CaMKⅡγ在慢性粒细胞白血病急变中的作用和机制研究
发布时间:2019-05-19 14:54
【摘要】:研究背景慢性粒细胞白血病(Chronic Myeloid Leukemia, CML),是由BCR-ABL融合基因引起的造血干细胞疾病。所有的慢粒均具有Ph染色体,即特异性的t(9;22)(q34;q11)核型。BCR-ABL融合基因编码的p210BCR-ABL蛋白具有极强的酪氨酸激酶活性,它将使一系列信号蛋白发生持续性磷酸化,影响细胞的增殖分化、凋亡及黏附,导致慢粒的发生。因此,BCR-ABL融合基因被认为是慢粒发病的分子基础,也是慢粒诊断、疗效观察、预后等的监测指标。慢性粒细胞白血病根据临床特征和实验室检查可分为三个阶段:慢性期,加速期和急变期,急变期是慢性粒细胞白血病进程的最后阶段,其表现类似急性白血病,进展迅速、生存期较短。目前的观点认为,BCR-ABL融合基因在慢粒的启动过程中起着重要作用,而后续继发性分子和遗传学异常可能导致CML急变期的进展。然而慢粒急变期进展的确切分子机制迄今仍不十分清楚,因此鉴定出控制慢粒从慢性期向急变期转变的分子开关显得尤为重要。以往系列研究已经表明,慢性粒细胞白血病从慢性期到急变期的进展需要大量白血病干细胞(Leukemia stem cells, LSCs)的自我复制,但有关慢性粒细胞白血病急变的关键驱动因子目前依然未知。我们以往的研究证实,钙调蛋白依赖性激酶Ⅱγ(CaMKⅡγ),是小檗胺清除伊马替尼耐药的慢性粒细胞白血病干细胞的一个关键靶分子,它在白血病细胞增殖中的多个信号通路起了关键调节作用。这一新的发现提示我们CaMKⅡγ可能参与了慢粒急变的分子机制。研究内容与结果1.CaMKⅡγ在慢粒急变和白血病干细胞的自我更新中的作用为了确定CaMKⅡγ是否在CML急变的发展中起作用,我们首先关注了CaMKⅡγ缺失对BCR-ABL诱导的小鼠CML模型的影响。CaMKⅡγ/-CML小鼠的外周血白细胞数量较WT组低得多,其存活时间显著延长。这一结果表明,CaMKⅡγ基因缺失会显著抑制CML进展,改善CML小鼠的存活。白血病干细胞在维持CML急变的关键作用促使我们研究CaMKⅡγ对CML的LSCs在体内作用的效果。我们检测了移植25天后CML小鼠骨髓和脾脏中CML LSC和正常造血干细胞的数量。流式细胞仪检测数据显示,CaMKⅡγ缺失组骨髓和臆脏中的LSC水平降低了21.5倍和5.1倍,但对正常HSC的影响不大。这些结果表明,CaMKⅡγ对LSCs的自我更新和复制是必需的。2.CaMKⅡγ在人慢粒白血病细胞克隆和CD34阳性细胞增殖中的作用为了确定CaMKⅡγ在人慢性粒细胞白血病细胞克隆形成中的作用,我们下调了人慢性粒细胞白血病急变细胞系K562细胞CaMKⅡγ的表达,结果显示,CaMKⅡγ特异性shRNA明显抑制了K562细胞的克隆形成能力。同时,CaMKⅡγ过表达导致的细胞克隆比对照组要大,白血病细胞有丝分裂的数目也比对照组显著增加。这些结果意味着,CaMKⅡγ可以通过促进白血病干细胞的自我更新来增强白血病细胞的集落形成能力。为了验证这一假设,我们接下来关注了CaMKⅡγ对K562细胞CD34表达水平的影响,结果显示,CaMKⅡγ的过表达显著增加了K562细胞CD34+细胞的数量,而下调CaMKⅡγ后,K562细胞CD34+细胞的数量减少,这表明CaMKⅡγ在白血病细胞的集落形成能力和CD34+细胞的自我复制中具有重要作用。3.CaMKⅡγ的异常激活与肿瘤的生长、进展和总生存率有关为了评估CaMKⅡγ在体内是否促进人类慢性粒细胞白血病的进展,我们构建了两种人慢粒急变异种移植瘤动物模型,一种是使用CaMKⅡγ高表达(K562/ ADR)构建的,另外一种是用CaMKⅡγ低表达(K562)所构建,然后比较了荷瘤老鼠肿瘤的生长和肿瘤的生存率。与体外实验观察一致,我们观察到,CaMKⅡγ高表达组显示出较高的肿瘤生长速率和更快的体重下降程度。另外,CaMKⅡγ表高达组的荷瘤小鼠组生存期显著缩短。这些结果表明CaMKⅡγ可能促进CML急变的进展。为了进一步验证在CML原代样本中的表达,我们接下来检测了CaMKⅡγ是否在人慢性粒细胞白血病的进展过程中异常活化。我们使用免疫印迹法检测了15个慢性期(CP),12个加速期(AP)和19个急变期(BC)原代样本中磷酸化CaMKⅡγ的表达,结果发现:在慢性粒细胞白血病急变期和加速期磷酸化CaMKⅡγ异常高表达,但在慢性期表达量低或不表达。为了确定CaMKⅡγ在CML急变中的异常活化是否与CML的LSCs相关联,我们还同时检测了一个公认的CML LSC自我更新中关键的调节分子β-catenin的表达水平。结果表明,CaMKⅡγ的表达与慢性粒细胞白血病患者样本中β-catenin的水平呈正相关,这表明CaMK Ⅱγ可能在β-catenin介导的LSC自我更新中发挥了重要作用。因此,高度活化的CaMKⅡγ的确参与CML慢性期到加速和急变期的进程。4.CaMKⅡγ通过降低细胞核p27kip1的表达导致慢粒急变为了阐明CaMKⅡγ导致慢粒急变的分子机制,我们接下来检测CaMKⅡγ表达是否对细胞周期蛋白依赖性激酶(CDK)抑制剂p27Kip1(p27蛋白)发生作用,p27在维持干细胞静止中是个关键的制动分子。结果表明,CaMKⅡγ的过表达显著增强了磷酸化p27(T187)蛋白水平,并降低细胞核p27的积聚。在293-T细胞转染了EGFP-CaMKⅡγ质粒后也观察到了类似的结果。这些数据表明,CaMKⅡγ通过磷酸化的p27,反过来通过蛋白酶体依赖性降解降低细胞核p27蛋白的表达。为了获得生化证据,我们进行了免疫共沉淀实验。从K562细胞分离的总蛋白,分别用flag-CaMKⅡγ抗体或磷酸化的p27抗体进行免疫共沉淀,通过SDS-PAGE,并用与对p27的抗体或标记抗体或CaMKⅡγ抗体进行Western印迹法分析。正向反向结果均证实,p27蛋白和CaMKⅡγ蛋白都存在于免疫沉淀复合物中。为了验证这些结果,我们关注了CaMKⅡγ过表达对静止期细胞的影响。结果显示,CaMKⅡγ过表达增加了有丝分裂期的细胞,静止细胞(Go/G1期)从43.9%减少到36.1%,而增殖期细胞(S+G2/M)从52.7%增加至62.2%,表明CaMKⅡγ可以通过终止p27介导的细胞静息期,使白血病细胞进入增殖期。为了进一步证实CaMKⅡγ在白血病细胞中的作用,我们分析了该激酶的亚细胞定位,并发现CaMKⅡγ蛋白在细胞周期的不同阶段中的表达水平是不同的。CaMKⅡγ存在于休眠细胞(G0期),但一旦细胞进入增殖状态,CaMKⅡγ蛋白表达水平显著增加,高峰出现在早期的S/G2期细胞,然后下降。这些结果表明,CaMKⅡγ可能在两个细胞周期Go-G1和S-G2/M起促进作用,这表明,CaMKⅡγ在促进CML细胞的生长过程中发挥重要作用。总之,我们已经确定CaMKⅡγ在慢粒急变中的重要作用。在我们的研究结果的基础上,我们推断,慢性粒细胞白血病是由BCR-ABL融合基因导致,但从慢性期为急变期可能通过CaMKⅡγ的异常活化促进白血病干细胞的自我更新和复制来实现的。此外,CaMKⅡγ通过磷酸化p27蛋白(T187),降低了细胞核p27的表达水平,并重新激活休眠期的白血病干细胞。因此,CML急变需要CaMKⅡγ的异常活化。针对CaMKⅡγ可能代表了一种新的诊断和治疗CML的方法。结论1.CaMKⅡγ的异常活化促进了白血病干细胞的自我更新与复制,在慢性粒细胞白血病从慢性期到急变期中发挥重要作用。2.CaMKⅡγ通过磷酸化p27,降低细胞核p27的表达水平,重新激活休眠期的白血病干细胞。3.CaMKⅡγ通过促进细胞周期的G0-G1期和S-G2/M期过渡来促进慢粒细胞生长。4.CaMKⅡγ可能成为一种诊断和治疗慢性粒细胞白血病的新靶点。
[Abstract]:Background Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease caused by BCR-ABL fusion gene. All the slow particles have the Ph chromosome, that is, the specific t (9;22) (q34; q11) karyotype. The p210BCR-ABL protein encoded by the BCR-ABL fusion gene has strong tyrosine kinase activity, which will cause persistent phosphorylation of a series of signal proteins, which can affect the proliferation and differentiation, apoptosis and adhesion of the cells, and lead to the occurrence of slow particles. Therefore, the BCR-ABL fusion gene is thought to be the molecular basis of the pathogenesis of slow-grain, and it is the monitoring index of slow-grain diagnosis, curative effect observation and prognosis. Chronic myeloid leukemia can be divided into three stages according to the clinical features and laboratory tests: the chronic stage, the acceleration phase and the acute phase, and the acute stage is the last stage of the chronic granulocytic leukemia process, which is similar to the acute leukemia, the progress is rapid, and the survival time is short. The present point of view is that the BCR-ABL fusion gene plays an important role in the initiation of CML, and the follow-up secondary and genetic abnormalities may lead to the progress of the acute phase of CML. However, the exact molecular mechanism of the progress of the slow-grain phase transition is still not clear so far, so it is very important to identify the molecular switches that control the transition of the slow-grain from the chronic stage to the acute stage. The past series of studies have shown that the progression of chronic myeloid leukemia from chronic to acute phase requires self-replication of a large number of leukemia stem cells (LSCs), but the key drivers of the acute change in chronic myeloid leukemia are still unknown. Our previous studies have confirmed that calmodulin-dependent kinase II (CaMK鈪,
本文编号:2480818
[Abstract]:Background Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease caused by BCR-ABL fusion gene. All the slow particles have the Ph chromosome, that is, the specific t (9;22) (q34; q11) karyotype. The p210BCR-ABL protein encoded by the BCR-ABL fusion gene has strong tyrosine kinase activity, which will cause persistent phosphorylation of a series of signal proteins, which can affect the proliferation and differentiation, apoptosis and adhesion of the cells, and lead to the occurrence of slow particles. Therefore, the BCR-ABL fusion gene is thought to be the molecular basis of the pathogenesis of slow-grain, and it is the monitoring index of slow-grain diagnosis, curative effect observation and prognosis. Chronic myeloid leukemia can be divided into three stages according to the clinical features and laboratory tests: the chronic stage, the acceleration phase and the acute phase, and the acute stage is the last stage of the chronic granulocytic leukemia process, which is similar to the acute leukemia, the progress is rapid, and the survival time is short. The present point of view is that the BCR-ABL fusion gene plays an important role in the initiation of CML, and the follow-up secondary and genetic abnormalities may lead to the progress of the acute phase of CML. However, the exact molecular mechanism of the progress of the slow-grain phase transition is still not clear so far, so it is very important to identify the molecular switches that control the transition of the slow-grain from the chronic stage to the acute stage. The past series of studies have shown that the progression of chronic myeloid leukemia from chronic to acute phase requires self-replication of a large number of leukemia stem cells (LSCs), but the key drivers of the acute change in chronic myeloid leukemia are still unknown. Our previous studies have confirmed that calmodulin-dependent kinase II (CaMK鈪,
本文编号:2480818
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