组细胞膜上CD41表面标记表达阳性率为76％。结论氨基甾体Z1能显著抑制MEG-01细胞增殖并诱导其向巨核细胞系分化,提示该药或可成为慢性粒细胞白血病的一种新型诱导分化剂。     

The Efficient Generation of Immunocompetent Dendritic Cells from Leukemic Blasts in Acute Myeloid Leukemia: A Local Experience

Dendritic cells (DCs) are the most important antigen presenting cells with potentially useful applications in cancer immunotherapy. Leukemic cells of patients with acute myeloid leukemia (AML) could be differentiated to DC-like cells possessing the ability of stimulating anti-leukemic immune response. Despite obvious progress in DC-based immunotherapy, some discrepancies were reported in differentiation potential of AML blasts from all patients toward DC like cells. The present study, as a local experience, was set up to generate DCs from AML blasts of various subtypes. Leukemic Blasts from 16 Iranian AML patients were differentiated into functional DCs by culturing in the presence of rhGM-CSF, rhIL-4 and TNF-alpha for 8 days. The morphology, expression of key surface molecules and allostimulatory activity of resultant DCs were compared with primary blasts and cultured but cytokine untreated control groups. The pattern of angiotensin-converting enzyme (ACE) expression was used to approve the leukemic origin of generated DCs. Neo-expression or upregulation of DC-associated markers were occurred during culturing period in cytokine treated cells compared with primary blasts and cultured but cytokine untreated control groups: CD1a (63.22% vs. 3.22% and 11.79%), CD83 (41.27% vs. 0.11% and 0.70%), CD40 (15.17% vs. 0.00% and 0.04%), CD80 (49.96 vs. 0.02% and 0.32%), CD86 (56.49% vs. 0.50% and 5.71%) and HLA-DR (52.52% vs. 14.32% and 2.49%) respectively. The potency of generated DCs to induce allogeneic T cell proliferation increased significantly compared to pre and post culture control groups (27,533.4 ± 2,548.3, 8,820.4 ± 1,639.4 and 3,200.35 ± 976 respectively). The expression pattern of ACE in AML-DCs, blast cells and DCs derived from normal monocytes (7.93%, 1.28% and 74.97% respectively) confirmed the leukemic origin of DCs. Our data confirmed the generation of sufficient AML-derived cells with the properties of DCs in all cases. This potency of AML blasts, offers a useful route for active immunotherapy of AML patients.     

孕酮对白血病细胞的抑制增殖和诱导分化作用

 目的探讨孕酮对K562和NB4白血病细胞系的抑制增殖和诱导分化作用。方法采用细胞计数,XTT实验观察孕酮对白血病细胞增殖能力的影响,采用Wright-Giemsa染色,联苯胺染色,NBT还原实验和流式细胞术观察孕酮对白血病细胞的诱导分化作用。结果不同浓度孕酮连续作用白血病细胞5天,液体培养显示细胞数明显减少,第5天处理组的XTT值显著低于对照组,并呈剂量依赖关系;液体培养和XTT均显示孕酮对NB4细胞的增殖并无显著影响。K562细胞联苯胺染色 OD值显著升高,与对照组比较差异有统计学意义（P<0.01）,NB4细胞NBT还原能力提高（P<0.01）,并呈剂量依赖性。形态学观察孕酮可诱导K562细胞和NB4细胞趋向成熟分化,流式细胞术检测孕酮处理后K562和NB4细胞膜CD71和CD11b分化抗原表达阳性率分别为85.72％和61.28％。结论孕酮可显著抑制K562白血病细胞增殖,诱导K562和NB4白血病细胞向成熟分化。     

A Dual Topoisomerase Inhibitor, TAS-103, Induces Apoptosis in Human Cancer Cells

TAS-103 (6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one dihydrochloride), a dual topoisomerase (topo) inhibitor, was developed as an anticancer agent by targeting topo I and topo II and has previously been shown to be effective against lung tumors. In this study, we investigated the cytotoxic activity of TAS-103 in various human cancer cell lines (including gastric, colon, squamous, lung, and breast cancer cells) and the induction of apoptosis by TAS-103. We next established stable transfectants of Bcl-2 in the gastric cancer cell line AZ521 and found that Bcl-2 blocked TAS-103-induced apoptosis. In addition, we demonstrated that the activities of ICE-like and CPP32-like proteases are involved in the signal transduction pathway of TAS-103-induced apoptosis. In summary, TAS-103 is a novel type of anticancer agent with a unique mechanism and could be useful as a lead compound for development of new drugs.     

Inhibition of Proliferation and Induction of Differentiation of Human and Mouse Myeloid Leukemia Cells by New Ethyleneglycol-type Nonphosphorus Alkyl Ether Lipids

A variety of ethyleneglycol-type nonphosphorus alkyl ether lipids, ether derivatives of diethylene-glycol in which the two hydroxyl groups were substituted with long chain alkyl and quaternary ammonioalkyl groups, were synthesized and their effects on proliferation and differentiation of cultured human (HL-60) and mouse (M1) myeloid leukemia cells were studied. Incubation with these compounds inhibited the cellular proliferation, and the cells differentiated into morphologically and functionally mature granulocytes. Of the compounds tested, 1-[2-[2-(octadecyloxy)ethoxy]ethoxy]-butylpyridinium mesylate (EG-6) was the most effective in inducing differentiation of HL-60 cells. Almost maximal induction of differentiation and inhibition of growth of HL-60 cells on day 6 were observed when the cells were treated with EG-6 for 1 day and then cultured without EG-6 for a further 5 days. The inhibitory effect of EG-6 on the leukemic cells was over 100 times more than that of 2-[2-(dodecyloxy)ethoxy]ethyl 2-pyridinioethyl phosphate, a potent antileukemic ether phospholipid.     

Topoisomerase expression in cancer cell lines and clinical samples

Topoisomerase II activity has been previously associated with chemosensitivity to cytotoxic agents in cell lines made resistant to drugs in vitro. Examination of unselected cancer cell lines, however, shows a relatively poor correlation between topoisomerase II content and intrinsic chemoresistance. Studies of topoisomerase II expression in clinical materials from human tumor biopsies also demonstrate a poor relationship with the response of the cancers to induction chemotherapy. A major problem with assessing topoisomerase II activity in clinical materials is the marked heterogeneity of the enzyme among the cells and the associated high proportion of tumor cells which are not traversing the cell cycle. While the activation of oncogenes may disregulate topoisomerase II expression in some experimental systems, there is currently no evidence that enzyme activity is disconnected from cell cycling in clinical cancer specimens. Novel techniques of topoisomerase II measurement may permit more accurate correlation of enzyme activity with clinical chemosensitivity.This work was supported by NIH grant CA 52178 and grant CN-58 from the American Cancer Society and was supported in part by the Bristol-Myers Company under a research grant program for studies of Tumor Cell Resistance to Chemotherapy     

Inhibitory Action of Transforming Growth Factor-β on Induction of Differentiation of Myeloid Leukemia Cells

The effect of transforming growth factor-β (TGF-β) on induction of differentiation of mouse myeloid leukemia M1 cells was investigated. TGF-β1 induced adherence of M1 cells to plastic dishes and inhibited their proliferation. However, it did not induce differentiation-associated properties, such as phagocytic activity, lysozyme activity or morphological maturation. TGF-β1 also caused dosedependent inhibition of dexamethasone-induced differentiation of M1 cells. The inhibitory activity of TGF-β1 was 20 times that of TGF-β2 on M1 cells. These results suggest that TGF-β1 inhibits proliferation and dexamethasone-induced differentiation of M1 cells by interacting with receptors that can distinguish between TGF-β1 and TGF-β2. TGF-β1 had a much lower inhibitory effect on the growth of a variant M1 cell clone, which was resistant to differentiation inducers, and it did not induce adherence of the resistant M1 cells.     

Biomodulation by Hyperthermia of Topoisomerase II-Targeting Drugs in Human Colorectal Cancer Cells

We examined whether heat stress could enhance the sensitivity of human colon cancer WiDr cells to topoisomerase II-targeting anticancer agents, etoposide (VP-16) and teniposide (VM-26), and also determined the most effective timing for the drug administration after exposure to hyperthermia. Both topoisomerase II contents and topoisomerase II activity were significantly increased in WiDr cells 3 to 12 h after heat stress at 43°C for 1 h, in comparison with those immediately after the heat stress. Cytotoxicity by VP-16 was most significantly enhanced 3 to 12 h after exposure to 43°C for 1 h, but no synergistic effect was observed when the drug was administered immediately after the heat stress. A combination of VM-26 with heat stress, but not that of a topoisomerase I-targeting camptothecin derivative (CPT-11), or vincristine, showed a synergistic cytotoxic effect on WiDr cells. VP-16 alone induced cellular accumulation at the G2+M phase, whereas the combination of VP-16 and heat stress further increased the cell population at the G2+M phase, and decreased S-phase cells. A possible application of the combination of VP-16 and hyperthermia in clinical use is discussed.     

Enhanced Topoisomerase I Activity and Increased Topoisomerase IIα Content in Cisplatin-resistant Cancer Cell Lines

Although the combined effects of cisplatin (CDDP) and DNA topoisomerase (Topo) inhibitors have been described in recent literature, little is known about the combined effects and their biological basis in CDDP-resistant cells. The aim of the present study was to elucidate the combined effect of CDDP and Topo inhibitors on CDDP-resistant cells as well as to investigate the biological factors involved in the sensitivity to these anti-cancer agents. We found synergistic actions between CDDP and SN-38 (a Topo I inhibitor) or VP-16 (a Topo II inhibitor) in KFr cells, a CDDP-resistant subline of the KF epithelial ovarian carcinoma cell line, but not in the parent KF cells. We subsequently assayed Topo protein levels and enzymatic activities in two sets of CDDP-sensitive and -resistant cell lines: KF and KFr, and HeLa and HeLa/CDDP. The levels of Topo I protein in the CDDP-resistant cells did not differ from those of their parent cell lines and were unaffected by exposure to CDDP. Topo I enzymatic activity, however, was 2- to 4-fold higher in the CDDP-resistant cell lines than in their respective parent cell lines. In contrast, higher levels of Topo lice protein were observed both before and after CDDP exposure in the CDDP-resistant cells than in their controls. However, no difference in Topo II catalytic activity was observed between the CDDP-resistant and -sensitive cells.     

Forsaken Pharmaceutical: Glasdegib in Acute Myeloid Leukemia and Myeloid Diseases

Advancements in the understanding of the pathogenesis of acute myeloid leukemia (AML) have led to the introduction and approval of a number of novel drugs in AML. Glasdegib, an oral hedgehog pathway inhibitor, was approved in 2018 in combination with low-dose cytarabine for the treatment of newly diagnosed AML in patients unfit for intensive chemotherapy. In this review, we discuss the preclinical rationale for glasdegib, important clinical trials that led to glasdegib’s approval, and future trials of glasdegib in AML and other myeloid diseases. Notably, 2 large randomized, placebo-controlled phase 3 trials (AML BRIGHT 1019) are currently recruiting patients with newly diagnosed AML to evaluate glasdegib in combination with intensive chemotherapy or azacitidine, depending on the patient’s ability to tolerate induction chemotherapy. While glasdegib and low-dose cytarabine have been eclipsed by venetoclax and hypomethylating agent combinations for newly diagnosed AML in the United States, we discuss other areas where glasdegib may still have an opportunity to improve outcomes in this devastating disease.     

慢性髓性白血病来源的树突状细胞诱导的CTL体外作用研究

 目的　研究慢性髓性白血病来源的树突状细胞的免疫功能。方法　分离慢性期CML患者外周血单个核细胞 (PBMNC) ,用细胞因子GM CSF及IL 4在体外诱导培养DC ,检测细胞表型 ,并观察其诱导的CTL体外抗肿瘤效应。用酶联免疫 (ELISA)方法测定CML DC混合淋巴细胞反应 (MLR)上清液中IL 12及IFN γ的量 ,并与正常DC进行比较。结果　联合GM CSF及IL 4可诱导CML细胞分化为CML DC ,CML DC的CD1a、CD80、CD83表达率均低于正常DC ;CML DC混合淋巴细胞反应上清IL 12及IFN γ含量均低于正常DC ;CML DC能诱导出对自身CML细胞有特异性杀伤作用的CTL。结论　CML DC具有抗原提呈细胞的免疫功能 ,能诱导抗白血病CTL反应。     

Combined Effects of Differentiation-inducing Factor and Other Cytokines on Induction of Differentiation of Mouse Myeloid Leukemic Cells

Mouse myeloid leukemic M1 cells are induced to differentiate into macrophage-like cells by differentiation-inducing factors (D-factors) and granulocyte colony-stimulating factor. We examined the effects of recombinant human tumor necrosis factor (rTNF), lymphotoxin (rLT) and interleukin 1 (rIL-1) on the induction of differentiation of M1 cells, compared with the effects of D-factor purified from the conditioned medium of mouse Ehrlich as cites tumor cells and recombinant human granulocyte colony-stimulating factor (rG-CSF). rIL-1 induced phagocytic activity, a typical marker of cell differentiation, in at most 30% of M1 cells at concentrations ranging from 10^-10 M to 10^-7 M . The differentiation-inducing activity of rIL-1 was similar to that of rG-CSF and less than that of D-factor. rTNF induced phagocytic activity in 14% of M1 cells only at a high concentration (10^-7 M ). rLT did not induce differentiation of the cells even at 10^-7 M . rTNF stimulated induction of differentiation of M1 cells by D-factor, rG-CSF or rIL-1 by two or three fold. The combination of any two of the cytokines D-factor, rG-CSF and rIL-1 induced differentiation of M1 cells more efficiently than any of these cytokines alone. Moreover, the combination of three cytokines rG-CSF, rIL-1 and rTNF, all of which are known to be produced by macrophages, was more effective than the combination of any two of these cytokines in induction of differentiation of M1 cells.     

Generation and Characterization of Lymphokine-activated Killer Cells against Fresh Human Leukemia Cells

Lymphokine-activated killer (LAK) cells generated from 15 acute leukemia patients in remission showed significant levels of cytotoxicity against Daudi 1A4, a natural killer-resistant cell line. This indicates that lymphocytes of leukemia patients in remission could respond to interleukin-2 to generate conventional LAK cells. However, LAK cells caused lysis of autologous leukemia cells at considerably lower levels in seven out of the 15 patients, with the exception of one case (48.6% cytolysis). None of the remaining eight patients exhibited LAK activity against autologous leukemia cells. On the other hand, patients' LAK could lyse allogeneic leukemia cells including those resistant to autologous LAK. Thus, patients' LAK seem not to be defective in lysis of leukemia cells. In the cold target competition analysis, the binding of patients' LAK to leukemia cells could be inhibited by autologous and allogeneic leukemia cell competitors, implying that almost all leukemia cells could be recognized by patients' LAK. Most LAK cells from normal donors showed significant lysis of allogeneic leukemia cells, but some leukemia cells were found to be resistant to lysis. LAK cells against both leukemia cells and Daudi 1A4 were phenotypically heterogenous, and were predominantly observed in the T3⁻ fraction in the precursor phase. In the effector phase, whereas LAK activity against leukemia cells was also predominantly shown in the T cell-depleted fraction, similar levels of LAK activity against Daudi 1A4 were found in both the T cell-depleted and -enriched fractions.     

Thrombopoietin, Interleukin-11, and Early-Acting Megakaryocyte Growth Factors in Human Myeloid Leukemia Cells

In this study we report our data on effects of early-acting megakaryocyte growth factors, particularly the c-mpl ligand also known as thrombopoietin (TPO) and interleukin-11 (IL-11), on cell proliferation and apoptosis (Apo) of primary acute myeloid leukemia (AML) cells. A proliferative response to TPO was noticed in the majority of AML samples (17/19) with an average increase of S-phase cells from 7.8% ± 1.5 to 14.5% ± 2.1 (p=0.0006). Resulting cell cycle activation did not always correlate with expression of the c-mpl receptor, although it was coupled, in the majority of samples, by an average decrease of apoptotic cells from 13% ± 0.7 to 8.8% ± 1.8 (p=0.05). Clonogenic cell growth (CFU-L) was confirmed in 5/17 of the samples with a mean colony number of 21.4 ± 9.6 × 10⁵ cells plated. Conversely, effects of IL-11 on AML cells demonstrated that cell cycle changes (recruitment from G₀ to S phase) were promoted only in a minority of samples (2/14) and there was little, if any, effect on CFU-L growth (mean colony number=17.5 ± 9.5) or Apo (from 13% ± 0.7 to 13.3±1.9). Combination of TPO with IL-11 induced a slight increase of clonogenic cell growth, while the addition of IL-3 or SCF to the c-mpl ligand significantly raised the mean colony numbers up to 119.2 ± 68.3 and 52.9±22.1 × 105 cells plated, respectively. In summary, TPO shows activity on AML cells by stimulating their proliferation in a significant proportion of cases and generally protecting the majority of AML blast cells from induction of Apo. Conversely, IL-11 exerts little effect on the cell cycle activation and Apo. These data help to understand regulation of myeloid leukemia cell growth and should be considered in the clinical use of early-acting megakaryocyte growth factors in acute leukemia.     

Phenotypic Screening Reveals Topoisomerase I as a Breast Cancer Stem Cell Therapeutic Target

Cancer stem cells (CSCs) are a subpopulation generally thought to be responsible for cancer initiation and progression. Because CSCs are often rare in the total tumor cell population and differentiate rapidly when grown in culture, it has been challenging to uncover compounds that selectively target CSCs. We previously described CSC-emulating cells derived from breast cancer cell lines that maintained a stable undifferentiated state. We optimized a phenotypic assay with these cells and screened 1,280-bioactive compounds, identifying five that preferentially inhibited CSC-like cell proliferation. Using a compound-guided target identification approach, we found high topoisomerase I (Topo I) expression levels in breast CSC-like cells and primary breast CSCs. Structurally unrelated small molecules targeting Topo I preferentially inhibited CSC-like cells. These results illustrate the substantial power of this CSC phenotypic screening platform and promote Topo I as a potential molecular therapeutic target for therapies aimed at expunging CSCs.     

Cell-free Entry of Human T-Cell Leukemia Virus Type 1 to Mouse Cells

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. Recently we infected newborn mice by inoculating HTLV-1-producing human cells, and found that T-cells, B-cells and granulocytes were infected in vivo. To understand the mechanism of viral-cell interaction and the pathogenesis of HTLV-1 using the mouse model, it is important to clarify the cellular tropism using a cell-free HTLV-1 transmission system. We employed a highly transmissible cell-free HTLV-1 produced by a feline kidney cell line, c77, and studied the susceptibility of 9 kinds of mouse cell lines, EL4, RLml, CTLL-2, J774.1, DA-1, Ba/F3, WEHI-3, NIH3T3 and Bl, and two kinds of human cell lines, Molt-4 and Hut78. HTLV-1 proviral sequence was found by PCR in all 9 mouse cell lines as well as in 2 human cell lines and viral entry was blocked with sera from an HTLV-1 carrier and an adult T-cell leukemia patient. Unexpectedly, mouse cell lines EL4 and RLml and human cell lines Molt-4 and Hut78 showed similar efficiency for viral entry. These results suggest a wide distribution of HTLV-1 receptor in mouse cells.     

伊马替尼治疗慢性粒细胞白血病的远期疗效观察

目的探讨伊马替尼治疗慢性粒细胞白血病(CML)的长期疗效及影响CML患者生存的因素。方法对66例CML患者应用伊马替尼治疗,检测血常规、染色体核型、bcr/abl转录本表达及不良反应情况。结果中位随访39(6～84)个月。CML慢性期患者完全血液学缓解(CHR)率为95.8%,主要细胞遗传学缓解(MCyR)率为75.0%,完全细胞遗传学缓解(CCyR)率为68.8%,完全分子学缓解(CM0R)率为41.7%,均显著高于加速期及急变期(P<0.01)。慢性期患者3、5和7年总生存(OS)率分别为95.3%、90.3%和87.5%,疾病无进展生存(PFS)率分别为91.6%、84.5%和81.3%;加速期患者1、2和3年OS率分别为90.5%、37.6%和37.6%;1、2和3年的预期PFS率分别为37.2%、28.4%和17.6%;急变期患者6、12、18个月的预期OS率分别为83.6%、32.3%和32.3%。慢性期患者与加速期、急变期患者OS及PFS比较差异有统计学意义(P<0.01);多因素分析结果显示,应用伊马替尼治疗前接受过其他治疗是影响PFS的不利因素。结论伊马替尼对CML慢性期疗效显著优于加速期及急变期;慢性期患者达到CCyR甚至CM0R,是获得长期生存的关键。