FHIT对急性髓系白血病细胞HL60影响的研究

目的:进行外源FHIT基因转染人白血病细胞缺乏FHIT表达的HL60,研究FHIT基因对转染细胞生长的生物学影响。方法:构建pEGFP-FHIT真核表达质粒(实验组)与质粒pEGFP-N1(对照组)。分别电穿孔法转染HL60细胞。应用荧光显微镜、RT-PCR、Westernblot检测转染基因的转录和表达情况。四甲基偶氮唑盐(MTT)法、流式细胞术研究转染基因对HL60细胞体外增殖、凋亡情况的影响,并与转染对照质粒的细胞株进行比较。结果:经PCR、酶切和DNA测序证实pEGFP-FHIT真核载体构建成功。荧光显微镜下实验组HL60细胞可见绿色荧光,转染率为40%。RT-PCR和Westernblot分别从mRNA和蛋白水平检测到FHIT的表达。MTT检测结果显示:转染后实验组抑制率明显升高,细胞增殖受到抑制(P<0.05)。流式细胞术结果显示:实验组细胞凋亡率明显增加(P<0.05)。结论:转染FHIT基因对白血病细胞HL60的生长有抑制作用,并能诱导其凋亡。     

FHIT对急性髓系白血病细胞HL60影响的研究

目的:进行外源FHIT基因转染人白血病细胞缺乏FHIT表达的HL60,研究FHIT基因对转染细胞生长的生物学影响。方法:构建pEGFP-FHIT真核表达质粒（实验组）与质粒pEGFP-N1（对照组）。分别电穿孔法转染HL60细胞。应用荧光显微镜、RT-PCR、Western blot检测转染基因的转录和表达情况。四甲基偶氮唑盐（MTT）法、流式细胞术研究转染基因对HL60细胞体外增殖、凋亡情况的影响,并与转染对照质粒的细胞株进行比较。结果:经PCR、酶切和DNA测序证实pEGFP-FHIT真核载体构建成功。荧光显微镜下实验组HL60细胞可见绿色荧光,转染率为40%。RT-PCR和Western blot分别从mRNA和蛋白水平检测到FHIT的表达。MTT检测结果显示:转染后实验组抑制率明显升高,细胞增殖受到抑制（P＜0.05）。流式细胞术结果显示:实验组细胞凋亡率明显增加（P＜0.05)。结论:转染FHIT基因对白血病细胞HL60的生长有抑制作用,并能诱导其凋亡。     

ENO1对急性髓系白血病细胞增殖与凋亡的影响

目的:探讨Enolase 1(ENO1)调节急性髓系白血病(AML)细胞的代谢及分子机制。方法:分析GEO、TCGA等数据库中AML患者ENO1表达情况;运用Kaplan-Meier分析基因表达水平与患者生存期之间的关系;构建ENO1 shRNA慢病毒质粒体系,感染MV4-11、NB4细胞系构建分组,应用Western blot法检测ENO1在AML两种细胞系中的敲降效率;CCK-8法检测其对AML细胞生长的影响;Annexin V-PE/7-AAD法检测细胞凋亡情况;应用GO、KEGG富集方法分析ENO1在细胞中的功能及相关信号通路。结果:通过GEO、TCGA数据库分析表明ENO1在多数AML患者体内显著高表达(P<0.05,P<0.01),而在其它类型白血病患者中无此变化;ENO1在AML细胞中,在白血病干细胞,在AML小鼠模型造血干细胞中均显著高表达(P<0.001)。Kaplan-Meier分析表明ENO1高表达AML患者生存期显著低于ENO1低表达患者(P<0.05,HR>1)。敲降ENO1显著抑制AML细胞生长,诱导AML细胞凋亡(P<0...     

凋亡素-2配体联合三氧化二砷对急性髓系白血病细胞生长和凋亡的影响

目的：探讨凋亡素．2配体(Apo2L)联合三氧化二砷(As2O3)诱导急性髓系白血病(AML)细胞凋亡的作用。方法：分离25例AML患者的骨髓单个核细胞,每例分为4组进行观察：空白对照组、Apo2L组、As2O3、组、Apo2L As2O3组,共同培养12h、24h和48h。通过MTT比色法测定细胞生长抑制率及流式细胞仪检测细胞凋亡。结果：Apo2L和As2O3可协同降低AML细胞活力,诱导细胞凋亡,其作用随培养时间延长及药物浓度增加而增强。结论：Apo2L与三氧化二砷具有协同作用,能高效杀灭白血病细胞。Apo2L是一种有望应用于临床的新型生物制剂。     

淋系分化抗原在急性髓细胞白血病及髓系分化抗原在急性淋巴细胞白血病的表达及疗效观察

目的　探讨淋系分化抗原在急性髓性细胞白血病及髓系分化抗原在急性淋巴细胞白血病的表达及疗效。方法　采用流式细胞术检测 70例急性白血病的免疫表型 ,其中急性髓细胞白血病 32例 ,急性淋巴细胞白血病 38例。 32例急性髓细胞白血病有 9例除表达髓系抗原外 ,尚有淋巴抗原表达。 38例急性淋巴细胞白血病有 11例除表达淋系抗原外 ,尚有髓系抗原表达。结果　 9例LY+ AmL病人用常规DA方案化疗仅 1例缓解 ,11例MY+ ALL病人用标准DVP方案化疗无一缓解。LY+ AmL的病人改用DOAP方案化疗 ,有 2例获缓解 ,缓解率 2 5 %。MY+ ALL改用QOAP方案化疗有 2例获缓解 ,1例获部分缓解 ,缓解率 18.1% ,有效率 2 7.2 %。结论　对于LY+ AmL及MY+ ALL病人其化疗方案的选择应AML与ALL两者兼顾方可提高缓解率。     

中药有效成分诱导急性髓系白血病细胞凋亡作用机制研究进展

目的 总结中药有效成分诱导急性髓系白血病(A ML)凋亡作用机制,为临床发现特异性的抗白血病的作用靶点提供参考.方法 以急性髓系白血病、细胞系、中药、作用机制为中文关键词,以acute myeloid leukemia、cell line、tra-ditional Chinese medicine、mechani...     

急性髓系白血病细胞异常免疫表型表达的研究

应用免疫酶标染色法检测了５９例急性髓系白血病患者的白血病细胞免疫表型，结果表明，ＣＤ２，ＣＤ５，ＣＤ７，ＣＤ１０，ＣＤ１９，ＣＤ２２淋系抗原的表达率分别为１６．９％，１１．９％，１６．９％，１５．３％，１０．２％和６．８％。进一步分析结果表明，在Ｍ３病例细胞中，ＣＥ２，ＣＤ１０和ＣＤ１９抗原表达阳性率明显高于Ｍ５组，而ＣＤ７抗原表达阳性率明显低于Ｍ５组。     

和厚朴酚诱导人急性髓性白血病 KG1a细胞凋亡

目的:探讨和厚朴酚(honokiol,HNK)对人急性髓性白血病KG1a细胞凋亡的影响及其可能的机制。方法:XTT法检测不同质量浓度HNK对KG1a细胞增殖的影响,流式细胞术检测不同质量浓度HNK作用后KG1a细胞的细胞周期及凋亡,RT-PCR法检测KG1a细胞Bcl-2、Bid、Bax、Bak、Bad、P53、NF-κB等凋亡相关基因的表达。结果:HNK(2.5、5、8、10、15、20、40μg/ml)对KG1a细胞的增殖有明显抑制作用,且呈时间和剂量依赖性(P<0.01),其中24、48 h的半数抑制浓度(IC50)分别为10.23、8.25μg/ml。流式细胞术结果显示,经HNK(5、10μg/ml)处理后,KG1a细胞被阻滞在G0/G1期,早期凋亡率分别为(11.16±1.27)%和(21.46±3.13)%,显著高于对照组的(6.03±1.10)%(P<0.01)。RT-PCR检测结果显示,HNK(10μg/ml)处理后KG1a细胞内促凋亡基因Bax表达显著上调,Bad轻度上调;抗凋亡基因NF-κB表达显著下调。结论:HNK能诱导人急性髓性白血病KG1a细胞凋亡,其机制可能与Bax、Bad基因表达上调及NF-κB基因表达下调有关。     

左旋棉酚对小鼠宫颈癌移植瘤细胞凋亡的影响及其机制

目的探讨左旋棉酚［(-)-gossypol,LGP］对顺铂诱导小鼠宫颈癌移植瘤凋亡的影响及可能的作用机制。方法建立小鼠宫颈癌U14细胞皮下移植瘤模型,将60只小鼠按随机原则分为左旋棉酚组、顺铂(Cisplatin,DDP)组、左旋棉酚+顺铂(LGP+DDP)组及模型对照（Model Control）组4组,用药干预8天后采用电子天平称量瘤重并计算抑瘤率;采用TUNEL法检测各组瘤组织的凋亡情况;采用免疫组织化学法检测各组肿瘤组织中Bcl-2、Bax和P-gp蛋白的表达水平。结果各干预组小鼠移植瘤瘤重均低于Model组,其中LGP+DDP组瘤重最低,其次为DDP组(P＜0.01);各组移植瘤组织内AI均高于Model组（P＜0.05）,且LGP+DDP组最高（P＜0.01）;含LGP组移植瘤组织内P-gp、Bcl-2蛋白的表达降低（P＜0.05）, Bax蛋白的表达升高（P＜0.05）,Bax/Bcl-2比值升高,LGP+DDP组的上述作用明显优于单药组（P＜0.01）;相关性分析表明P-gp蛋白的表达量与Bcl-2的表达量呈正相关,与Bax的表达量呈负相关（P＜0.01）。结论 LGP能抑制小鼠宫颈癌移植瘤的生长,且与DDP联合后抑制作用更显著;其可能的机制:LGP可能是通过下调Bcl-2、上调Bax蛋白诱导小鼠宫颈癌细胞凋亡,降低P-gp蛋白的表达,增加肿瘤组织对DDP的化疗敏感度。     

Targeting Apoptosis Pathways in Acute Myeloid Leukemia

The anti-apoptotic protein BCL2 is overexpressed in hematological malignancies, including acute myeloid leukemia (AML), favoring tumor survival and chemoresistance. BCL2 inhibits BIM and BAX, effector proteins necessary for the formation of pores in the mitochondrial outer membrane, and its inhibition primes cells for the release of cytochrome c and subsequent apoptosis. Such priming can be facilitated by venetoclax, an oral BCL2 inhibitor, therefore representing an ideal strategy to induce

References

• 1.Letai A, Bassik MC, Walensky LD, Sorcinelli MD, Weiler S, Korsmeyer SJ. Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell. 2002;2(3):183-192.
• 2.Pan R, Hogdal LJ, Benito JM, et al. Selective BCL-2 inhibition by ABT-199 causes on-target cell death in acute myeloid leukemia. Cancer Discov. 2014;4(3):362-375.
• 3.Konopleva M, Pollyea DA, Potluri J, et al. Efficacy and Biological Correlates of Response in a Phase II Study of

     

Antitumor Agent-Induced Apoptosis in Myeloid Leukemia Cells: A Controlled Suicide

Traditional antitumor research has generally believed that the cytotoxicity of antitumor agents was directly correlated with the amount of drug-induced cellular lesions. Accordingly, oncologists have tried to improve anticancer agent / target interactions by increasing the intracellular dose of active effectors. However, a growing body of evidence stemming from both clinical and experimental observations, strongly suggests that similar anticancer-induced lesions may result in different cellular responses, greatly influencing cytotoxicity. For example, it has been shown that in some but not all cellular models, antitumor agents trigger apoptosis, an irreversible process which leads to a rapid and complete elimination of tumor cells. Several of these studies also demonstrated that apoptosis induced by antitumor agents is highly regulated by multiple signaling pathways which are themselves influenced by oncogenes, protein kinase activities, external stimuli and the oxidative balance. Therefore, it appears that cell death commitment is controlled by both external and internal factors which interfere downstream of drug-or ionizing radiation-target interaction. The characterization of these mediators may provide novel strategies for modulating intracellular signaling pathways in order to promote apoptosis in drug-resistant tumor cells.     

抗CD44抗体HI44a对白血病细胞分化与凋亡作用的体外研究

背景与目的探讨抗CD44抗体HI44a对新鲜白血病细胞分化及凋亡的作用。材料与方法从细胞形态学、四氮唑蓝(NBT)还原反应和细胞分化特异性抗原CD11b,CD14和CD15的变化,体外研究HI44a对31例急性髓系白血病患者白血病细胞的诱导分化作用。并利用Annexin-Ⅴ试剂盒检测HI44a对白血病细胞的凋亡诱导,RT-PCR方法检测其对细胞分化相关因子G-CSF,M-CSF及原癌基因c-myc表达的影响。结果经HI44a作用后,白血病细胞形态向成熟方向转变;M2~M5各亚型的NBT还原反应阳性率分别升高到31%(对照为9%)、55%(对照为10%)、25%(对照为12%)和32%(对照为11%),与对照组相比,差异均有统计学意义(P<0.01)。CD11b,CD14和CD15表达分别由对照组的9.65%、27.40%、57.38%升高到19.29%、40.60%和66.82%(P均<0.01)。细胞的早期凋亡率由对照组的26.21%升高到41.18%。RT-PCR检测发现HI44a作用后,M-CSF表达增强,而原癌基因c-myc表达降低。结论HI44a能够有效的诱导白血病细胞分化及凋亡,为治疗急性髓性白血病提供了一条新思路。     

体外化疗药物诱导白血病细胞凋亡预测临床疗效

[目的]探讨急性髓系白血病(AML)患者体外化疗药物诱导白血病细胞凋亡在化疗疗效预测中的价值。[方法]应用TdT介导的脱氧核苷酸切口和末端标记法(Tunel)、单克隆抗体免疫组化检测等方法研究42例初治AML患者体外化疗药物诱导白血病细胞凋亡、bcl鄄2表达与临床化疗疗效的关系。[结果]42例AML患者中,30例获完全缓解(CR)者,bcl鄄2表达显著低于12例未缓解(NR)者(P<0.05);CR患者柔红霉素(DNR)和阿糖胞苷(Ara鄄C)体外诱导白血病细胞凋亡率均分别高于NR患者,差异有显著性(P均<0.05);体外DNR和Ara鄄C两药诱导白血病细胞的总凋亡率,可以作为临床预测AML患者DA方案疗效的定量指标。[结论]体外化疗药物能否有效地诱导白血病细胞凋亡是判断AML患者化疗敏感性的重要指标。     

Accuracy of Immunohistochemistry and Flow Cytometry in Estimating the Proportion of Leukemic Cells in S Phase in Chronic Myeloid Leukemia Patients

We compared the proportion of S phase cells in bone marrow and peripheral blood samples obtained from 17 patients with chronic myeloid leukemia (CML). Before sampling all patients received a one hour IV infusion of iododeoxyuridine (IdUrd). The proportion of S phase cells was studied by immunohistochemistry (IHC) in bone marrow biopsies, and by flow cytometry (FCM) in bone marrow aspirates and peripheral blood samples. The IdUrd labelling index (LI) in bone marrow biopsy sections (27.5 ± 1.8%) was significantly higher than the proportion of IdUrd labelled cells in bone marrow aspirate (15.1 ± 2.0%). The percentage of S phase cells in peripheral blood was approximately the same as that in the aspirate (12.4 ± 1.3%) and was correlated with that of bone marrow aspirate indicating a high degree of the aspirate dilution by peripheral blood. It is likely that the differences in % S phase cells in the aspirate and the biopsy result from this dilution. Estimates of the % S phase cells in the peripheral blood study by IHC and FCM were essentially the same. Samples labelled for one hour in vitro resulted in 1.5 fold higher LI than the same samples labelled in vivo. We conclude that estimates of the 8% S phase cells in the bone marrow of patients with CML should be made by infusing patients with IdUrd or BrdUrd with immunohistochemical evaluation of a marrow biopsy. Additionally in vitro labelling is not reflective of the percent S phase cells in vivo in patients.     

Clonality Switch in Acute Myeloid Leukemia

We have previously described a case of clonality switch in a female patient with acute myeloid leukemia (AML) by X-chromosome inactivation analysis. She presented with refractory anemia with excess blasts in transformation but soon progressed to overt AML. Following induction chemotherapy, she went into complete remission but later relapsed into a second myelodysplastic phase. Analysis of her X-linked DNA polymorphism patterns at presentation and relapse showed that hematopoiesis was clonal, but the genotypes of the two clones was different. She remains clinically well and has a virtually normal blood count more than 5 years from presentation. We now report.an update of this unique case and discuss the implications of this finding within the context of a multicellular origin of leukemia.     

Comparison of Three Doses of Cytarabine Consolidation for Intermediate- and Adverse-risk Acute Myeloid Leukemia: Real World Evidence From Thai Acute Myeloid Leukemia Registry

BackgroundIntermediate or high doses of cytarabine (IDAC or HiDAC) were recommended as postremission chemotherapy for acute myeloid leukemia (AML). This retrospective study investigated the real-world outcomes of 3-different cytarabine doses from the multicenter Thai AML registry database.Patients and MethodsThe intermediate- and adverse-risk AML patients (N = 258) who achieved complete remission and proceeded to single-agent cytarabine consolidation were enrolled.ResultsThe median relapse-free survival (RFS) using IDAC 1.5 g/m², high-dose cytarabine (HiDAC) 2 g/m², and HiDAC 3 g/m² were 12.6, 11.7, and 13 months, respectively. The median overall survival (OS) using IDAC 1.5 g/m², HiDAC 2 g/m², and HiDAC 3 g/m² were 34.9, 22.7, and 23.7 months, respectively. No significant difference in RFS and OS was detected between the 3 doses. Secondary AML, white blood cell > 100×10⁹/L and the adverse-risk AML were independent prognostic factors for inferior survival (P= .008, P < .001, P= .014). Patients who completed 3 to 4 cycles of consolidation had significantly superior RFS and OS (P< .001, P< .001). Febrile neutropenia occurred in 72.9% of IDAC, 73.8% of HiDAC 2 g/m², and 78.1% of HiDAC 3 g/m² without statistical significance. However, the incidence of septic shock was significantly higher after HiDAC 3 g/m² compared to IDAC regimen (8% vs. 3%, P= .037).ConclusionIDAC is an appropriate regimen for postremission chemotherapy for intermediate- and adverse-risk AML. The higher dosing levels may not produce any benefits to patients and may increase incidence of septic shock. The number of consolidation cycles may impact on survivals rather than the intensity of cytarabine.     

Interleukins and Colony Stimulating Factors in Human Myeloid Leukemia Cell Lines

The present review has summarized the expression, production and effects of the human interleukins (IL) 1-11 and myelopoietic colony stimulating factors (CSF) in the established myeloid leukemia cell lines and in cells from patients with acute myeloid leukemia as well as the oncogene expression reported in these myeloid leukemia cell lines. The genetic dissection of leukemic myelopoiesis may provide new perspectives for the control of myeloid leukemias. Based on their expression of phenotypic markers (e.g., surface antigens, cytochemical staining, etc.), myeloid cell lines can be further subdivided into myelogenous, monocytic, erythroid and megakaryoblastic leukemia cell lines. Due to the close relationship of erythroid and megakaryoblastic progenitor cells and to the existence of a probably common precursor cell giving rise to these two different cell lineages, many megakaryoblastic cell lines express erythroid markers (e.g., expression of hemoglobin or glycophorin A) and conversely cell lines with a predominant erythroid profile might display megakaryoblastic features (e.g., platelets peroxidase or glycoproteins CD41, CD42b or CD61). The recent cloning of the specific cytokine: thrombopoietin (TPO) and its receptor generated a strong interest in these particular myeloid cell lines that are discussed in more detail in the present review. Both normal and leukemic megakaryocytopoiesis are stimulated by granulocyte-macrophage colony stimulating factor (GM-CSF), IL-3, GM-CSF/IL-3 fusion protein, IL-6, IL-11 and TPO but inhibited by IL-4, interferon-alpha (IFN-alpha) and IFN-gamma. Human megakaryoblastic leukemia cell lines have common biological features: high expression of the megakaryocytic specific antigen (CD41); high expression of early myeloid antigens (CD34, CD33 and CD13); constitutive expression of IL-6 and platelet-derived growth factor; a complex karyotype picture; expression of c-kit (the stem cell factor receptor); growth-dependency or-stimulation by IL-3 and/or GM-CSF; and in vivo tumorigenicity in mice associated with marked fibrosis. Whereas numerous chemical and biologic agents induce granulocytic and/or monocytic differentiation of myeloid leukemia cell lines, only a few agents including phorbol myristate acetate, vitamin D3, IFN-alpha, IL-6 and thrombin have been reported to induce megakaryocytic differentiation in the megakaryoblastic leukemia cells.     

The Binding of Acute Myeloid Leukemia Blast Cells to Human Endothelium

AML blast cell adhesion to endothelium is in all likelihood a prerequisite for blast cell migration across the vascular wall in the periphery and the subsequent establishment of leukemic extravascular disease. A general feature of malignant cells is their acquisition of altered or aberrant adhesive capabilities which appear to be associated with their ability to metastasize. Aberrant expression of integrin adhesion molecules and of membrane oligosaccharide structures is found in AML and various solid tumors. With respect to AML, these alterations in adhesive phenotype may confer a proliferative advantage on the malignant cells in the marrow, may facilitate egress from the bone marrow into the peripheral vasculature and may enable AML blast cells to traverse the vessel wall and so establish extravascular disease. Oncogenes may be directly involved in the acquisition of such aberrant adhesive phenotypes. Neutrophil extravasation is described as a model for leukocyte migration across the vessel wall and brief summaries of experimental work involving aspects of AML blast cell and normal CD34+ bone marrow cell adhesion to endothelium in vitro are described.