巨核白血病细胞系(HI—Meg)对抗癌药物敏感性的测定及其意义

应用~3H—TdR掺入、死活细胞计数和集落形成试验三种体外药物检测方法,测定了人巨核白血病细胞系(HI—Meg)对6种临床抗癌药物的敏感性,并用人早幼粒白血病细胞系(HL—60)作比较。结果表明6种药物对两株细胞均有较好的剂量效应曲线和较高的敏感性。HI—Meg的建立为抗癌药物筛选提供了一个新的模型,并可用于寻找和筛选有效的抗巨核细胞白血病的药物。     

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.     

急性髓系白血病的bcl-2基因表达及其临床意义

目的：探讨bcl—2基因在急性髓系白血病(AML)中的表达与预后的关系。方法：应用链亲和素-胶体金原位杂交(ISH—SAG)法检测57例AML患者单个核细胞的bcl—2基因表达水平。结果：57例AML患者不同程度表达bcl—2基因，范围从0—98％，其中初治组的阳性率为46．2％(24／52)，缓解组的阳性率为40．7％(11／27)，难治复发组的阳性率为100％(15／15)；难治复发组与初治组及缓解组之间差异均具有显著性(P＜0．01)，AML各亚型之间表达无显著性差异(P＞0．05)；疗效分析发现bcl-2基因表达与临床缓解密切相关，阴性组缓解率(76．2％)显著高于阳性组(42．1％)(P＜0．05)。结论：bcl—2基因过度表达对AML的预后有重要关系，可作为判断预后和合理制定治疗方案的重要依据。     

白血病细胞和正常细胞生成树突状细胞的比较研究

目的体外诱导髓系白血病原代细胞分化生成树突状细胞(dendritic cell，DC)．并与正常的DC比较。方法分离初诊24例急性髓细胞性白血病和10例慢性髓细胞性白血病患的骨髓单个核细胞以及5例正常人外周血的单个核细胞，用rhGM—CSF 1000U／ml、rhIL-4500U／ml和TNF—α 50U／ml联合培养10天；形态学(Wright染色、倒置显微镜、透射电镜)、免疫学(CD80、CD86、CD83、CD1a、HLA—DR)鉴定，RT—PCR检测白血病克隆以及混合淋巴细胞反应(MLR)检测抗原递呈功能。结果细胞因子诱导后，正常细胞和白血病细胞均出现典型形态的树突状细胞，CD80、CD86、CD83、CD1a的表达明显上调(正常和CML—DC的HLA—DR也明显上调)，急性和慢性髓细胞性白血病以及正常细胞来源的树突状细胞具有不同程度的刺激异基因T淋巴细胞增殖的能力。结论无论是急性和慢性白血病细胞均能诱导分化成DC，并且特征完全相似；白血病与正常骨髓的DC在形态和免疫学表达方面相似，但功能较弱。     

ATM在人胃癌细胞系的表达及其在细胞DNA损伤中的作用

目的：研究DNA损伤剂对ATM基因缺陷和正常的胃癌细胞系的作用机制。方法：Western—blot方法检测6株胃癌细胞系ATM蛋白的表达情况,并用DNA损伤剂顺铂作用于ATM基因缺陷的MKN28细胞系和ATM基因正常的BGC823细胞系,采用Western—blot、DNAladder、Hoechest33258／PI双荧光染色等方法观察凋亡相关激酶的表达和细胞的应答反应。结果：Western—blot显示,ATM蛋白在胃癌细胞系MGC803、MKN28、MKN45、SGC7901的表达水平显著低于BGC823和AGS细胞系,BGC823细胞在CDDP作用后,ATM蛋白表达水平升高,磷酸化chkl和chk2活性24小时后增强,而Cdc25C表达减少;MKN28细胞中G2期检测点蛋白ATM、P—chkl、P—chk2表达较低,Cdc25C表达较高,但在CDDP作用前后无明显变化。MKN28细胞在DNA损伤剂作用后出现显著凋亡,而BGC823细胞在DNA损伤剂作用48小时后未见显著凋亡。结论：ATM在细胞周期的多个环节均有凋控作用,特别是在细胞周期检测点和DNA损伤的修复中有重要的监视和启动作用。     

癌灵Ⅰ号对急性早幼粒细胞性白血病(APL)患者白血病细胞抗癌活性的作用

作者前已报导了癌灵Ⅰ号体外抗癌(人早幼粒细胞株 HL—60,人慢粒急变白血病细胞株 K562)活性检测及癌灵Ⅰ号体内抗白血病(小鼠 T 淋巴性白血病细胞L_(651)作用检测。现将应用癌灵Ⅰ号对4例急性早幼粒细胞性白血病患者的白血病细胞抗癌作用报告如下:     

转染抗VEGF发夹状核酶基因白血病细胞模型的建立

 目的 建立转染抗血管内皮生长因子（VEGF）发夹状核酶基因的白血病细胞模型,探讨发夹状核酶对白血病细胞系K562 VEGF基因表达的效应。方法 采用脂质体介导的方法将抗VEGF发夹状核酶基因真核表达载体（pcDNA3-RZ）转染入人白血病细胞系K562,G418抗性筛选获得阳性克隆,转染VEGF pcDNA3-RZ和空载体（pcDNA3）的细胞组均有抗性细胞生长,分别命名为K562-RZ和K562-PC;抽提基因组DNA,用PCR方法验证核酶基因已转染入K562细胞,荧光定量PCR和western blot方法分别检测白血病细胞VEGF mRNA和蛋白的表达量;同时测定K562-RZ,K562-PC和 K562三组培养上清刺激内皮细胞生长的情况。结果 抗VEGF pcDNA3-RZ成功转入白血病细胞系K562,G418筛选2周获得阳性克隆,PCR检测证实核酶基因整合入白血病细胞基因组DNA;与K562及K562-PC细胞相比,转染VEGF核酶基因的K562-RZ细胞VEGF mRNA和蛋白的表达量明显降低;K562-RZ组培养上清对内皮细胞的刺激作用明显弱于K562-PC组。结论 建立了转染VEGF发夹状核酶基因的白血病细胞模型,抗VEGF发夹状核酶基因可明显下调白血病细胞VEGF的表达,为抗肿瘤治疗提供了新的线索。     

miRNA-335基因启动子区甲基化与胃癌细胞不良生物学行为的关系

目的:探讨miR-335基因启动子区异常甲基化状态对胃癌细胞系中miR-335表达水平的影响,以及miR-335基因启动子区甲基化状态对胃癌细胞侵袭,迁移,以及增殖能力的影响。方法:1株永生化胃黏膜上皮细胞系(GES-1)和4株胃癌细胞系(SGC-7901,MKN-45,BGC-823和AGS)。实时荧光定量PCR(qRT-PCR)检测胃癌细胞株miR-335及CRKL的表达水平。甲基化特异性PCR(MSP)方法检测胃癌细胞株miR-335的基因启动子区甲基化状态。应用MTT方法检测恢复miR-335表达对胃癌细胞增殖能力的影响,Transwell侵袭迁移实验及划痕愈合实验分析恢复miR-335表达对胃癌细胞系侵袭及迁移能力的影响。结果:MSP实验结果表明,MKN-45、SGC-7901和BGC-823细胞系均存在基因启动子区异常的高甲基化状态,AGS细胞系基因启动子区亦呈部分高甲基化状态。去甲基药物5-aza-2′-deoxycytidine处理后胃癌细胞miR-335的表达水平可升高2~3倍。Transwell侵袭迁移实验及划痕愈合实验表明miR-335表达水平恢复后SGC-7901细胞的侵袭和迁移能力明显降低。MTT实验结果表明5-aza-2′-deoxycytidine处理后的SGC-7901细胞系与对照组相比,增殖能力显著降低。结论:miR-335启动子区的异常高甲基化状态抑制了miR-335在胃癌细胞系中的表达,恢复miR-335的表达水平可以抑制胃癌细胞的增殖,迁移和侵袭能力。miR-335为胃癌的肿瘤抑制因子。     

miR-152在急性髓系白血病患者骨髓和细胞系中的表达及生物学功能的初步研究

目的:检测miR-152在急性髓系白血病(AML)患者骨髓和细胞系中的表达水平。初步研究miR-152在急性髓系白血病中的生物学功能。方法:收集急性髓系白血病患者40例,非恶性血液病对照组20例,提取骨髓有核细胞;培养U937、Kasumi-1、THP-1三种急性髓系白血病细胞系。RT-PCR检测miR-152的表达水平。分别对U937细胞系和Kasumi-1细胞系转染miR-152 mimics和inhibitor,CCK-8法检测U937细胞及Kasumi-1细胞增殖情况,Annexin V/PI流式实验检测细胞凋亡,流式细胞术检测细胞周期。结果:miR-152在急性髓系白血病患者骨髓及细胞系中较非恶性血液病对照组表达明显下降。U937细胞系转染miR-152 mimics后,增殖受抑制,细胞凋亡增加,细胞阻滞在G0-G1期;Kasumi-1细胞系转染miR-152 inhibitor后,增殖增加,细胞凋亡减少,G0-G1期细胞减少。结论:miR-152在急性髓系白血病患者骨髓及细胞系中的表达水平均较正常对照组显著降低。miR-152在急性髓系白血病中起抑癌基因的作用,过表达miR-152可以抑制细胞增殖,促进凋亡。     

hCLP46基因在不同侵袭转移潜能乳腺癌细胞表达中的差异及其意义

目的 探讨hCLP46基因在MCF-7和MDA-MB-231乳腺癌细胞中表达的差异性及其意义。方法 采用RT-PCR的方法检测2个细胞系(乳腺癌低度侵袭转移细胞系MCF-7、中度侵袭转移细胞系MDA-MB-231)中hCLP46 mRNA的表达差异。取MCF-7和MDA-MB-231细胞株进行培养,分别加入100 μmol/L的转化生长因子-β(TGF-β)并设对照组,培养72 h,用Western-blot方法分析P15蛋白表达。结果 RT-PCR检测hCLP46结果显示,在MCF-7和MDA-MB-231细胞系中,内参基因GAPDH的表达量相近,hCLP46基因在两个细胞系中均表达,其中MDA-MB-231细胞系中的表达高于MCF-7细胞系。两个细胞系分别加入TGF-β培养72 h后,与相应的对照细胞系相比,P15的表达均有降低,hCLP46基因高表达的MDA-MB-231细胞中P15表达较MCF-7细胞显著降低。结论 hCLP46基因过表达可能抑制TGF-β对MDA-MB-231和MCF-7乳腺癌细胞P15基因的上调,hCLP46可能在乳腺癌的发病中起到一定作用。     

Detection of low abundance mRNA of myeloid specific genes in cells of acute and chronic lymphoid leukemias by cRNA hybridization

The hybridization to a complementary RNA (cRNA) probe both in situ and in solution was used to assay tiny amounts of mRNA of the lactoferrin (LF) and myeloperoxidase (MPO) genes in normal bone marrow cells and in acute and chronic lymphoid leukemias. Evidence is reported that this technique is much more sensitive than the standard Northern blot technique. The LF mRNA was detectable in three of seven cases of acute lymphoblastic leukemia (ALL) and in three of seven cases of chronic lymphocytic leukemia (CLL). Four cases of ALL were also positive when tested with the MPO cRNA. It is apparent from these results that myeloid specific mRNA, different from MPO, may be detected in leukemic cells with lymphoid phenotype using a method more sensitive than the Northern blot technique. Whether or not the molecular events observed in these cell populations reflect events physiologically occurring rather than a deregulation of gene expression associated to leukemogenesis remains to be established.     

Immunophenotype and Ultrastructural Studies in Blast Crisis of Chronic Myeloid Leukemia

Thirty-four patients with chronic myeloid leukemia in blast crisis (CML-BC) were evaluated for lineage differentiation, with immunological markers and the presence of ultrastructural peroxidase. Eighteen (52.9%) were found to have myeloid blast crisis. Cytochemically, myeloperoxidase (MPO) could bedetectedonly in six patientson light microscopy while in the remaining 12 patients, myeloid differentiation was confirmed only by demonstration of MPO either at ultrastructural level or by the reactivity with anti myeloperoxidase (anti MPO) antibody. Six (17.6%) had lymphoid blast crisis as identified by lymphoid specific markers (CD 19; CD 10; CD7; CD4) along with the absence of myeloid markers. Heterogenous blast cell populations with mixed lineage differentiation were seen in 4 (I I .7%) patients. These cases showed both lymphoid (CD 19, CD 10) and myeloid (anti MPO and ultrastructural MPO) characteristics. A single case of megakaryoblastic blast crisis was identitied with positivity for CD41 and CD42 along with the presence of platelet peroxidase at the ultrastructural level. Five cases (148) of CML blast crisis remained unclassifiable. These results suggest that blast crisis in CML show an arrest of differentiation at an early stage when compared to de novo acute leukemias. This is particularly evident from the fact that MPO could only be demonstrated ultra-StNCtIIrally or with anti MPO antibody in the majority of patients with myeloid differentiation. It is expected that utilisation of molecular studies including immunoglobulin and T-cell receptor gene rearrangement and m-RNA expression for myeloperoxidase will provide a better insight into the level of differentiation for the presently unclassifiable cases of CML-blast crisis.     

Alterations of the PPP1R3 gene in hematological malignancies

PPP1R3 (protein phosphatase 1, regulatory subunit 3) is a candidate tumor suppressor gene at chromosome 7q31, since nonsense and missense mutations of the PPP1R3 gene have been detected in a variety of human cancers. Loss of chromosome 7q is a recurrent abnormality in hematological malignancies, especially of myeloid lineage, and a common region of 7q deletions has been mapped to 7q31. Thus, it has been suggested that 7q31 harbors a tumor suppressor gene whose functional loss contributes to leukemogenesis. To evaluate the possible involvement of the PPP1R3 gene in the development of hematological malignancies, we examined 72 leukemia and lymphoma cell lines for alterations of the PPP1R3 gene by PCR-SSCP and direct sequence analyses. Mutations were detected in 1 (2.8%) of 36 myeloid cell lines, 4 (20.0%) of 20 B-lineage lymphoid cell lines and none of 16 T-lineage lymphoid cell lines. All the mutations were heterozygous, and they consisted of two missense mutations and three silent mutations. The PPP1R3 gene was expressed in cell lines of various cell lineages. These results indicate that PPP1R3 is not a major target of 7q deletions in myeloid leukemia, however, alterations of the PPP1R3 gene may contribute to the development of a subset of hematological malignancies.     

Frequent microsatellite instability and BAX mutations in T cell acute lymphoblastic leukemia cell lines

Allelic status of the BAT26 and BAT25 loci was examined in 117 leukemia/lymphoma cell lines consisting of 44 B-lymphoid lineage cell lines, 30 T-lymphoid cell lines and 43 myeloid cell lines to define the lineage specificity of microsatellite instability (MSI) in hematological malignancies. Seventeen (15%) cell lines were defined as having MSI. The incidence of MSI was significantly (P < 0.01) higher in cell lines of lymphoid lineage (15/74; 20%) than in those of myeloid lineage (2/43; 5%). In the cell lines of lymphoid lineage, the incidence of MSI in T cell acute lymphoblastic leukemia (T-ALL) (11/30; 37%) was significantly (P < 0.01) higher than those in B-lineage malignancies (4/44; 9%). The 17 cell lines with MSI were subjected to the mutation analysis of the coding microsatellites in 13 candidate genes. Frameshift mutations were most frequently detected in the BAX gene (14/17, 82%), while the hMSH3, hMSH6, TGFbetaRII, DRP and IGFIIR genes were less frequently mutated (24-47%). The present result indicates that MSI is involved in the development and/or progression of lymphoid malignancies, especially of T-ALL, through the inactivation of BAX and several other genes.     

Receptor binding of human granulocyte colony-stimulating factor to the blast cells of myeloid leukemia.

Human granulocyte colony-stimulating factor (G-CSF) rapidly loses the biological activity and the receptor binding capacity following radioiodination. We have made a mutein of human G-CSF, KW-2228, in which Thr-1, Leu-3, Gly-4, Pro-5, and Cys-17 were respectively substituted with Ala, Thr, Tyr, Arg, and Ser; showed more potent G-CSF activity; and retained full biological activity and receptor binding capacity at least 2 weeks of radioiodination. G-CSF is an effective growth factor for the blasts of myeloid leukemia. Radioiodinated KW-2228 was prepared using solid-phase glucose oxidase-lactoperoxidase. Human leukemia cell lines and the blast cells from leukemia patients were examined for binding. High affinity binding sites were identified on myeloid cell lines and on the blasts obtained from acute myeloid leukemia patients. Scatchard analysis showed that a single binding site for G-CSF was observed (361-1688 receptors/cell; Kd 128-1400 pM). In contrast, specific binding of 125I-KW-2228 was not demonstrated on lymphoblastic cell lines or the blast cells of acute lymphoid leukemia or lymphoma. This difference was reflected in the effectiveness of G-CSF to stimulate colony formation in acute myeloid leukemia blasts, while G-CSF did not stimulate colony formation of the blast cells from acute lymphoid leukemia.     

Expression of myeloid cell phenotypes by a novel adult T-cell leukemia/lymphoma cell line.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) can infect a number of cells of different lineages in vitro, yet the immunophenotypes of most adult T-cell leukemia/lymphomas (ATLs) are restricted to CD4+. The apparent discrepancy between these findings is still largely unknown. PURPOSE: We report on a unique case of ATL in which the leukemia cells were positive for both T-cell and myeloid cell antigens. To characterize these cells, we isolated cell lines from this patient with ATL. METHODS: The fresh leukemia cells were cultured without the addition of interleukin-2. Cell cloning was carried out by limiting dilution. RESULTS: A cell line (MU) and its clonal sublines were established. MU cells showed the same chromosomal abnormalities and T-cell receptor beta-chain gene rearrangement pattern as those of fresh leukemia cells. MU cells were exclusively positive for a myeloid cell marker (CD13) but not for T-cell markers, despite the presence of T-cell receptor gene rearrangement. CONCLUSION: The established ATL cell line showed both T-cell and myeloid cell characteristics, which seems to be the first evidence for the close association of ATL cells with both lymphoid and myeloid features. The cell line may provide a new insight for the targets of HTLV-1 infection and transformation in vivo.     

Cell surface characteristics of human histiocytic lymphoma lines-I. Surface glycoprotein patterns

The galactose oxidase tritiated sodium borohydride cell surface labelling technique has been used to study the expression of major surface glycoproteins on 6 human diffuse histiocytic lymphoma (HL) cell lines. The cell surface glycoprotein profiles were compared with that of normal human blood monocytes and granulocytes and 1 myeloid leukemia cell line. Two major types of patterns, one ‘monocytic’ and one ‘lymphoid’ were found among the lines: (1) One line, the U-937, shared the basic surface glycoproteins with normal monocytes and strongly expressed the glycoprotein (GP 130) characteristic for normal granulocytes and myeloid leukemic cells. The U-937 has other phenotypic characteristics strongly suggesting a monocyte derivation. (2) All other lines expressed major surface glycoproteins dissimilar from monocytes and granulocytes but with some resemblance to that of some lymphoid cell populations previously examined by the same surface radiolabelling technique. However, some heterogeneity with regard to the expressed major surface glycoproteins was found within that group of HL lines. The SKW-4 and the Su-DHL-2 both expressed a prominent GP 85 but failed to express detectable GP 24 and GP 31 (Ia-like antigens) while the other three lines (Su-DHL-4; Su-DHL-7 and Su-DHL-9) all had weak GP 85 but identifiable GP 24 and GP 31. The results suggest that galactose oxidase surface radiolabelling technique is useful for distinguishing monocytoid and lymphoid types of HL and that the group of lymphoid HL is heterogenous.     

Expression of the myeloperoxidase gene in acute and chronic myeloid leukemias: relationship to the expression of cell cycle-related genes

The expression of the myeloperoxidase (MPO) gene was studied, by means of Northern blot analysis in 14 cases of acute myeloid leukemia (AML), 11 cases of chronic myeloid leukemia (CML), and 6 cases of CML blast crisis, and in HL60 cells before and after induction of terminal differentiation with retinoic acid (RA), phorbol esters (TPA), or vitamin D. The expression of a panel of cell cycle-related genes, namely C-MYC, histone H3, ornithine decarboxylase, P53, vimentin, and calcyclin, was also studied in the same cell populations. Our results indicate that: (a) MPO gene expression (steady state mRNA levels) is strictly confined to the first stages of myeloid differentiation, reaching its peak at the promyelocyte stage and becoming undetectable in mature granulocytes and monocytes; (b) cells devoid of any detectable MPO enzymatic activity such as leukemic basophils have a high content of MPO mRNA; and (c) MPO gene expression is not related to the growth activity of the cell population. Finally, our results show that the pattern of expression of growth-regulated genes in the neoplastic myeloid disorders AML, CML, and CML blast crisis is remarkably different.     

Myeloperoxidase gene expression and regulation by myeloid cell growth factors in normal and leukemic cells

Myeloperoxidase (MPO) is present in azurophilic granules which appear in the promyelocyte stage of differentiation, and is the most common functional protein of myeloid cells. With progress in molecular biology, the expression and regulation of MPO have been clarified in normal myeloid and leukemic cells, not only by enzymatical activity but at the gene level MPO expression is affected by the differentiation of myeloid cells, and has been suggested to be regulated by myeloid cell growth factors, such as granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor and interleukin-3. In the past decade the signal transduction from their receptors has been clarified. This review describes the expression and regulation of the MPO gene in myeloid cells including myeloid disorders, such as myeloid leukemia or myelodysplastic syndromes, The effects on MPO by myeloid growth factors and signal transduction from their receptors are also presented.