小檗胺诱导K562细胞凋亡及其与bcr/ablgene及P210表达的关系

目的:探讨小檗胺(BER)诱导K562细胞凋亡及其可能的机制。方法:以流式细胞仪(FCM)分析、电镜观察细胞微结构变化及基因组DNA电泳等方法检测细胞凋亡。以RT-PCR及Westernblot方法检测K562细胞BCR/ABLmRNA和蛋白质水平表达。结果:FCM检测见到典型的凋亡峰,8.0mg/LBER作用24-72h,随药物作用时间延长,细胞凋亡率由(29.20±3.82)%上升至(61.77±4.35)%(P＜0.01);以2.0-8.0mg/LBER作用24h,随药物浓度增加,K562细胞的凋亡率也增加。电镜下可见明确的细胞凋亡的形态学改变。DNA电泳呈现典型的梯形条带。16.0mg/LBER处理24h,K562细胞bcr/ablmRNA相对表达量及BCR/ABL融合蛋白质P210水平均明显低于对照组,分别为0.73±0.02vs1.19±0.02(P＜0.01)和0.63±0.01vs1.04±0.02(P＜0.01),呈时间-浓度依赖效应。经BER作用后,K562细胞bcr/ablmRNA表达强度与P210水平呈正相关(r=0.928,P＜0.05),且细胞凋亡率与bcr/ablmRNA表达呈负相关(r=-0.997,P＜0.01)。结论:在体外BER对K562细胞有明显的促凋亡作用,抑制bcr/ablmRNA表达及其蛋白质水平可能是其作用的重要机制之一。     

The Synergistic Inhibitory Effect of STI571 in Combination with Arsenic Trioxide (As2O3) on Multidrug-Resistant Leukemic Cells

OBJECTIVE To study the synergistic effect of STI571, an inhibitor of tyrosine kinase, in combination with arsenic trioxide As2O3 on a multidrug-resistant leukemia cell line expressing bcr-abl.METHODS The cytotoxic effect of STI571 alone or in combination with different concentrations of As2O3 on the bcr-abl and mdr1 -positive leukemia cell line, K562-n/VCR, was examined by the MTT method.RESULTS One μmol/L of STI571 alone had no significant cytotoxic effect on K562-n/VCR cells. However the cytotoxic effect increased markedly when combined with As2O3 at concentrations of 10-5, 10-6, 10-7 and 10-8 mol/L. The IC50 of K562-n/VCR cells in As2O3 group was 1.879 μmol/L, with. Upon addition of STI571, the IC50 decreased to 0.155 μmol/L resulting in a synergistic cytotoxic effect on K562-n/VCR ceils that was increased 12.1 times.CONCLUSION A combination of STI571 with As2O3 has a more powerful inhibitory effect on leukemia cells expressing positive bcr-abl and positive mdrl compared to the effect with As2O3 alone.     

CD66 expression in acute leukaemia

Antibodies against CD66 identify antigens from the carcinoembryonic antigen (CEA) family of proteins, which belong to the immunoglobulin gene superfamily. Despite being usually restricted to cells of myeloid or monocytic origin, CD66 expression has also been reported in blasts from children with B-cell lineage acute lymphocytic leukaemia (ALL). An analysis of the CD66 expression was undertaken in a series of acute leukaemia patients. Antigenic expression was analysed using triple combinations of monoclonal antibodies (mAbs) in forty-five patients. The CD66 Kat4 fluorescein isothiocyanate clone was purchased from Dako (Glostrup, Denmark). CD66 was expressed in 2 of 29 patients with AML (acute myeloblastic leukemia) (6.8%) and in 8 of 12 patients with B-cell lineage ALL (66.7%; P <0.001); in blast crisis (BC) of chronic myelocytic leukaemia (CML), CD66 was expressed in two patients with lymphoid BC but not in the two with myeloid BC. The co-expression of CD66 with other myeloid antigens was observed in all CD66+ ALL/Ly-BC cases tested: CD 13 in six patients, CD33 in seven and CD117 in two patients. The CD66 expression is more frequent in ALL than in AML. Furthermore, we analysed minimal residual disease (MRD) in eight patients in complete remission. CD66 expression was associated with an abnormal B-cell differentiation pattern and with increases in CD34/CD19+ cells in all but one case. These findings suggest that an aberrant expression of CD66 could be used to investigate MRD in ALL. The association between CD66 reactivity and bcr-abl in adult ALL remains to be investigated. Received: 31 May 1999 / Accepted: 10 November 1999     

BCR-ABL influences the antileukaemic efficacy of alkylphosphocholines

We have compared the antileukaemic efficacy of a series of new i.v. injectable alkylphosphocholines (APC) with their clinically used congeners miltefosine and perifosine. The test system consisted of four leukaemic cell lines carrying the bcr-abl rearrangement (K-562, LAMA-84, CML-T1 and BV-173) and two other leukaemic cell lines (HL-60 and SKW-3) without this genetic alteration. The prototype of i.v. injectable APC, erucylphosphocholine, was more active against BCR-ABL-positive cell lines than the two reference APC. It induced programmed cell death in HL-60 and SKW-3 cells after exposure for 24 h, and in bcr-abl expressing cells after a prolonged incubation period (48 h). LAMA-84 cells responded to i.v. injectable APC with increased conversion to an adherent, fibroblast-like phenotype. Experiments with a cell-free system showed that the target structures of APC are localized within the cytoplasmic compartment. Blockade of ceramide synthase by fumonisin B1 was insufficient to prevent oligonucleosomal DNA fragmentation. Using RT-PCR we confirmed that K-562 and LAMA-84 cells carry the b3a2 fusion type, and CML-T1 and BV-173 the b2a2 variant. BV-173 cells had the lowest level of bcr-abl mRNA which correlated with their increased sensitivity. Transfection of K-562 cells with antisense oligonucleotides directed against bcr-abl caused a specific suppression of K-562 clonogenicity. Our data indicated that i.v. injectable alkylphosphocholines are potent inducers of apoptosis and display increased antileukaemic efficacy against BCR-ABL-positive blasts as compared with miltefosine and perifosine. The expression of BCR-ABL cannot prevent apoptosis but delays erucylphosphocholine-induced programmed cell death. Transfection with bcr-abl directed antisense oligonucleotides reduces the clonogenicity of K-562 cells.     

大黄素诱导耐药白血病细胞株K562/Adr凋亡及下调P210表达

 目的 研究大黄素对多药耐药白血病细胞株K562／Adr（KAR）增生、凋亡的影响及探讨bcr-abl、mdr-1基因在其中的变化。方法 应用四甲基偶氮唑蓝（MTT）比色法、DNA片段化分析及TdT酶介导的末端缺失原位标记（TUNEL）法检测大黄素对KAR细胞增生及凋亡的影响；RT-PCR法检测大黄素对KAR细胞bcr-abl、mdr-1基因mRNA表达的影响；Western blotting法检测大黄素对KAR细胞bcr-abl融合蛋白P210表达的影响。结果 大黄素能有效抑制KAR细胞的增生，作用72 h的IC50约为20μmol／L，并能诱导其凋亡,随药物作用浓度的增加，凋亡率也逐渐上升；大黄素下调KAR细胞bcr-abl、mdr-1基因mRNA的表达；也下调了KAR细胞P210蛋白的表达。结论 大黄素能有效抑制KAR细胞增生，诱导其凋亡；并可能通过下调bcr-abl和mdr-1 的表达起作用。     

Role of Dok-1 and Dok-2 in myeloid homeostasis and suppression of leukemia

Dok-1 and Dok-2 are closely related rasGAP-associated docking proteins expressed preferentially in hematopoietic cells. Although they are phosphorylated upon activation of many protein tyrosine kinases (PTKs), including those coupled with cytokine receptors and oncogenic PTKs like Bcr-Abl, their physiological roles are largely unidentified. Here, we generated mice lacking Dok-1 and/or Dok-2, which included the double-deficient mice succumbed to myeloproliferative disease resembling human chronic myelogenous leukemia (CML) and chronic myelomonocytic leukemia. The double-deficient mice displayed medullary and extramedullary hyperplasia of granulocyte/macrophage progenitors with leukemic potential, and their myeloid cells showed hyperproliferation and hypo-apoptosis upon treatment and deprivation of cytokines, respectively. Consistently, the mutant myeloid cells showed enhanced Erk and Akt activation upon cytokine stimulation. Moreover, loss of Dok-1 and/or Dok-2 induced blastic transformation of chronic phase CML-like disease in mice carrying the bcr-abl gene, a cause of CML. These findings demonstrate that Dok-1 and Dok-2 are key negative regulators of cytokine responses and are essential for myeloid homeostasis and suppression of leukemia.     

STI571与三氧化二砷对多药耐药bcr-abl阳性白血病细胞的协同抑制作用

目的　研究酪氨酸激酶抑制剂STI5 71与三氧化二砷 (As2 O3 )联合应用对多药耐药bcr abl阳性白血病细胞的协同效应。方法　采用MTT法比较STI5 71单独或与不同浓度的As2 O3 联合应用对bcr abl和mdr1共同阳性的白血病细胞系K5 6 2 n/VCR的抑制作用。结果　 1μmol/L的STI5 71对K5 6 2 n/VCR细胞无明显细胞毒作用 ,与 10 -5,10 -6,10 -7,10 -8mol/L的As2 O3 联合应用 ,细胞毒作用明显增强。As2 O3 单独对K5 6 2 n/VCR细胞的IC50 为 1.879μmol/L ,加STI5 71后 ,IC50 为 0 .15 5 μmol/L ,协同抑制作用为 12 .12倍。结论　STI5 71与As2 O3 联合应用对于bcr abl与mdr1共同阳性的白血病细胞有更强的抑制作用     

Activation of Bcr-abl Fusion Gene and Ras Oncogenes in Chronic Myelogenous Leukemia

We attempted to detect the bcr-abl fusion gene and ras gene family in CML by the in vitro focus forming assay and the tumorigenicity assay. Eight of 14 chronic phase and both of two blastic phase cases showed transforming activity in the tumorigenicity assay. However, only one chronic phase sample was positive in the in vitro focus forming assay. Among these 10 transformants, we found N-ras activation in one chronic phase, and K-ras activation in another chronic phase case. The bcr-abl fusion gene was activated in one chronic phase and all of the blastic phase cases by the tumorigenicity assay. The present result showed that the bcr-abl fusion gene transfected N1H3T3 cells formed tumors in nude mice in contrast to the in vitro focus forming assay. The bcr-abl fusion gene may play important roles in the progression as well as the pathogenesis of CML.     

NGAL在慢性粒细胞性白血病中的表达及其诊断意义

目的探讨NGAL在慢性粒细胞性白血病(下称慢粒)患者骨髓中的表达及其临床意义,为深入研究NGAL在慢粒发生、发展过程中所发挥的作用提供科学依据。方法运用免疫组化技术,对42例慢粒患者及健康对照组骨髓样品中的NGAL表达情况进行检测,并结合ELISA分析方法检测慢粒患者和健康对照组血清中NGAL的表达。结果通过免疫组化染色,NGAL在42例慢粒患者及健康对照组骨髓中表达皆为阳性,但是慢粒组NGAL表达量明显高于健康对照组,差异具有统计学意义(P0.05)。同时,通过ELISA分析方法测出慢粒患者血清中NGAL含量明显高于健康对照组,差异具有统计学意义(P=0.001)。结论 NGAL在慢粒的发生、发展过程中起着重要作用。