氯化锂对白血病THP-1细胞生长的抑制作用及其对Wnt通路的影响

摘 要：目的 研究氯化锂（LiCl）在体外对白血病细胞THP-1增殖、凋亡、周期以及Wnt通路的影响。 方法 氯化锂不同浓度不同时间作用THP-1细胞后,用MTT法检测细胞增殖,流式细胞术检测细胞凋亡和周期分布,Western blot检测细胞内Wnt通路的变化。 结果 氯化锂能抑制白血病THP-1细胞的增殖,诱导细胞凋亡,使细胞周期阻滞于G2/M期,且均呈浓度和时间依赖性。氯化锂不同浓度不同时间作用THP-1细胞后,t-GSK3β、cyclinD1表达不变,p-GSK3β、β-catenin表达升高,短时间低浓度作用后c-myc表达升高,但随作用时间延长和浓度增加,c-myc表达降低。 结论 氯化锂对THP-1细胞有显著的增殖抑制、凋亡诱导、细胞G2/M期阻滞作用,并激活Wnt通路,影响Wnt通路下游蛋白c-myc、cyclinD1的表达。     

Twist-1基因在髓系白血病细胞中的表达及其作用

目的:检测Twist-1基因在白血病患者和造血系统恶性肿瘤细胞系中的表达情况,并探讨其高表达对髓系白血病细胞增殖和凋亡的影响.方法:用Real-time PCR检测急性髓系白血病(acute myeloid leukemia,AML)、急性淋巴细胞白血病(acute lymphoid leukemia,ALL)、慢性粒细胞白血病(chronic myeloid leukemia,CML)患者和正常人的骨髓单个核细胞对照以及造血系统恶性肿瘤细胞系中Twist-1 mRNA表达情况.构建Twist-1过表达及干扰载体,制备慢病毒并感染髓系白血病细胞系K562、U937、KG-1a,通过细胞计数实验、集落形成实验、流式细胞术、Annexin V/PI方法评价Twist-1对白血病细胞增殖、集落形成能力、周期、凋亡的影响.结果:Twist-1在AML及CML患者中的表达水平显著高于对照(均P＜0.05),而ALL患者与对照组没有显著差异(P＞0.05).在K562、U937、KG-1a中过表达及干扰实验证实,Twist-1高表达促进肿瘤细胞增殖、集落形成并抑制细胞凋亡;干扰Twist-1表达则效果相反.结论:Twist-1高表达于AML、CML髓系白血病细胞,并促进白血病细胞的增殖和抑制凋亡.     

Wnt信号通路与慢性粒细胞白血病

 慢性粒细胞白血病作为血液系统的恶性增殖性肿瘤，其主要发病机制为BCR-ABL1融合基因形成，因此针对BCR-ABL1基因的靶向药物TKI为其治疗带来了希望，但TKI无法完全消除CML患者体内的LSCs，CML患者容易出现TKI药物耐药及治疗后复发。随着Wnt信号通路在实体瘤中的应用，大量研究发现Wnt信号通路同样在CML中起到重要作用，我们就Wnt信号通路对CML的影响作一综述。     

Ph样急性淋巴细胞白血病的研究进展

Ph样急性淋巴细胞白血病(philadelphia chromosome-like acute lymPhoblastic leukemia,Ph-like ALL)是近年来提出的一种新的急性淋巴细胞白血病亚型,其特点是Ph染色体或者BCR-ABL1融合基因阴性,但基因表达谱与Ph阳性的ALL类似,且与不良预后相关.Ph-like ALL通常可引起细胞因子受体和/或酪氨酸激酶信号的激活并级联放大,导致下游相关信号通路的活化.临床前研究显示此类白血病可能对酪氨酸激酶抑制剂(tyrosine kinase inhibitors,TKIs)敏感.对Ph-like ALL这一高危亚型患者群体的识别,可以通过个体化的靶向治疗来改善预后,因此意义重大.本文对Ph样ALL的研究进展做一概述.     

GATA-3 基因在白血病骨髓基质细胞中的表达

 目的　了解转录因子GATA-3 基因在白血病和正常骨髓基质细胞(BMSC) 中表达的情况。方法　体外扩增培养BMSC ,运用RT-PCR-EL ISA 方法检测GATA-3 基因的表达情况,并对其相对表达水平进行半定量比较。结果　GATA-3 基因在正常和白血病的BMSC 中均有一定的表达。BMSC 中急性髓性白血病(AML) 和慢性粒细胞白血病(CML) 组的GATA-3 基因表达率与正常组比较无明显差异,而急性淋巴细胞白血病(ALL) 组的GATA-3 表达率比正常组低,且AML BMSC 中GATA-3 基因表达水平> ALL > 正常> CML 。结论　转录因子GATA23 基因可在正常和白血病骨髓基质细胞中表达,并可能影响骨髓微环境的造血调控作用,其表达模式的改变可能与白血病发生发展有一定的关系。     

Wnt/β-catenin信号通路在髓系白血病细胞中的研究进展

Wnt通路在进化过程中高度保守,在人体中发挥着重要的生理功能,与肿瘤的发生发展密切相关.髓系白血病患者细胞中存在Wnt/β-catenin信号通路的异常活化,参与调控急性白血病的发生及发展.本文拟从Wnt通路的组成及机制,Wnt通路在不同类型髓系白血病中的作用等方面综述这一途径在髓系白血病方面的研究进展.     

吲哚美辛抑制慢性粒细胞白血病急变期CD34~+细胞增殖及其机制探讨

目的:观察吲哚美辛对急变期慢性粒细胞白血病患者骨髓CD34~+细胞增殖和自我更新能力的影响,并从Wnt/β-catenin信号通路的角度初步探讨吲哚美辛作用的分子机制。方法:采用免疫磁珠分选技术分选获得慢性期和急变期慢性粒细胞白血病患者骨髓以及健康人脐带血标本中的CD34~+细胞,并采用FCM和实时荧光定量PCR法检测CD34~+细胞中β-catenin和Bcr-Abl的表达水平。用吲哚美辛或(和)伊马替尼处理CD34~+细胞后,采用FCM法检测细胞中β-catenin的表达水平,MTT法检测细胞增殖情况,克隆形成实验检测细胞克隆形成能力,实时荧光定量PCR法检测c-Myc和cyclin D1的mRNA水平。结果:成功分选出各标本中的CD34~+细胞,其中急变期慢性粒细胞白血病患者的CD34~+细胞中β-catenin和Bcr-Abl均高表达(P值均<0.05)。吲哚美辛作用后,急变期慢性粒细胞白血病患者的CD34~+细胞中β-catenin表达显著下调(P<0.05),细胞增殖和克隆形成能力均被明显抑制(P值均<0.05),并且β-catenin下游靶基因c-Myc和cyclin D1的mRNA水平也被明显下调(P值均<0.05);而吲哚美辛与伊马替尼联合用药后,以上作用效果更加明显(P值均<0.05)。结论:吲哚美辛可能通过影响Wnt/β-catenin信号通路,抑制CD34~+细胞的增殖和自我更新能力,从而增强伊马替尼对急变期慢性粒细胞白血病患者中CD34~+细胞的杀伤力。     

Wnt/β-catenin信号通路在白血病中的研究进展

Wnt/ β-catenin信号通路在胚胎发生和器官发育中发挥着重要作用.该信号通路与多种实体肿瘤的发生密切相关.新近研究发现,Wnt/β-catenin信号通路在多种白血病中异常激活,提示其可能参与了白血病的发生、发展.针对Wnt/β-catenin信号通路的靶向治疗可能成为白血病治疗的新方向.本文就Wnt/β-catenin信号通路在白血病中的研究进展作一综述.     

miR-181a诱导慢性淋巴细胞白血病细胞周期阻滞及细胞凋亡的分子机制探讨

目的:探讨miR-181a在慢性淋巴细胞白血病细胞周期阻滞及细胞凋亡中的作用并初步阐明其调控机制。方法:利用荧光定量PCR检测慢性淋巴细胞白血病患者肿瘤细胞中miR-181a的表达水平;利用双荧光素酶试验验证miR-181a的靶基因MCL-1和BCL-2,利用免疫印迹试验检测过表达miR-181a对p53通路中关键蛋白MCL-1和BCL-2的影响,利用流式细胞仪检测过表达miR-181a对慢性淋巴细胞白血病细胞凋亡和周期的影响。结果:RT-qPCR结果显示,慢性淋巴细胞白血病细胞中miR-181a的表达下调;双荧光素酶试验验证发现,p53通路下游基因MCL-1和BCL-2均是miR-181a的靶基因;免疫印迹试验结果显示,过表达miR-181a抑制了MCL-1和BCL-2蛋白的表达;流式细胞仪检测结果显示,过表达miR-181a阻滞细胞周期G1/S期转换,促进细胞凋亡。结论:miR-181a 通过靶向作用p53通路中关键基因MCL-1和BCL-2,诱导细胞周期阻滞及细胞凋亡,可能在慢性淋巴细胞白血病中扮演着抑癌基因的角色。     

丙戊酸钠诱导急性淋巴细胞白血病原代细胞凋亡机制的研究

目的 探讨丙戊酸钠（VPA）对急性淋巴细胞白血病（ALL）原代细胞凋亡现象与去甲基化机制之间的联系.方法 选取10例AIL患者原代细胞,MTT法检测VPA对ALL原代细胞增殖抑制作用,DNA ladder试验观察细胞凋亡,Annexin-V-FITC/PI检测细胞早期凋亡,半巢式甲基化特异PCR检测p15INK4B基因甲基化,反转录PCR方法检测p15基因表达水平.实验数据采用SPSS 16.0统计软件处理.结果 VPA可明显抑制细胞增殖,对原代细胞IC50为1.898 mmol/L;DNA ladder试验显示VPA对原代ALL细胞的凋亡作用明显.Annexin-V-FITC/PI检测1.0、2.0、4.0 mmol/LVPA组的凋亡率分别是（5.80±0.65）％、（48.46±2.49）％、（76.45±2.98）％,与未用药组的（0.44±0.04）％比较差异有统计学意义（P＜0.05）.原代ALL细胞经VPA作用后,p15INK4B甲基化水平明显下降,2.0、4.0 mmol/LVPA组与未用药组相比,p15 mRNA表达水平增强,差异有统计学意义（P＜0.05）.结论 VPA通过使p15INK4B基因去甲基化,促进p15基因表达上调,从而促进原代ALL细胞凋亡.     

Acute lymphocytic leukemia-associated cell membrane antigen.

Antisera were raised in New Zealand White rabbits against non-B, non-T acute lymphocytic leukemia (ALL) cells coated with antilymphocyte serum. Following minimal absorption with chronic lymphocytic leukemia (CLL) cells, the antiserum reacted mainly with non-B, non-T ALL cells. The following numbers of patients had leukemia cells that reacted with the ALL antisera: 13 of 18 with ALL, 3 of 27 with acute myelocytic leukemia, 1 of 8 with chronic myelocytic leukemia (CML), and 0 of 12 with CLL. The positive CML was a patient in CML blast crisis. Normal peripheral blood B- and T-lymphocytes and normal bone marrow were negative. Reactions of the anti-ALL serum (136K) were compared with the reactions of a rabbit anti-B-cell antiserum (63K) that reacted with approximately 70% of leukemia cells. Cultured lymphoblastoid cell lines from normal donors were negative by both cytotoxicity and immunofluorescence tests. However, by immunofluorescence testing, 8 of 17 known malignant lines from a variety of lymphoproliferative disorders were positive; 4 of these lines were of T-cell origin. By immunoprecipitation and polyacrylamide gel electrophoresis, the ALL antigen appeared to consist of a single polypeptide chain of approximately 98,000 daltons. The anti-ALL antiserum was not cytotoxic for normal myeloid stem cells (colony-forming units).     

Loss of beta-catenin impairs the renewal of normal and CML stem cells in vivo

A key characteristic of stem cells and cancer cells is their ability to self-renew. To test if Wnt signaling can regulate the self-renewal of both stem cells and cancer cells in the hematopoietic system, we developed mice that lack beta-catenin in their hematopoietic cells. Here we show that beta-catenin-deficient mice can form HSCs, but that these cells are deficient in long-term growth and maintenance. Moreover, beta-catenin deletion causes a profound reduction in the ability of mice to develop BCR-ABL-induced chronic myelogenous leukemia (CML), while allowing progression of acute lymphocytic leukemia (ALL). These studies demonstrate that Wnt signaling is required for the self-renewal of normal and neoplastic stem cells in the hematopoietic system.     

Wnt5a enhances the response of CML cells to Imatinib Mesylate through JNK activation and γ-catenin inhibition

Imatinib Mesylate is widely used for the treatment of chronic myelogenous leukaemia (CML), and its effects on CML cells are influenced by several signalling proteins. The research is aimed at determining whether Wnt5a affects the effects of Imatinib Mesylate against BCR-ABL positive CML cells (K562 cells and KU812 cells) and which signalling proteins are involved in. The results showed that Wnt5a augmented the effects of Imatinib Mesylate on inhibiting CML cells proliferation and inducing apoptosis in vitro; Wnt5a enhanced the inhibition effect of Imatinib Mesylate on the growth of K562 cells xenograft tumour in an animal model. Furthermore, Wnt5a inhibited β-catenin and its target gene Survivin, increased the activity of JNK and suppressed γ-catenin expression. When inhibiting the activity of JNK, the influence of Wnt5a on the effects of Imatinib Mesylate was attenuated. Moreover, JNK suppressed β-catenin and its target gene Survivin, and enhanced the effects of Imatinib Mesylate. These results suggest that Wnt5a can enhance the efficacy of Imatinib Mesylate through JNK/β-catenin/Survivin and γ-catenin/β-catenin/Survivin pathways.     

Clinical Significance of BCR-ABL Fusion Gene Subtypes in Chronic Myelogenous and Acute Lymphoblastic Leukemias

Background: Some reports have suggested that chronic myeloid leukemia (CML) patients have a higherprevalence of M-bcr than acute lymphoblastic leukemia (ALL) patients, which show a higher prevalence ofm-bcr. However, the relationship between BCR-ABL subtypes and progression of CML and ALL remains unclear.Materials and Methods: 354 CML chronic phase (CML-CP) patients, 26 CML blastic phase (CML-BP) patientsand 72 ALL patients before treatment with BCR-ABL positive were recruited for blood routine examinationand bone marrow smear cytology. Some 80 CML-CP and 32 ALL patients after imatinib (IM) treatment werefollowed-up for BCR-ABL relative concentrations detected after treatment for 3, 6 and 9 months and 1 year.Results: Before treatment, CML-CP patients showed lower BCR-ABL relative concentrations with a higherproportion of M-bcr (42.7%) compared to CML-BP and ALL patients while ALL patients had a higher BCR-ABLrelative concentration with high expression of m-bcr (51.4%). Patients with M-bcr demonstrated higher WBCcounts than those with m-bcr and the mixed group and higher PLT counts were noted in the CML-CP and ALLgroups. After imatinib (IM) treatment, patients with m-bcr showed higher BCR-ABL relative concentrations inboth CML-CP and ALL groups. Conclusions: This study identified the BCR-ABL gene as an important factor inCML and ALL cases. The M-bcr subtype was associated more with CML while the m-bcr subtype was associatedmore with ALL. Patients with m-bcr seem to have a poorer response to IM in either CML or ALL patientscompared to M-bcr patients.     

Molecular construction of Philadelphia chromosome and its relation to the clinical features

In 1960, Nowell and Hungerford found, for the first time, a minute chromosome at the metaphase in chronic myelocytic leukemia (CML) cells, which was called Philadelphia chromosome (Ph1 chromosome) later. Ph1 chromosome was considered to be specific for the disease and was frequently used as an important marker for the definite diagnosis. However, in mid-1970s, some cases with acute lymphocytic leukemia (ALL) were also found to have Ph1 chromosome in the leukemic cells. Therefore, Ph1 chromosome seemed to be non-specific for the diagnosis of CML. In 1980s, molecular-biology techniques were applied in the fields of leukemia research. As a result, clear differences were demonstrated between the two diseases (CML and ALL with Ph1 chromosome, respectively) at the molecular level using oncogene concept. In this review, molecular-genetic constructions of ABL, BCR and BCR-ABL hybrid genes in CML, as well as m-BCR-ABL hybrid gene in Ph1 positive ALL are focused in detail. Relationship between these molecular-genetic changes with the clinical features and the mechanism of cell growth in these cells with BCR-ABL or m-BCR-ABL hybrid genes are also discussed.     

Heteroantiserum against acute lymphocytic leukemia raised to the lymphoblastoid cell line NALM-1

Antisera have been raised in rabbits to the lymphoblastoid cell line NALM 1 precoated with antilymphocyte serum (ALS). Following absorption with chronic lymphocytic leukemia cells (CLL) the antisera reacted mainly with acute lymphocytic leukemia (ALL) cells, and were very similar in specificity to antisera raised to ALL cells precoated with ALS. Leukemia cells from the following numbers of patients were positive for the anti-NALM 1 sera in a complement-dependent cytotoxicity test; 11/14 ALL, 3/15 acute myelocytic leukemia (AML), 1/5 chronic myelocytic leukemia (CML) and 0/8 CLL. Normal B and T peripheral blood lymphocytes were negative. The titer of the anti-NALM 1 sera against positive cells was 1:64 to 1:256 whereas the undiluted sera did not react with negative cells. Ten out of 11 of the positive ALL cells were of the non-B non-T type. However, cells from 1/4 T ALL patients and a cultured T ALL line 8402 were also positive. Six of 12 cultured lymphoblastoid cell lines were positive, all of which were of malignant origin. The molecular weight of the ALL antigen detected by anti-NALM-1 serum was determined by immunoprecipitation and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) to be approximately 98,000 daltons.     

Wnt pathway contributes to the protection by bone marrow stromal cells of acute lymphoblastic leukemia cells and is a potential therapeutic target

Leukemia cells are protected by various components of their microenvironment, including marrow stromal cells (MSCs). To understand the molecular mechanisms underlying this protection, we cultured acute lymphoblastic leukemia (ALL) cells with MSCs and studied the effect of the latter on the molecular profiling of ALL cells at the mRNA and protein levels. Our results indicated that activated Wnt signaling in ALL cells is involved in MSC-mediated drug resistance. Blocking the Wnt pathway sensitized the leukemia cells to chemotherapy and improved overall survival in a mouse model. Targeting the Wnt pathway may be an innovative approach to the treatment of ALL.     

Sprycel for chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia resistant to or intolerant of imatinib mesylate.

PURPOSE: On June 28, 2006, the U.S. Food and Drug Administration approved dasatinib (Sprycel; Bristol-Myers Squibb), a new small-molecule inhibitor of multiple tyrosine kinases, for the treatment of adults with chronic phase, accelerated phase, or myeloid or lymphoid blast phase chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) with resistance or intolerance to prior therapy including imatinib. This summary reviews the database supporting this approval. EXPERIMENTAL DESIGN: Four single-arm multicenter studies supported the efficacy and safety of dasatinib. The primary efficacy end point in chronic phase CML was major cytogenetic response. The primary end point in accelerated phase, myeloid phase, and lymphoid blast phase CML, and Ph(+) ALL was major hematologic response. RESULTS: The four studies combined enrolled 445 patients. In patients with chronic phase CML, the major cytogenetic response rate was 45% with a complete cytogenetic response rate of 33%. Major hematologic response rates in patients with accelerated phase CML, myeloid CML, lymphoid blast CML, and Ph(+) ALL were 59%, 32%, 31%, and 42%, respectively. Median response durations in chronic phase, accelerated phase, and myeloid phase CML had not been reached. The median durations of major hematologic response were 3.7 months in lymphoid blast CML and 4.8 months in Ph(+) ALL. Common toxicities with dasatinib included myelosuppression, bleeding, and fluid retention. CONCLUSIONS: This report describes the Food and Drug Administration review supporting the approval of dasatinib for CML and Ph(+) ALL based on the rates and durability of cytogenetic and hematologic responses.     

Relation of "lymphoid" phenotype and response to chemotherapy incorporating vincristine-prednisolone in the acute phase of Ph1 positive leukemia.

Forty-four patients with Ph positive leukemia (36 developing blast crisis after chronic phase and eight presenting in acute leukemia) were classified into subgroups on the basis of reactivity of blasts with an anti-serum made against non-T,non-B acute lymphoid leukemia (ALL+), levels of terminal transferase enzyme (TdT+) and morphology. Positivity with anti-ALL serum was the most sensitive and reliable marker, and TdT was an important aid. The presence of "lymphoid" blasts in blast crisis of CML was related to the response to chemotherapy incorporating Vincristine and Prednisolone (VP). Patients with ALL+ blasts frequently (14 of 15 cases) responded to therapy while 21 of 25 patients who had no ALL+ blasts failed to respond. The clinical course of the ALL+ patients was variable: eight patients remitted with return to the appearances of the chronic phase; four patients demonstrated elimination of the Ph1 positive clone with hypoplasia and this was followed by normal (Ph1 negative) marrow regeneration in two. Subsequent relapse was of either the ALL+ "lymphoid" or the ALL-myeloid type. A regimen incorporating VP should be the treatment of choice in "lymphoid" blast crisis of CML.