多单位核酶对慢性粒细胞白血病细胞的增殖抑制及凋亡诱导效应研究

目的：探讨多单位核酶体外净化慢性粒细胞白血病（慢粒）骨髓的可能性,研究多单位核酶的体外切割活性及对慢粒细胞恶性表型的逆转作用。方法：针对慢粒发病中起重要作用的bcrabl融合基因,在融合位点两侧44碱基范围内设计,合成3个核酶,其中2个切割位点位于bcr基因,1个位于abl基因。通过基因重组构建多单位核酶体外转录载体及逆转病毒表达载体,鉴定其体外切割活性。将多单位核酶逆转录病毒表达载体转染K562细胞,应用MTT,3H－TdR掺入,RT－PCR,斑点杂交,流式细胞仪,透射及扫描电镜等方法,检测多单位核酶对慢粒细胞增殖活性及细胞超微结构,细胞周期,凋亡等的影响。结果：多单位核酶的体外切割活性为70．8％,逆转录病毒载体转染慢粒酶转染细胞后能够有效切割细胞RNA,使转染细胞RNA减少1000倍左右,流式细胞仪检测显示多单位核酶转染72h后,18．4％的细胞发生凋亡,大多数细胞被阻滞于G期,分裂期（S期）细胞数减少约41．9％,透射,扫描电镜见转染细胞出现核固缩,凋亡小体等细胞凋亡的特异性表现。结论：多单位核酶不仅具有较高的体外切割活性,而且其逆转录病毒载体能够有效转染慢粒细胞系,在细胞内持续表达,切割细胞RNA,诱导细胞凋亡,抑制细胞增殖,逆转慢粒细胞的恶性表型,在慢粒基因治疗中具有潜在应用的价值。     

PRDX1抑制蛋白酶体抑制剂诱导人甲状腺癌细胞凋亡机制探讨

目的：初步探讨过氧化还原蛋白1（peroxirodoxin1,PRDX1）抑制蛋白酶体抑制剂诱导人甲状腺癌细胞凋亡的机制。方法：选取人甲状腺癌细胞,设立随机序列核酸siRNA、siASK1、siPRDX1和siASK1＋siPRDX1组,分别用培养液、lactacystin和MG132培养细胞;用蛋白印迹法检测PRDX1、ASK1、p38、JNK1／2、p-ASK1、p-P38和p-JNK1／2在各组甲状腺癌细胞中的表达;用微小RNA干扰技术转染细胞;应用流式细胞仪检测细胞凋亡率。结果：MG132处理组中,p-ASK1是对照组的1．5倍（F=214．14,P〈0．001）,其下游效应子p-p38和p-JNK1／2分别为对照组的1．34（F=216．75,P〈0．001）和1．94倍（F=1601．68,P〈0．001）;siASK1下调MG132介导的p-ASK1为随机序列组的0．58倍（F=1136．82,P〈O．001）,p-p38为随机序列组的0．73倍（F=507．00,P〈0．001）,p-JNK1／2为随机序列组的0．51倍（F=6087．48,P〈0．001）。甲状腺癌细胞凋亡率降低为34．25％,显著低于于随机序列组的51.98％,F=18．39,P=0．013。MG132处理组中,siPRDX1组甲状腺癌细胞凋亡率为68．99％显著高于随机序列组的49．58％和siPRDX1＋siASK1联合组的41．28％,而siASK1组的甲状腺癌细胞凋亡率为31．85％,显著低于上述两组,组间差异有统计学意义,F=30-13,P〈0．001。结论：PRDX1通过负向调节ASK1活性及ASK1-p38和ASK1-JNK通路,抑制ASK1介导的细胞凋亡进而消减MG132对甲状腺癌细胞毒性效应。     

细胞外信号调节激酶1/2参与三氧化二砷诱导的胶质瘤细胞凋亡

目的:探讨细胞外信号调节激酶1/2(ERK1/2)信号通路在三氧化二砷诱导的胶质瘤细胞U251凋亡中的作用,为应用三氧化二砷治疗胶质瘤奠定基础。方法:50μmol/L三氧化二砷作用U251细胞,不同时间检测胶质瘤细胞增殖活性,半定量PCR检测ERK1/2mRNA表达,Western blot检测ERK1/2蛋白表达;Ho-echst33258染色及流式细胞术(FCM)检测细胞凋亡;转染ERK1/2上游激酶MEK1,应用ERK1/2激酶抑制剂U0126观察ERK1/2通路在肿瘤凋亡及增殖中的作用;比色分析法检测Caspase-3活性的变化。结果:三氧化二砷诱导胶质瘤细胞发生明显凋亡,抑制肿瘤细胞增殖;增加ERK1/2蛋白的表达,呈时间依赖性;阻断ERK1/2信号通路后胶质瘤细胞凋亡受到抑制,Caspase-3活性下降。结论:ERK1/2信号通路在三氧化二砷诱导的胶质瘤细胞凋亡中起重要作用。     

慢性粒细胞白血病伴戈谢细胞增多一例

患者男性,45岁,2004年7月21日因发现白细胞高18年就诊。患者18年前因皮肤感染迁延不愈,外院查血常规示白细胞高于40×109/L,血小板和红细胞具体数值不详,骨髓细胞检查示:骨髓有核细胞增生极度活跃,粒∶红=6.5∶1,粒细胞系占0.775,NAP:阴性,考虑为慢性粒细胞白血病(CML),给予白消安、靛玉红交替服用,白细胞控制在10×109/L以下。于1997年10月复查骨髓细胞示:骨髓有核细胞增生明显活跃,粒∶红=5.4∶1,粒系明显增生,占0.76,中幼粒比值明显增高,杆状核粒细胞胞体增大,其他细胞形态大致正常;红细胞系增生偏低,全片见巨核细胞7个,后改用羟基脲…     

FLT3靶向抑制诱导急性髓细胞白血病细胞凋亡的实验研究

目的通过检测FMS样酪氨酸激酶3（FLT3）靶向抑制对急性髓细胞白血病（AML）细胞株THP-1、HL-60凋亡的作用,探讨FLT3异常表达在AML细胞凋亡中的作用。方法用FLT3靶向短发夹状干扰RNA (FLT3-shRNA)特异性下调THP-1、HL-60细胞中FLT3的表达。用Annexin V-FITC检测早期凋亡细胞比例,用 DNA Ladder检测细胞凋亡的特征条带,用TUNEL细胞原位杂交的方法检测细胞凋亡晚期形态学变化和比例,用流式细胞法（FCM）检测细胞周期的变化。结果用Annexin V-FITC检测FLT3-shRNA处理48 h的THP-1、HL-60细胞的早期凋亡率与对照组相比都有增加（P＜0.01）;两细胞株都检测到了凋亡细胞梯状条带（DNA Ladder）;细胞周期都出现G0/G1期细胞比例的增加（P＜0.01）,S期细胞比例的下降（P＜0.05）。另外在THP-1细胞TUNEL细胞原位杂交也观察到晚期凋亡细胞比例的明显增加（P＜0.01）。结论shRNA介导的FLT3抑制可诱导THP-1、HL-60细胞凋亡,支持FLT3过表达具有抗AML细胞凋亡的作用。     

HLA-A0201阳性慢性粒细胞白血病PR1特异性细胞毒性T细胞的初步研究

目的 探讨PR1特异性细胞毒性T细胞(CTL)与HLA-A0201阳性慢性粒细胞白血病(CML)患者疗效及预后的关系,探讨是否可以应用PR1肽作为疗效达到停药试验标准患者的后续免疫治疗,从而维持长期缓解状态.方法 收集28例HLA-A0201阳性CML患者外周血,利用可溶性PR1-MHC-Ⅰ类四聚体技术及流式细胞术检测PR1特异性CTL存在与否、频率高低;比较不同治疗时间CML患者PR1特异性CTL表达的差异.结果 PR1特异性CTL频率与同期PCR(bcr-abl/abl)IS(IS:国际标准值)之间呈负相关(r=-0.658,P＜0.001).接受治疗3、6、9个月及1、2、3、4、5、6年的PR1特异性CTL频率分别为(0.06±0.02)％、(0.10±0.02)％、(0.14土0.02)％、(0.16±0.02)％、(0.20±0.03)％、(0.18±0.03)％、(0.18±0.01)％、(0.17±0.05)％、(0.18±0.03)％.治疗3、6、9个月的频率分别与其他时间点频率相比,差异均有统计学意义(均P＜ 0.05).治疗1年及以上的频率两两相比,差异均无统计学意义(均P＞0.05).伊马替尼400 mg 1次/d治疗3、6、9、12个月,Sokal评分高危组患者PR1特异性CTL频率低于低危组、中危组.结论 治疗效果显著的CML患者体内可持续检测到PR1特异性CTL,其与肿瘤负荷呈负相关,提示PR1特异性CTL可能与抗白血病细胞作用相关,为疗效获得持续稳定MR4-5及MR5.0的CML患者使用PR1肽疫苗作为后续免疫治疗提供了依据.     

慢性中性粒细胞白血病1例

患者男性,60岁,于2013年12月就诊于山东大学齐鲁医院,行右侧疝气修补术时发现脾肿大、白细胞升高,外周血白细胞波动在20×109/L左右,分叶核粒细胞约占85%,血小板和红细胞计数正常,未见幼稚细胞.2014年1月行骨髓穿刺检查:骨髓增生活跃,以粒系增生为主,无骨髓纤维化(图1~3), NAP积分为310分,阳性率92%.无Ph染色体,BCR-ABL1融合基因(-),JAK2-V617F突变(-),单克隆免疫球蛋白检查正常,全身CT扫描排除肿瘤和感染性疾病.初步诊断:骨髓增殖性疾病,未予治疗.2014年10月患者出现消化道出血,初步诊断为乙肝肝硬化、门脉高压、脾肿大,遂行脾脏切除+贲门胃底静脉离断术.术中见肝脏轻度结节性肝硬化表现,脾脏大小为20 cm×20 cm×15 cm.术后2 d患者出现高热、呼吸困难,白细胞升高至105×109/L,中性粒细胞占90%,血小板165× 109/L.考虑患者白细胞升高是术后感染和手术应激导致的类白血病反应,给予抗感染和支持治疗.     

苦参碱通过bcr-abl介导的MEK-ERK通路抑制人慢性粒细胞白血病K562细胞增殖

目的：探讨MEK-ERK通路在苦参碱抑制人慢性粒细胞白血病K562细胞增殖中的分子机制。方法采用Western blot法检测K562细胞内MEK-ERK通路关键分子MEK1、ERK1／2及其上游接头分子Shc、SHP2的总蛋白和磷酸化蛋白的表达;采用反转录聚合酶链反应（RT-PCR）和Western blot检测bcr-abl及有丝分裂原激活的蛋白激酶（MAPK）下游靶蛋白bcl-xL、Cyclin D1、c-myc及p27在转录及蛋白水平的表达。结果苦参碱可明显抑制K562细胞内MEK1、ERK1／2及Shc、SHP2的磷酸化表达,并可在转录及蛋白水平抑制bcr-abl分子表达。同时,RT-PCR和Western blot实验证实,苦参碱处理后,K562细胞内bcl-xL、Cyclin D1、c-myc表达均明显抑制,细胞周期负调控蛋白p27的表达增加。结论苦参碱对K562细胞的抑制效应与bcr-abl介导的MEK-ERK信号通路活性抑制有关,对信号通路中磷酸化蛋白或激酶分子活性的调控可能是其调控MEK-ERK通路活性的重要分子机制。     

抑制吲哚胺2,3-双加氧酶活性促进慢性粒细胞白血病源树突状细胞的功能

目的:研究吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)在慢性粒细胞性白血病源性树突状细胞(dendritic cells derived from chronic myeloid leukemia,CML-DCs)中的表达,及抑制IDO活性对CML-DCs免疫刺激功能的影响.方法:RT-PCR检测17例患者CML-DCs的IDO mRNA的表达情况,流式细胞仪检测CML-DCs免疫表型.在有或无IDO抑制剂1-甲基色氨酸(1-methyltroptophan,1-MT)作用下,分别以不成熟CML-DCs(imDCs)和成熟CML-DCs(mDCs)为刺激细胞,完全缓解期(complete remission,CR)CML患者外周T淋巴细胞为反应细胞建立混合淋巴细胞反应体系,ELISA法检测CML-DCs上清液IL-12水平,MTT法检测CML-DCs刺激自体T淋巴细胞的增殖能力.结果:随着CML-DCs的诱导分化和成熟,IDO mRNA表达逐渐上调;经TNF-α诱导的DCs免疫表型除CD1a外,CD80、CD86、CD83、HLA-DR的表达均明显上调(P<0.05),且上述分子的表达不受1-MT的影响.用1-MT抑制IDO活性后的imDCs和mDCs,其IL-12水平均明显增加(P<0.05,P<0.01),且激发自体T淋巴细胞增殖的能力也明显增强(P<0.05,P<0.01).结论:抑制IDO活性可提高CML-DCs的IL-12分泌水平,增强其对自体T细胞增殖的刺激能力,IDO对DCs的负性调节为白血病生物治疗提供了新的思路.     

PDK1 induces JunB,EMT, cell migration and invasion in human gallbladder cancer

The protein 3-phosphoinositide-dependent protein kinase 1 (PDK1) is upregulated in cancer. Here we showed that PDK1 stimulated cell proliferation, invasion and metastasis in gallbladder cancer (GBC), by inducing JunB and epithelial–mesenchymal transition. JunB levels were increased in GBC samples and positively correlated with PDK1 levels in tumors. High levels of JunB predicted poor overall survival in GBC patients. Thus, PDK1 functions as a tumor promoter in human GBC by upregulating JunB.     

Leukemia inhibitory factor protects cholangiocarcinoma cells from drug-induced apoptosis via a PI3K/AKT-dependent Mcl-1 activation

Cholangiocarcinoma is an aggressive, strongly chemoresistant liver malignancy. Leukemia inhibitory factor (LIF), an IL-6 family cytokine, promotes progression of various carcinomas. To investigate the role of LIF in cholangiocarcinoma, we evaluated the expression of LIF and its receptor (LIFR) in human samples. LIF secretion and LIFR expression were assessed in established and primary human cholangiocarcinoma cell lines. In cholangiocarcinoma cells, we tested LIF effects on proliferation, invasion, stem cell-like phenotype, chemotherapy-induced apoptosis (gemcitabine+cisplatin), expression levels of pro-apoptotic (Bax) and anti-apoptotic (Mcl-1) proteins, with/without PI3K inhibition, and of pSTAT3, pERK1/2, pAKT. LIF effect on chemotherapy-induced apoptosis was evaluated after LIFR silencing and Mcl-1 inactivation.Results show that LIF and LIFR expression were higher in neoplastic than in control cholangiocytes; LIF was also expressed by tumor stromal cells. LIF had no effects on cholangiocarcinoma cell proliferation, invasion, and stemness signatures, whilst it counteracted drug-induced apoptosis. Upon LIF stimulation, decreased apoptosis was associated with Mcl-1 and pAKT up-regulation and abolished by PI3K inhibition. LIFR silencing and Mcl-1 blockade restored drug-induced apoptosis.In conclusion, autocrine and paracrine LIF signaling promote chemoresistance in cholangiocarcinoma by up-regulating Mcl-1 via a novel STAT3- and MAPK-independent, PI3K/AKT-dependent pathway. Targeting LIF signaling may increase CCA responsiveness to chemotherapy.     

Statins induce apoptosis in ovarian cancer cells through activation of JNK and enhancement of Bim expression

Purpose  Ovarian cancer is the leading cause of death among all gynecological malignancies in Western countries. Although therapy for ovarian cancer has been greatly improved in the past 20 years, the overall survival for patients with advanced ovarian cancer has not changed significantly. The poor survival rates in patients with advanced ovarian cancer are due both to late diagnosis and to lack of effective drugs for the majority of patients who have a relapse and develop resistance to current chemotherapy agents used for ovarian cancer. Thus, developing and discovering effective novel drugs with different molecular structures from conventional chemotherapy agents have become an urgent clinical need. Methods  Ovarian cancer cells were treated with lovastatin and atorvastatin. Apoptosis in these cells and tumor formation in soft agar were determined. The molecular mechanism by which statins suppress ovarian cancer cell growth was evaluated. Results  Both lovastatin and atorvastatin effectively induced apoptosis in ovarian cancer cells and suppressed anchorage-independent growth of these cells in soft agar. Further investigation of the molecular mechanism has revealed that the expression of Cdc42 and Rac1, small GTPase family members, was highly induced in the cells by these statins along with the activation of Jun N-terminal kinases (JNK). In addition, Bim, a proapoptotic protein, was significantly induced by these statins. Conclusions  Our findings provide new insight into the molecular mechanism by which statins induce apoptosis in ovarian cancer cells and may lead to novel therapies for advanced ovarian cancer.     

EFFECTS OF IFN-a COMBINED WITH IL-6 ON GROWTH AND EXPRESSION OF THE GENES RELATED TO CELL-GROWTH AND APOPTOSIS OF BONE MARROW CELLS FROM PATIENTS WITH CML

CML is a clonal myeloproliferative disorder. The Philadelphia chromosome (ph) is the cytogenetic hall-mark of this disease.[1] The ph chromosome translocation [t(9; 22) (q34; qll)] results in the creation of a hybrid bcr-abl fusion gene and forms an abnormal 210-kD protein (p210bcr-abl) with increased tyrosine kinase activity.[2,3] These molecular changes were thought to play a significant role in leukemogenesis. p210bcr-abl confers a growth advantage on ph-positive CML cells over normal he…     

Simvastatin inhibition of mevalonate pathway induces apoptosis in human breast cancer cells via activation of JNK/CHOP/DR5 signaling pathway

Simvastatin (SVA) was shown to up-regulate expression of death receptor-5 (DR5), CCAAT/enhancer binding protein homologous protein (CHOP) and phosphorylated c-Jun N-terminal kinase (pJNK) in human breast cancer cell lines. siRNA knockdown of DR5, CHOP or JNK significantly blocked SVA-induced apoptosis, demonstrating the importance of JNK/CHOP/DR5 signaling pathway in SVA-induced apoptosis. Exogenous addition of either mevalonate or geranylgeranyl pyrophosphate (GGPP) inhibited SVA activation of JNK/CHOP/DR5 pro-apoptotic pathway, indicating that activation of JNK/CHOP/DR5 pro-apoptotic pathway is dependent on SVA inhibition of 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase and its intermediate GGPP. Data provide novel insight into better understanding the anticancer mechanisms of SVA.     

The activation of ERK1/2 via a tyrosine kinase pathway attenuates trail-induced apoptosis in HeLa cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) serves as an extracellular signal that triggers apoptosis in tumor cells. To characterize the molecular events involved in TRAIL-induced apoptotic signaling, we investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) in HeLa cell death. Here we show that TRAIL-activated ERK1/2 through a tyrosine kinase-dependent pathway, subsequently elevated anti-apoptotic Bcl-2 protein levels. ERK1/2 inhibition with PD98059 promoted apoptotic cell death through the downregulation of ERK1/2 activity and Bcl-2 protein levels. Moreover, tyrosine kinase inhibition with Genistein in TRAIL-induced apoptosis effectively attenuated ERK1/2 activity and enhanced apoptotic cell death. Taken together, our results indicate that ERK1/2 activation via tyrosine kinase pathway plays a protective role as the cellular defense mechanism through the upregulation of Bcl-2 protein levels in TRAIL-induced apoptosis.     

Inhibition of apoptosis by bcr-abl fusion gene in K562 cells

Chronicmyel0idleukemia(CML)isahematologicalmalignancycharacterizedbyaninitialchronicphaseofexpandedclonalhematop0iesiswithcontinueddifferentiationintomaturemyeloidcells.Itscyt0genetichallmarkisthephiladelphiachromosome(Ph),ort(9;22)(q34.l;qll.2);thisbalancedtranslocati0nresultedinthecreationofachimericbcr-ablgene,whichisthemolecularbiologyfeatureofCML.Thebcr-ablhasbeenprovedtocausehumanCML-likesyndromeinmice.Toelucidatetheeffectofachimericbcr-ablgeneinCML,weobservedtheapopt0sisofK562cell…     

Arsenic trioxide induces apoptosis of glucocorticoid-resistant acute lymphoblastic leukemia CEM-C1 cells

Objective  To explore the effects of arsenic trioxide (ATO) on the apoptosis of glucocorticoid (GC)-resistant T-acute lymphoblastic leukemia (ALL) CEM-C1 cells and its possible mechanisms. Methods  Different concentrations of ATO (0.25 μmol/L-5 μmol/L) were used to induce the apoptosis of CEM-C1 cells. The inhibition rate of cell proliferation and apoptosis were detected by MTT test, Annexin V-FITC/PI flow cytometry and optical microscopy, respectively. RT-PCR was applied to semi-quantitatively analyze the mRNA expression of pro-apoptotic proteins (Bad and PDCD4) and anti-apoptotic proteins (XIAP and MCL-1) induced by different concentrations of ATO at different time points. Results  ATO could inhibit proliferation and induce apoptosis of CEM-C1 cells at a concentration and time dependent manner. Low-dose ATO mildly inhibited the proliferation of CEM-C1 cells while higher concentrations (1 μmol/L and 5 μmol/L) had strong anti-tumor effect with the inhibiting rates of 40.07±7.98% and 88.67±2.88%, respectively. Annexin V-FITC/PI flow cytometry showed that the apoptotic rates of CEM-C1 cells were significantly increased after 48 hours treatment of different concentrations of ATO. RT-PCR demonstrated up-regulated mRNA expression of pro-apoptotic protein Bad and PDCD4 but down-regulated mRNA expression of anti-apoptotic protein XIAP when CEM-C1 cells were treated with different concentrations of ATO at different time points. The MCL-1 mRNA expression was down-regulated only after the treatment of 5 μmol/L ATO. Conclusion  ATO can inhibit cell proliferation and induce cell apoptosis in GC-resistant CEM-C1 cells. The molecular mechanisms might involve the increased mRNA expression of pro-apoptotic protein Bad and PDCD-4, and rapid down-regulation of XIAP mRNA expression. This work was supported by a grant from the Science and Technology Committee of Sichuan Province (No. 2008JY0029-1).     

Molecular analysis of the BCR-ABL1 kinase domain in chronic-phase chronic myelogenous leukemia treated with tyrosine kinase inhibitors in practice: Study by the Nagasaki CML Study Group

An appropriate trigger for BCR-ABL1 mutation analysis has not yet been established in unselected cohorts of chronic-phase chronic myelogenous leukemia patients. We examined 92 patients after 12 months of tyrosine kinase inhibitor (TKI) treatment in Nagasaki Prefecture, Japan. Univariate analysis revealed that significant factors associated with not attaining a major molecular response (MMR) were the presence of the minor BCR-ABL1 fusion gene, a low daily dose of TKI, and the emergence of BCR-ABL1 kinase domain mutations conferring resistance to imatinib. Factors associated with the loss of sustained MMR were a low daily dose of TKI and the emergence of alternatively spliced BCR-ABL1 mRNA with a 35-nucleotide insertion. Taken together, our results suggest that the search for BCR-ABL1 mutations should be initiated if patients have not achieved MMR following 12 months of TKI treatment.     

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10 mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10 mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing apoptosis.