Comparative proteome profiling and functional analysis of chronic myelogenous leukemia cell lines

The aim of the present study was the molecular profiling of different Ph+ chronic myelogenous leukemia (CML) cell lines (LAMA84, K562, and KCL22) by a proteomic approach. By employing two-dimensional gel electrophoresis combined with mass spectrometry analysis, we have identified 191 protein spots corresponding to 142 different proteins. Among these, 63% were cancer-related proteins and 74% were described for the first time in leukemia cells. Multivariate analysis highlighted significant differences in the global proteomic profile of the three CML cell lines. In particular, the detailed analysis of 35 differentially expressed proteins revealed that LAMA84 cells preferentially expressed proteins associated with an invasive behavior, while K562 and KCL22 cells preferentially expressed proteins involved in drug resistance. These data demonstrate that these CML cell lines, although representing the same pathological phenotype, show characteristics in their protein expression profile that suggest different phenotypic leukemia subclasses. These data contribute a new potential characterization of the CML phenotype and may help to understand interpatient variability in the progression of disease and in the efficacy of a treatment.     

Cepharanthine activates caspases and induces apoptosis in Jurkat and K562 human leukemia cell lines.

Cepharanthine (CEP) is a known membrane stabilizer that has been widely used in Japan for the treatment of several disorders such as anticancer therapy-provoked leukopenia. We here report that apoptosis was induced by low concentrations (1-5 microM) of CEP in a human leukemia T cell line, Jurkat, and by slightly higher concentrations (5-10 microM) in a human chronic myelogenous leukemia (CML) cell line K562, which expresses a p210 antiapoptotic Bcr-Abl fusion protein. Induction of apoptosis was confirmed in both Jurkat and K562 cells by DNA fragmentation and typical apoptotic nuclear change, which were preceded by disruption of mitochondrial membrane potential and were induced through a Fas-independent pathway. CEP treatment induced activation of caspase-9 and -3 accompanied by cleavage of PARP, Bid, lamin B1, and DFF45/ICAD in both Jurkat and K562 cells, whereas caspase-8 activation and Akt cleavage were observed only in Jurkat cells. The CEP-induced apoptosis was completely blocked by zVAD-fmk, a broad caspase inhibitor. Interestingly, CEP treatment induced remarkable degradation of the Bcr-Abl protein in K562 cells, and this degradation was prevented partially by zVAD-fmk. When used in combination with a nontoxic concentration of herbimycin A, lower concentrations (2-5 microM) of CEP induced obvious apoptosis in K562 cells with rapid degradation or decrease in the amount of Bcr-Abl and Akt proteins. Our results suggest that CEP, which does not have bone marrow toxicity, may possess therapeutic potential against human leukemias, including CML, which is resistant to anticancer drugs and radiotherapy.     

The growth of K562 human leukemia cells was inhibited by therapeutic neural stem cells in cellular and xenograft mouse models

To confirm the anti-tumor effect of engineered neural stem cells (NSCs) expressing cytosine deaminase (CD) and interferon-β (IFN-β) with prodrug 5-fluorocytosine (FC), K562 chronic myeloid leukemia (CML) cells were co-cultured with the neural stem cell lines HB1.F3.CD and HB1.F3.CD.IFN-β in 5-FC containing media. A significant decrease in the viability of K562 cells was observed by the treatment of the NSC lines, HB1.F3.CD and HB1.F3.CD.IFN-β, compared with the control. A modified trans-well assay showed that engineered human NSCs significantly migrated toward K562 CML cells more than human normal lung cells. In addition, the important chemoattractant factors involved in the specific migration ability of stem cells were found to be expressed in K562 CML cells. In a xenograft mouse model, NSC treatments via subcutaneous and intravenous injections resulted in significant inhibitions of tumor mass growth and extended survival dates of the mice. Taken together, these results suggest that gene therapy using genetically engineered stem cells expressing CD and IFN-β may be effective for treating CML in these mouse models.     

Proteomic analysis of an imatinib-resistant K562 cell line highlights opposing roles of heat shock cognate 70 and heat shock 70 proteins in resistance

Understanding the molecular basis of resistance to imatinib, a tyrosine kinase inhibitor used as front-line therapy in chronic myeloid leukemia, remains a challenge for successful treatment. In an attempt to identify new mechanisms of resistance, we performed a comparative proteomic analysis of an imatinib-resistant cell line generated from the erythroblastic cell line K562 (K562-r) for which no known mechanism of resistance has been detected. Bidimensional gel electrophoresis was carried out to compare the protein expression pattern of imatinib-sensitive and of imatinib-resistant K562 cells. Among the 400 matched spots on five pairs of gels, only 14 spots had a significantly increased or decreased expression leading to the identification of 24 proteins identified as scaffold proteins, metabolic enzymes, DNA translation and maturation, and chaperon proteins. Among the chaperon family, only Hsp70 and Hsc70 are overexpressed in K562-r, results confirmed by Western blotting. We recently reported the participation of Hsp70 overexpression in imatinib resistance whereas a role for Hsc70 has yet to be determined. Hsc70 is not involved in imatinib resistance as the inhibition of its expression by siRNA does not restore sensitivity to imatinib. In contrast, the induced decreased expression of Hsc70 was accompanied by a greater overexpression of Hsp70. This proteomic study therefore suggests opposing roles of Hsp70 and Hsc70 in imatinib resistance.     

A proteomic investigation into adriamycin chemo-resistance of human leukemia K562 cells

This study aimed to explore the mechanism of adriamycin resistance in human chronic myelogenous leukemia cells. Proteomic approach was utilized to compare and identify differentially expressed proteins between human chronic myelogenous leukemia K562 cells and their adriamycin-resistant counterparts. The differentially expressed proteins were analyzed by 2-DE (two-dimensional gel electrophoresis), and protein identification were performed on ESI-Q-TOF MS/MS instrument. Out of the 35 differentially expressed proteins between the two cell lines, 29 were identified and grouped into 10 functional classes. Most of identified proteins were related to the categories of metabolism (24%), proteolysis (13%), signal transduction (21%) and calcium ion binding (6%), suggesting that alterations of those biological processes might be involved in adriamycin resistance of K562 cells. We believe this study may provide some clues to a better understanding of the molecular mechanisms underlying adriamycin resistance.     

Disialoganglioside GD3 synthase expression recruits membrane transglutaminase 2 during erythroid differentiation of the human chronic myelogenous leukemia K562 cells

By employing proteomics analysis tool, we examined the effects of GD3 synthase expression on the differentiation properties of chronic myelogenous leukemia (CML)-derived leukemia cells K562. Forced expression of GD3 synthase induced erythroid differentiation as determined by an increase in glycophorin A expression and synthesis of hemoglobins. The proteomic analysis revealed that 15 proteins were increased by GD3 synthase. In contrast, we observed three protein gel spots decreased in contents in the cell membranes of GD3 synthase-transfected K562 cells. Among the increased proteins, membrane transglutaminase 2 (TG2) was specifically increased in the cell membrane of GD3 synthase-transfected K562 cells. Then, we generated the GD3 synthase-transfected cells in the K562 cells. Interestingly, the TG2 level was increased in GD3 synthase-transfected cells compared with vector- and plasma membrane-associated ganglioside sialidase (Neu3)-transfected cells. In addition, its ability to be photoaffinity-labeled with [alpha-(32)P]GTP was also increased in the GD3 synthase- and TG2-transfected cells. Moreover, small interfering RNA (siRNA) analysis for the GD3 synthase showed the decrease or abolishment of the membrane TG2. Finally, GD3 synthase-transfected cells accelerated the erythroid differentiation. Therefore, we propose that the recruitment of TG2 into membranes by GD3 might play an important role in the erythroid differentiation in K562 cells.     

RPS27a promotes proliferation,regulates cell cycle progression and inhibits apoptosis of leukemia cells

Ribosomal protein S27a (RPS27a) could perform extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. The high expression level of RPS27a was reported in solid tumors, and we found that the expression level of RPS27a was up-regulated in advanced-phase chronic myeloid leukemia (CML) and acute leukemia (AL) patients. In this study, we explored the function of RPS27a in leukemia cells by using CML cell line K562 cells and its imatinib resistant cell line K562/G01 cells. It was observed that the expression level of RPS27a was high in K562 cells and even higher in K562/G01 cells. Further analysis revealed that RPS27a knockdown by shRNA in both K562 and K562G01 cells inhibited the cell viability, induced cell cycle arrest at S and G2/M phases and increased cell apoptosis induced by imatinib. Combination of shRNA with imatinib treatment could lead to more cleaved PARP and cleaved caspase-3 expression in RPS27a knockdown cells. Further, it was found that phospho-ERK(p-ERK) and BCL-2 were down-regulated and P21 up-regulated in RPS27a knockdown cells. In conclusion, RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells. It appears that drugs targeting RPS27a combining with tyrosine kinase inhibitor (TKI) might represent a novel therapy strategy in TKI resistant CML patients.     

Negative Regulation of Erythroid Differentiation via the CBX8-TRIM28 Axis

Although the mechanism of chronic myeloid leukemia (CML) initiation through BCR/ABL oncogene has been well characterized, CML cell differentiation into erythroid lineage cells remains poorly understood. Using CRISPR-Cas9 screening, we identify Chromobox 8 (CBX8) as a negative regulator of K562 cell differentiation into erythrocytes. CBX8 is degraded via proteasomal pathway during K562 cell differentiation, which activates the expression of erythroid differentiation-related genes that are repressed by CBX8 in the complex of PRC1. During the differentiation process, the serine/threonine-protein kinase PIM1 phosphorylates serine 196 on CBX8, which contributes to CBX8 reduction. When CD235A expression levels are analyzed, the result reveals that the knockdown of PIM1 inhibits K562 cell differentiation. We also identify TRIM28 as another interaction partner of CBX8 by proteomic analysis. Intriguingly, TRIM28 maintains protein stability of CBX8 and TRIM28 loss significantly induces proteasomal degradation of CBX8, resulting in an acceleration of erythroid differentiation. Here, we demonstrate the involvement of the CBX8-TRIM28 axis during CML cell differentiation, suggesting that CBX8 and TRIM28 are promising novel targets for CML research.     

A comparative proteomic study identified LRPPRC and MCM7 as putative actors in imatinib mesylate cross-resistance in Lucena cell line

Background

Although chronic myeloid leukemia (CML) treatment has improved since the introduction of imatinib mesylate (IM), cases of resistance have been reported. This resistance has been associated with the emergence of multidrug resistance (MDR) phenotype, as a BCR-ABL independent mechanism. The classic pathway studied in MDR promotion is ATP-binding cassette (ABC) family transporters expression, but other mechanisms that drive drug resistance are largely unknown. To better understand IM therapy relapse due to the rise of MDR, we compared the proteomic profiles of K562 and Lucena (K562/VCR) cells.

Results

The use of 2-DE coupled with a MS approach resulted in the identification of 36 differentially expressed proteins. Differential mRNA levels of leucine-rich PPR motif-containing (LRPPRC) protein, minichromosome maintenance complex component 7 (MCM7) and ATP-binding cassette sub-family B (MDR/TAP) member 1 (ABCB1) were capable of defining samples from CML patients as responsive or resistant to therapy.

Conclusions

Through the data presented in this work, we show the relevance of MDR to IM therapy. In addition, our proteomic approach identified candidate actors involved in resistance, which could lead to additional information on BCR-ABL-independent molecular mechanisms.     

Mechanism of MK-0457 efficacy against BCR-ABL positive leukemia cells

Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance. MK-0457 is a small molecule inhibitor of the Aurora kinase family, but the mechanism of MK-0457 has not been evaluated. In this study, the gene expression profiles and intracellular signaling of chronic myeloid leukemia (CML) cell line K562 exposed to imatinib or MK-0457. MK-0457 induced cell growth inhibition in K562 cells. In gene expression profiles, there was an increase of 938 genes in imatinib and 895 genes in MK-0457 and 638 genes overlapped. In contrast, there was a decrease of 597 genes in imatinib and 582 genes in MK-0457 and 406 genes overlapped. These down-regulated genes include heat shock proteins (HSPs). These results indicate that MK-0457 is effective in CML cells by the down-regulation of HSPs which may relate to BCR-ABL stability, and offer new information regarding the molecular basis of strategy against to CML.     

K562耐药细胞的建立及相关蛋白表达改变的研究

为研究肿瘤细胞发生多药耐药后蛋白整体水平表达的改变,将K562细胞与阿霉素(ADR)共同培养,逐渐增加药物浓度,最后建立耐阿霉素的细胞K562／ADR株。MTT法检测阿霉素(ADR)、顺铂(DDP)、5-氟尿嘧啶(5-FU)和长春新碱(VCR)对K562和K562／ADR细胞的半数抑制率(IC50)。利用蛋白质组学技术,通过双向凝胶电泳分离K562和K562／ADR细胞的总蛋白,银染显色,分析差异蛋白,对部分蛋白点进行胶内酶解,用基质辅助激光解析飞行时间质谱法得肽质量指纹图谱,用AutoMS-Fit软件查询NCBInr数据库鉴定蛋白质。结果表明K562细胞经ADR诱导后出现多药耐药现象,ADR、DDP、5-FU和VCR对K562／ADR细胞的IC50明显高于K562。将K562和K562／ADR的双向电泳图谱进行差异比较,初步鉴定仅在K562／ADR图谱上出现的蛋白是一些与细胞分裂、基因转录有关的蛋白质。这些蛋白的变化与耐药细胞的特性相关,可能与临床化疗的多药耐药现象有联系。     

亚砷酸诱导分化的急性早幼粒细胞白血病细胞浸润人脑膜组织的体外模拟及其分子机制研究

目的:探讨急性早幼粒细胞白血病(Acute promyelocytic leukemia,APL)合并中枢神经系统白血病的发病机制。方法:采用流式细胞术检测亚砷酸(Arsenious acid, ATO)诱导分化前后的APL细胞及人APL细胞株NB4细胞表面CD56、CXCR4的表达;用荧光染料-羧基荧光素二醋酸盐琥珀酰亚胺酯标记ATO分化的APL(APL/ATO)、NB4细胞(NB4/ATO);用微重力旋转培养法体外模拟APL细胞浸润人脑膜组织,观察组织学及超微结构。结果:ATO诱导后,APL/ATO细胞表面CXCR4的表达明显高于诱导前(35.2±9.5%vs. 18.6±4.9%);NB4/ATO细胞表面CXCR4的表达明显高于诱导前(39.6±2.6%vs. 21.0±7.3%);APL/ATO细胞表面CD56的表达明显高于诱导前(36.6±8.9%vs. 25.8±5.15%);NB4/ATO细胞表面CD56的表达明显高于诱导前(44.6±8.4%vs. 25.6±2.4%)。组织学实验结果显示对照组脑膜组织未见NB4、APL细胞浸润,实验组可见APL/ATO、NB4/ATO细胞浸润到人脑膜组织中;荧光显微镜下可见被标记的APL/ATO、NB4/ATO细胞浸润到人脑膜组织中,扫描电镜见APL/ATO、NB4/ATO细胞浸润到脑膜组织中。结论:本研究采用微重力旋转培养系统体外模拟了ATO诱导分化的异常早幼粒细胞浸润人脑膜组织,APL细胞和NB4细胞CXCR4、CD56的表达升高可能是ATO诱导治疗APL所致的中枢神经系统浸润的分子机制之一。     

Differential gene expression in retinoic acid-induced differentiation of acute promyelocytic leukemia cells, NB4 and HL-60 cells

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation t(15;17), which results in the fusion of the promyelocytic leukemia gene (PML) and retinoic acid receptor alpha gene (RARalpha). APL can be effectively treated with the cell differentiation inducer all-trans retinoic acid (ATRA). NB4 cells, an acute promyelocytic leukemia cell line, have the t(15;17) translocation and differentiate in response to ATRA, whereas HL-60 cells lack this chromosomal translocation, even after differentiation by ATRA. To identify changes in the gene expression patterns of promyelocytic leukemia cells during differentiation, we compared the gene expression profiles in NB4 and HL-60 cells with and without ATRA treatment using a cDNA microarray containing 10,000 human genes. NB4 and HL-60 cells were treated with ATRA (10(-6)M) and total RNA was extracted at various time points (3, 8, 12, 24, and 48h). Cell differentiation was evaluated for cell morphology changes and CD11b expression. PML/RARalpha degradation was studied by indirect immunofluoresence with polyclonal PML antibodies. Typical morphologic and immunophenotypic changes after ATRA treatment were observed both in NB4 and HL-60 cells. The cDNA microarray identified 119 genes that were up-regulated and 17 genes that were down-regulated in NB4 cells, while 35 genes were up-regulated and 36 genes were down-regulated in HL60 cells. Interestingly, we did not find any common gene expression profiles regulated by ATRA in NB4 and HL-60 cells, even though the granulocytic differentiation induced by ATRA was observed in both cell lines. These findings suggest that the molecular mechanisms and genes involved in ATRA-induced differentiation of APL cells may be different and cell type specific. Further studies will be needed to define the important molecular pathways involved in granulocytic differentiation by ATRA in APL cells.     

Proteomic investigation of 5-fluorouracil resistance in a human hepatocellular carcinoma cell line

Multi-drug resistance (MDR) is a major obstacle towards a successful treatment of hepatocellular carcinoma (HCC). The mechanisms of MDR are intricate and have not been fully understood. Therefore, we employed a cell-line model consisting of the 5-fluorouracil (5-FU) resistant BEL7402/5-FU cell line and its parental BEL7402 cell line. Using relative and absolute quantification (iTRAQ)-coupled 2D LC-MS/MS, a successfully exploited high-throughput proteomic technology, in total, 660 unique proteins were identified and 52 proteins showed to be differentially expressed in BEL7402/5-FU compared with BEL7402. Several differentially expressed proteins were further validated by Western blot and real-time quantitative RT-PCR analysis. Furthermore, the association of MDR with ANXA3, one of the highly expressed proteins in BEL7402/5-FU, was verified. Our study represents the first successful application of iTRAQ technology for MDR mechanisms analysis in HCC. Many of the differentially expressed proteins identified had not been linked to MDR in HCC before, which provide valuable information for further understanding of MDR.     

Identification of differentially expressed proteins in imatinib mesylate-resistant chronic myelogenous cells

Resistance to imatinib mesylate (also known as Gleevec, Glivec, and STI571) often becomes a barrier to the treatment of chronic myelogenous leukemia (CML). In order to identify markers of the action of imatinib mesylate, we used a mass spectrometry approach to compare protein expression profiles in human leukemia cells (K562) and in imatinib mesylate-resistant human leukemia cells (K562-R) in the presence and absence of imatinib mesylate. We identified 118 differentially regulated proteins in these two leukemia cell-lines, with and without a 1 microM imatinib mesylate challenge. Nine proteins of unknown function were discovered. This is the first comprehensive report regarding differential protein expression in imatinib mesylate-treated CML cells.     

Proteomic identification of differentially expressed plasma membrane proteins in renal cell carcinoma by stable isotope labelling of a von Hippel‐Lindau transfectant cell line model

The von Hippel‐Lindau (VHL) tumour suppressor gene plays a central role in development of clear cell renal cell carcinoma (RCC). Using a cell line pair generated from the VHL‐defective RCC cell line UMRC2 by transfection with vector control or VHL (?/+VHL) and stable isotope labelling with amino acids in cell culture (SILAC) followed by enrichment of plasma membrane proteins by cell surface biotinylation/avidin‐affinity chromatography and analysis by GeLC‐MS/MS, VHL‐associated changes in plasma membrane proteins were analysed. Comparative analysis of ‐/+VHL cells identified 19 differentially expressed proteins which were confirmed by reciprocal SILAC labelling. These included several proteins previously reported to be VHL targets, such as transferrin receptor 1 and the α3 and β1 integrin subunits and novel findings including upregulation of CD166 and CD147 in VHL‐defective cells. Western blotting confirmed these changes and also revealed VHL‐dependent alterations in protein form for CD147 and CD166, which in the case of CD166 was shown to be due to differential glycosylation. Analysis of patient‐matched normal and malignant renal tissues confirmed these differences were also present in vivo in a subset of clear cell RCCs. These results illustrate the potential of this approach for identifying VHL‐dependent proteins that may be important in tumorigenesis.