整合素α5β1介导的PI3K-AKT信号分子在CML发病机制中的作用

摘 要　目的：研究整合素α5β1介导的PI3K-AKT信号转导通路在慢性髓细胞白血病（chronic myeloid leukemia,CML）发病机制中的作用。方法：流式细胞术检测不同剂量抗整合素α5β1单抗（Anti-α5β1）作用后白血病K562细胞的凋亡率, Western blotting检测健康志愿者、CML急变期患者、K562细胞和Anti-α5β1处理的K562细胞中PI3K和AKT蛋白的表达水平。结果：Anti-α5β1可诱导K562细胞凋亡,并呈剂量依赖性。与健康志愿者比较,CML急变期患者骨髓单个核细胞和K562细胞中PI3K和AKT蛋白的表达水平明显增高（P<0.05）;Anti-α5β1处理后,K562细胞内PI3K、AKT蛋白表达水平明显降低,其磷酸化水平也下降（P<0.05）。结论: 整合素α5β1可能通过影响PI3K-AKT信号转导通路中关键分子PI3K和AKT的表达水平诱导CML祖细胞凋亡耐受,参与了CML的发生、发展过程。     

野生型p53对白血病K562细胞中心体过度复制的抑制作用

背景与目的：肿瘤组织细胞中p53基因突变或缺失是导致非整倍体的发生和基因组不稳定的主要原因之一：最近研究发现慢性粒细胞白血病（chronic myelogenous leukemia．CML）各期患者均有中心体异常,且异常的程度与临床分期有关。急变期显示更为严重的中心体异常。本研究建立携野生型p53基因的CML急变K562细胞株．以研究该细胞内野生型p53基因表达后p53信号转导通路对K562细胞中心体的影响。方法：用HEK293细胞扩增重组p53野生型、突变型及空载腺病毒载体,联合polybrene分别感染K562细胞;未接受感染的细胞作为空白对照。流式细胞术检测重组腺病毒载体感染效率,Western blot检测P53蛋白表达。间接免疫荧光染色后用激光共聚焦计数K562细胞中心体的变化。Westernblot检测p53信号转导通路下游效应分子生长阻滞和DNA损伤应答基因Gadd45a（growtharrest and DNA damage）、BubRl（Bublrelated）、AuroraA的表达。结果：成功建立携野生型p53基因的K562细胞株．腺病毒载体感染效率达60％以上,野生型p53可在K562细胞中持续表达。感染72h后,携野生型p53基因的K562细胞中中心体数量异常（n〉2）的细胞比例降至（0．38＋0．02）％,与空白对照组（0．71＋0．14）％比较其差异有统计学意义（P〈0．05）;Westernblot结果发现p53信号转导通路下游效应分子Gadd45a、BubRl表达分别上调93％、88％．而AuroraA的表达下降56％（P值均〈0．05）。结论：重组腺病毒介导的野生型p53基因能够在白血病K562细胞中持续表达：野生型P53蛋白可能通过转录激活-依赖途径上调Gadd45a、BubR1表达以及转录激活-非依赖途径使AuroraA的表达下降,从而抑制K562细胞中心体的过度复制。     

ERK参与裸鼠体内辛伐他汀诱导K 562细胞凋亡的调控作用

目的：探讨辛伐他汀（simvastatin）作用于裸鼠体内K562细胞后Ras-MAPK信号转导通路胞外信号调节激酶（extracellular signal—regulated protein kinase，ERK）分子水平的变化，以说明ERK参与裸鼠体内辛伐他汀诱导K562细胞凋亡的调控作用。方法：体外培养慢性髓细胞白血病（CML）细胞株K562细胞，构建BALB／c-nu／nu裸鼠的K562细胞移植瘤模型。流式细胞术（FCM）检测对照组和两个辛伐他汀处理组的K562细胞周期变化，TUNEL法检测K562细胞晚期凋亡情况。采用RT-PCR检测K562细胞中Ras-MAPK信号通路N-Ras、ERK1mRNA的差异表达。免疫组织化学标记葡聚糖聚合物（labbled dextran polymer，LDP）法检测P-ERK蛋白水平变化。结果：辛伐他汀能够明显抑制裸鼠K562移植瘤组织的增长，随着辛伐他汀剂量的增加，K562细胞移植瘤体积和质量明显减小（P〈0．05，P〈0．01）。每次注射0．05mg的辛伐他汀能够诱导裸鼠体内K562细胞发生明显的G0／G1期停滞，不同剂量的辛伐他汀能够诱导K562细胞发生明显的凋亡，并随剂量的增加，凋亡率逐渐增高（P〈0．01）；不同剂量的辛伐他汀能够引起N-Ras、ERK1mRNA的表达下调（P〈0．01）。与对照组相比，两个处理组的p-ERK蛋白分别出现表达下调（P=0．01，P〈0．01）。结论：辛伐他汀在体内可能依赖Ras—MAPK信号转导通路ERK基因和蛋白水平的表达下调诱导K562细胞凋亡发生。     

蛋白激酶C对Pgp介导的K562细胞耐药性的影响

目的：研究蛋白激酶C（PKC）在P－糖蛋白（Pgp)介导的人类白血病K562细胞多药耐药中的作用,方法：用苔盼蓝拒染法检测药物敏感性,用免疫组织化学法检测Pgp的表达,用流式细胞仪检测细胞内柔红霉素（DNR）积聚,用Takai法及Western blot法分别检测PKC活性和PKCα含量。结果：耐药株K562／D细胞对DNA及多种抗 药物显示多药耐药性,并存在Pgp过度表达,K562／D与敏感株K562／S细胞相比,PKC总活性无明显差异,但PKCα含量显著增高,PKC激活剂TPA增加K562／S和K562／D细胞的PKC活性,使PKC和PKCα由胞质向胞膜转位,K562／S和K562／D细胞的PKC活性及PKCα含量,增加K562／D细胞内DNA积聚。结论：PKCα在D562／D细胞中显著增高,上调PKC,使K562。D细胞内DNA积聚减小,下调PKC,使K562／D细胞内的DNA积聚增加,提示PKC可能通过调节Pgp的功能参与Pgp介导的多药耐药。     

木黄酮作用于K562细胞机制的初步研究

目的：探讨酪氨酸激酶抑制剂木黄酮（Genistein)作用于慢性粒细胞性白血病（CML）的机制。方法：通过细胞增殖、活力检测、形态学观察、半固体集落培养及DNA凝胶电泳和流式细胞术,观察木黄酮对K562细胞生长的影响。结果：（1）经≥5mg/L木黄酮处理2d的K562细胞,增殖抑制率达到50％以上,且与作用剂量和时间呈正相关;形态渐趋向凋亡但体积涨大;(2)K562细胞集落抑制达50％时的木黄酮浓度为10mg/L,也与时间和剂量呈正相关;（3）K562细胞经10mg/L木黄酮处理4d,DNA凝胶电泳可见清晰的梯状条带;（4）K562细胞分别经2．5mg/L、5mg/L和10mg/L木黄酮处理1d和2d后,流式细胞术检测细胞凋亡率分别为0．62％、1．64％、2．71％和0．68％、4．09％、8．4％;2d后G2期细胞分别占总细胞数的17％、34．5％和79．5％,而G1期和S期细胞逐渐明显减少。结论：木黄酮对K562细胞具有显的抑制增殖作用,其机制与诱导凋亡有关。     

姜黄素诱导人慢粒白血病K562细胞凋亡与线粒体途径的关系

目的研究姜黄素（Cur）对慢性粒细胞白血病（CML）细胞株K562的作用，并且探讨该作用与线粒体凋亡途径的关系。方法应用MTT法检测Cur对细胞增殖的影响，AO／EB荧光染色法、细胞光度术、DNA凝胶电泳等方法观察细胞凋亡，分光光度法检测Caspase-3的活性，用蛋白免疫印迹法检测细胞色素C的含量。结果Cur对K562细胞抑制作用呈量效、时效关系；Cur对人正常骨髓单个核细胞与K562细胞之间有一定的选择性，对K562细胞的敏感性约为人正常骨髓单个核细胞的5倍，在对K562细胞抑制超过95％时，对人正常骨髓有核细胞几无毒性。Cur5．0，10．00g／mL作用24h，可诱导K562细胞凋亡，促进细胞色素C释放，激活caspase-3。结论Cur可通过线粒体途径诱导人慢性粒细胞白血病K562细胞凋亡。     

K562细胞硫氧还蛋白还原酶活力测定及其抑制剂体外抗白血病的初步研究

 目的　探讨慢性粒细胞白血病（CML）细胞株K562中硫氧还蛋白还原酶（TrxR）的活力及其新型抑制剂乙烷硒啉（BBSKE）体外抗白血病作用。方法　应用胰岛素还原法检测K562细胞株及健康人骨髓单个核细胞中TrxR的活力。运用CCK-8法测定BBSKE对K562细胞的增殖抑制率。应用激光共聚焦显微镜、琼脂糖凝胶电泳以及Annexin Ⅴ-FITC／PI双标记流式细胞术观察BBSKE的抗白血病作用。结果　K562细胞中TrxR的活性明显高于健康人骨髓单个核细胞,10 μmol／L BBSKE与K562细胞作用24 h,激光共聚焦显微镜可见典型的细胞凋亡表现,琼脂糖凝胶电泳后可见典型的DNA“梯”条带出现,流式细胞术检测凋亡率为（10.28±2.74）%;10 μmol／L BBSKE对CML患者原代细胞有诱导凋亡的作用,凋亡率为（5.70±0.48）%。结论　慢性粒细胞白血病细胞株K562中TrxR活力高于健康人骨髓单个核细胞,BBSKE有抑制TrxR活力、抑制K562细胞增殖和诱导凋亡的作用,是治疗CML潜在的有效药物。     

CRKL基因反义寡核苷酸诱导K562细胞的凋亡

背景与目的：近几年研究发现,CRKL（CT10regulator of kinase like)基因编码的蛋白在慢性粒细胞白血病（chronic myeloid leukemia,CML)发病中起重要作用。本实验研究CRKL基因反义寡核苷酸对慢性粒细胞白血病细胞系K562细胞凋亡的影响。方法：以脂质体为载体,用CRKL基因反义寡核苷酸转染K562细胞,通过活细胞计数,透射电镜,流式细胞术,DNALadder测定观察K562细胞形态学,细胞周期,基因表达等的变化。结果：在CRKL基因反义寡核苷酸作用下,K562细胞的生长明显出现抑制;流式细胞仪检测见二倍体峰前出现凋亡峰;在电镜下可见处于凋亡各期的K562细胞;提取其基因组DNA进行琼脂糖凝胶电泳,可见明显的细胞凋亡梯状条带,而对照组K562细胞未观察到上述变化。结论：CRKL基因是Ph染色体阳性CML发病的重要因素。     

丝裂原活化蛋白激酶信号转导系统对Vp-16诱导分化作用的影响

目的：探讨丝裂原活化蛋白激酶（mitogen-activated protein kinases，MAPKs）信号转导系统对依托泊苷（Vp-16）诱导K562细胞分化作用的影响。方法：采用四甲基偶氮唑盐（MTT）法测定细胞增殖活性；流式细胞仪解析细胞周期；硝基四氮唑蓝（NBT）还原实验检测细胞向单核／巨噬系统分化。结果：0．1～0．8μg／mL的Vp-16抑制K562细胞增殖，引起细胞G2/M期阻滞，诱导细胞向单核／巨噬系统分化；细胞外信号调节激酶（extracellular signal-regulated kinases，ERK）抑制剂PD98059降低Vp-16的诱导分化作用。P＜0．05；p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinases，p38 MAPK）抑制剂SB203580增强Vp-16的作用，P＜0．05；而C-JUN氨基末端激酶（c-jun N-terminal kinascs，JNK）抑制剂SP600125对Vp-16的诱导分化作用无明显影响，P〉0．05。结论：在Vp-16诱导K562细胞向单核／巨噬系统分化过程中，ERK正向，p38MAPK负向调节Vp-16的诱导分化作用。     

反义寡聚脱氧核苷酸抗K562细胞的实验研究

针对慢性粒细胞白血病(CML)特征基因bcr/abl的嵌合位点和CML相关癌基因c-myb的第2外显子分别设计、合成的反义寡聚脱氧核苷酸asODN及其硫代磷酸化修饰物s-asODN分别或联合作用于慢性粒细胞白血病细胞株K562.结果表明各种asODN可显著抑制K562细胞的存活,最大抑制率为64.7%±3.2%,可显著抑制K562细胞的DNA合成,最大抑制率为85.8%±4.1%,可显著降低甚至几乎完全抑制bcr/abl的转录,可显著诱导K562发生凋亡.s-asODN的作用效果显著优于未加修饰的对应asODN.联合使用bcr/abl与c-myb的ASO比单独使用其中之一效果更佳.各种作用效果均表现出片断、时间相关性.这些说明,bcr/abl在CML发病机制中起双重作用,不仅刺激了CML细胞的过度增殖,而且抑制了细胞的凋亡.c-myb可能主要通过对bcr/abl的调控而与CML相关.asODN修饰物及多个癌基因的asODN联合使用可能是反义基因治疗的发展方向.     

Aminopeptidase inhibitors inhibit proliferation and induce apoptosis of K562 and STI571-resistant K562 cell lines through the MAPK and GSK-3beta pathways

A tyrosine kinase inhibitor, STI571, has been demonstrated to be effective for the treatment of chronic myelogenous leukemia (CML). STI571 inhibits tyrosine kinase activity of ABL and induces apoptosis of CML cells. However, drug resistance develops commonly in patients with blast phase CML, and has become a significant therapeutic problem. We examined the effects of aminopeptidase inhibitors on CML cell line (K562) and a STI571-resistant subline of K562. Ubenimex and the more potent aminopeptidase inhibitor, actinonin, inhibited proliferation of both K562 cells and STI571-resistant K562 cells and also induced their apoptosis in dose- and time-dependent manners. Ubenimex and actinonin induced the activation of caspase-3, and the induction of apoptosis was inhibited by pan-caspase inhibitor, indicating this apoptosis is caspase-dependent. We found that serine phosphorylation of both MAPK and glycogen synthase kinase-3β were suppressed by aminopeptidase inhibitors in parent K562 and STI571-resistant K562 cells. The expression level of cyclin D1 protein was also reduced by ubenimex and actinonin in both cell lines. These results indicated STI571-resistance does not confer the cross-resistance to aminopeptidase inhibitors in K562 cells and revealed the new findings of aminopeptidase inhibitor-induced intracellular signaling pathways.     

Rapamycin provides a therapeutic option through inhibition of mTOR signaling in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a neoplasm of myeloid progenitor cells expressing Bcr-Abl fusion protein. However, some patients with CML are less likely to respond to imatinib, the inhibitor of Bcr-Abl kinase. Recent studies showed that mTOR pathway can increase responses to imatinib. The analysis of mTOR pathway in CML may provide new insights into possible targets of novel therapies. Therefore, we examined the expression of mTOR pathway molecules in bone marrow cells from CML patients and effect of rapamycin on K562 cells in?vitro. Western blot analysis showed the visibly higher phosphorylation of mTOR (70.6%), 4E-BP1 (76.5%) and p70S6K (73.5%) in bone marrow cells from CML patients. Moreover, treatment of CML cell line (K562) with rapamycin resulted in a decrease of phosphorylation of mTOR, 4E-BP1 and p70S6K. In?vitro, the cell viability in groups with rapamycin treatment displayed a significant decrease in a dose-dependent manner by MTT. The data presented an increase of G0/G1 phase cells and decrease of S phase cells after rapamycin treatment, and the decreased expression of cyclinD1, higher expression of p21 at mRNA level was also detected in K562 with rapamycin. Treatment with 20 nmol/l or more rapamycin could increase apoptotic cells, decrease expression of bcl-2 and activate caspase-3. In conclusion, the mTOR pathway might be involved in chronic myelogenous leukemia. Inhibition of mTOR pathway could interfer with cell proliferation and increase cell apoptosis in K562 cells. It suggested that mTOR might be an important therapeutic target for myelogenous leukemia.     

Phosphotyrosine phosphatase activity prevents the detection of P210bcr/abl protein in mature cells in chronic myelogenous leukemia even by an immunoblotting technique

P210bcr/abl protein with tyrosine protein kinase has been implicated in the proliferation and differentiation of chronic myelogenous leukemia cells. Using an immunoblotting technique with antiphosphotyrosine (anti-P-Tyr) antibodies, we examined whether P210bcr/abl protein was expressed in chronic phase cells in patients with chronic myelogenous leukemia (CML). We could detect P210bcr/abl protein in blast cells regardless of myeloid or lymphoid lineage but not in chronic phase cells from patients. However, in a patient with both blast cells and chronic phase cells, we could identify the protein only after the enrichment of the blast crisis cells by Percoll gradient centrifugation. When K562 cells were mixed with mature granuloid cells, the P210bcr/abl in K562 cells detected by immunoblotting was decreased. Using phosphotyrosyl proteins in K562 cells as substrates, high phosphotyrosyl (P-Tyr) phosphatase activity was observed, not only in the lysate of chronic phase cells from CML patients but also in the lysate of neutrophils from normal subjects. These findings suggest the possibility that high P-Tyr phosphatase activity prevents the detection of P210bcr/abl in CML cells in the chronic phase. The activity may be characteristic of mature cells and may regulate cellular events through dephosphorylation of P210bcr/abl.     

In vitro effect of adenovirus-mediated human Gamma Interferon gene transfer into human mesenchymal stem cells for chronic myelogenous leukemia

For developing gene therapy for chronic myelogenous leukemia (CML), we evaluated the feasibility of using autologous bone marrow stromal cells (BMSCs) of one CML patient as a target cell population and studied the efficiency of recombinant adenovirus-mediated human Gamma Interferon (hIFN-gamma) gene transfer into BMSCs. BMSCs can be readily obtained, expanded, and successfully transduced with adenoviral vectors in vitro. We studied the in vitro expression of hIFN-gamma in human BMSCs following transduction with Ad/hIFN-gamma. On transduction of BMSCs at a MOI of 50, the expression and secretion of hIFN-gamma were achieved as high as 5492 +/- 660 approximately 50647 +/- 4049 ng/10(6) cells per 24 h over the course of 3 weeks. We further studied the effects of hIFN-gamma produced by transduced BMSCs on the proliferation of the human leukemia cell line K562 cells in vitro, proliferation of K562 cells was markedly inhibited in the experimental groups as compared with the other two control groups after 5 days of coculture. We also found that the percentage of K562 cells in the G(1) phase of cell cycle can be increased by treatment of hIFN-gamma produced by Ad/hIFN-gamma transduced BMSCs, but the percentage of K562 cells in the S phase of cell cycle can be decreased in the same time. Apoptosis rate of K562 cells in the experimental groups was 30.8 +/- 8.5%, as compared with the other two control groups (5.6 +/- 1.3% and 5.5 +/- 0.8%, respectively) (p < 0.01). Our results indicate that hIFN-gamma gene engineered BMSCs of CML donors could be successfully established and that local production of hIFN-gamma is sufficiently to inhibit the proliferation of K562 cells and induce apoptosis of K562 cells in vitro, suggesting an important potential use in the clinical gene therapy of CML.     

Notch signaling inhibits the growth of the human chronic myeloid leukemia cell line K562

Notch signaling functions in the development of some types of leukemia and lymphoma, but the relationship between Notch signaling and chronic myeloid leukemia (CML) remains to be elucidated. In this study, we examined the expression of Notch receptors and ligands in the human CML cell line K562. When the active form of Notch1, the Notch intra-cellular domain (NIC), was over-expressed in K562, the proliferation of K562 was mildly but significantly inhibited, accompanied by increased Hes1 mRNA level. On the other hand, when Notch signaling was attenuated by over-expression of a dominant-negative RBP-J, RBP-J(R218H), in K562 cells, the proliferation of K562 was increased. Moreover, we found that activation of Notch signaling inhibited while repression of Notch signaling promoted the colony-forming activity of K562 cells. We examined cell cycle-related molecules in K562 transfected with NIC or RBP-J(R218H), and found that the protein level of the retinoblastoma gene product (the Rb protein) was induced in K562 expressing NIC, and down-regulated in K562 expressing RBP-J(R218H). These data suggest that the Notch signaling may function as a tumor inhibitor in human CML cells.     

Cell adhesion-mediated drug resistance (CAM-DR) protects the K562 chronic myelogenous leukemia cell line from apoptosis induced by BCR/ABL inhibition, cytotoxic drugs, and gamma-irradiation.

Integrin-mediated cellular adhesion to extracellular matrix (ECM) components is an important determinant of chemotherapeutic response of human myeloma cells. Here, we demonstrate that when K562 chronic myelogenous leukemia (CML) cells are adhered to fibronectin (FN), they become resistant to apoptosis induced by the BCR/ABL inhibitors AG957 and STI-571, as well as DNA damaging agents and gamma-irradiation. This phenomenon, termed cell adhesion-mediated drug resistance (CAM-DR), was induced by adhesion through the alpha5beta1 (VLA-5) integrin. Phosphotyrosine analysis demonstrates that anti-apoptotic signaling through integrins in K562 cells is independent of the tyrosine kinases activated by BCR/ABL, with the possible exception of an unknown 80 kDa protein. Cytoprotection of FN-adhered CML cells indicates that tumor-ECM interactions may be critical for the emergence of drug-resistant tumor populations and treatment failure in this disease. Antagonists of beta1 integrin-mediated adhesion or corresponding signal transduction elements may sensitize CML cells to chemotherapy and prevent resistance to the novel BCR/ABL kinase inhibitors being used for the treatment of this disease.     

A small molecule significantly inhibits the bcr/abl fusion gene at the mRNA level in human chronic myelogenous leukemia

Bcr/abl fusion gene is the marker gene in chronic myelogenous leukemia (CML) and becomes the target for CML therapy. Although Imatinib opened a new way to treat CML, the resistance to the drug caused by bcr/abl fusion protein mutation stimulated search for new molecules to inhibit bcr/abl expression. In our research, it was found that a novel 2-aminosteroid (H89465) possessed special mechanism in treating CML. H89465 inhibits the proliferation of both non-resistant and resistant CML cells such as K562, Meg-01 and clinical primary CML cells. It prolongs the survival time of NOD/SCID mice inoculated with K562 leukemia cells. The mechanism underlying the effects is concerned with down-regulation of bcr/abl mRNA expression followed by decreasing the BCR/ABL protein expression and tyrosine kinase activity in CML cells. Our results demonstrate that H89465 possesses the therapeutic potential in treating human CML.     

小分子化合物S1抑制K562细胞增殖及其机理研究

目的:探讨小分子化合物S1对K562细胞的抑制作用及机理,为S1治疗慢性粒细胞白血病（chronic myeloid leukemia,CML）提供理论与实验基础。方法:MTT法检测不同浓度S1对K562细胞增殖的抑制作用;应用Chou-Talalay计算联合指数,观察单独应用S1与S1联合阿糖胞苷对K562细胞增殖抑制的协同作用;Western-blotting方法检测不同浓度S1作用后K562细胞系中Bcl-2蛋白表达水平的变化。结果:与对照组相比,S1可以抑制K562细胞的增殖（P<0.05）;S1与阿糖胞苷联合应用对K562细胞的生长抑制具有协同作用,联合指数CI<1;S1可下调K562细胞系中抗凋亡蛋白Bcl-2的表达。结论:小分子化合物可通过下调抗凋亡蛋白Bcl-2的表达抑制K652细胞的增殖,并与阿糖胞苷具有协同作用。