Retinoic acid enhances differentiation of v-myb-transformed monoblasts induced by okadaic acid

Differentiation of various leukemic cells can be induced by liganded retinoic acid receptors and protein phosphatase inhibitors. In this study, we explored the effects of okadaic acid (OA), the phosphatase inhibitor, and retinoic acid (RA) in v-myb-transformed monoblasts BM2. OA induced differentiation of BM2 monoblasts into macrophage-like cells, as documented by analyses of cell morphology, cell cycle, phagocytic activity, non-specific esterase activity, production of reactive oxygen species and expression of vimentin and Mo-1. In contrast to many other leukemic cell lines, BM2 cells do not respond to retinoic acid. However, once exposed to OA and RA simultaneously, BM2 cells differentiate along monocyte/macrophage pathway more efficiently. We conclude that RA enhances differentiation of v-myb-transformed monoblasts induced by protein phosphorylation.     

Functional proteomic analysis of melanoma progression

Functional proteomics provides a powerful approach to screen for alterations in protein expression and posttranslational modifications under conditions of human disease. In this study, we use protein screening to examine markers of melanoma progression, by profiling melanocyte versus melanoma cell lines using two-dimensional electrophoresis and mass spectrometry. Eight candidate markers were identified as differentially regulated in transformed cells. In particular, hepatoma-derived growth factor (HDGF) and nucleophosmin B23 were strongly correlated with melanoma. Nucleophosmin B23 is a nucleolar and centrosome-associated protein, which has been implicated as a target for cyclin E/cyclin-dependent kinase 2 (CDK2) in modulating centrosome duplication and cell cycle control. Western blotting of one-dimensional and two-dimensional gels showed that the form of nucleophosmin B23 that is up-regulated in melanoma represents a posttranslationally modified form, most likely reflecting enhanced phosphorylation in the tumor-derived cells. In contrast, Western analysis of HDGF demonstrated increased expression of all forms in melanoma cell lines compared with melanocytes. Immunohistochemical analysis of human tissue biopsies showed strong expression of HDGF in early and late stage melanomas and low expression in melanocytes and nontumorigenic nevi. Interestingly, biopsies of nevi showed a graded effect in which HDGF immunoreactivity was reduced in nevoid nests penetrating deep into the dermis compared with nests at the epidermal-dermal junction, suggesting that HDGF expression in nevi is dependent on epidermal cell interactions. In contrast, biopsies of melanoma showed strong expression of HDGF throughout the tumor, including cells located deeply within dermis. Thus, expression of this antigen likely reports a reduced dependence of protein expression on epidermal interactions.     

Application of quantitative proteomic analysis for cancer therapy using "reverse-phase" protein lysate microarrays

Proteomic analysis using quantitative high-throughput technology can provide new insights in cancer therapeutics. It can reveal how cancer cells respond to given therapies by measuring multiple dimensions of information, from which dynamic proteomic responses can be observed. A lack of high throughput proteomic technologies has previously limited such multi-dimensional approaches. We have developed a high-throughput, "reverse-phase" protein microarray system which can handle more than 20,000 lysate features on a single glass slide. Subsequent immunochemical detection methods allow us to monitor protein expression in a quantitative manner as a function of both time and drug dosage. The data generated using this RPA technology has proved to be an excellent reference for theoretical protein network modeling in vitro. Clinical evaluation of drug efficacy based on the data generated by this technology may provide a means to accurately predict the effectiveness of cancer therapies.     

胆囊癌组织的比较蛋白质组学分析

目的 通过分离、鉴定胆囊癌和胆囊良性组织的差异表达蛋白质,探讨可能用于早期诊断或治疗胆囊癌的肿瘤标志物.方法 对6例人胆囊癌组织和胆囊良性组织进行比较蛋白质组学研究,采用双向凝胶电泳分离蛋白,经图像分析识别差异表达的蛋白.应用基质辅助激光解析电离飞行时间质谱(MALD-TOF-MS)鉴定差异蛋白质.采用免疫组化技术对差异蛋白磷脂酰乙醇胺结合蛋白1(PEBP1)进行验证.结果 获得了分辨率和重复性均较好的凝胶蛋白图谱.共筛选出46个在胆囊癌组织中明显差异表达的蛋白点,其中17个蛋白质鉴定成功.在这17个蛋白质中,胆囊癌组织中高表达9个,低表达8个.PEBP1在胆囊癌组织中的阳性表达率为74.0%.结论 成功鉴定了17个胆囊癌相关蛋白,为进一步筛选胆囊癌的诊断、治疗和预后评估的分子标志物奠定了基础.     

人肺腺癌细胞株A549和A549/DDP的比较蛋白质组学研究

目的:比较人肺癌细胞株A549和顺铂(cisplatin,DDP)耐药细胞株A549/DDP中的蛋白表达差异.方法:DDP诱导A549细胞建立DDP耐药细胞株A549/DDP.应用蛋白质组学技术分离鉴定A549和A549/DDP细胞中差异表达的蛋白质,通过real-time PCR、蛋白质印迹法和免疫细胞化学法对部分差异蛋白进行验证;应用生物学信息检索分析差异蛋白的功能.结果:A549和A549/DDP细胞中有8个蛋白质点的表达差异＞5倍,分别为POTE、FH (fumarate hydratase)、PDE (phosphodiesterase)、AKR1C1 (aldo-keto reductase family 1,member C1)、DDH2 (dihydrodiol dehydrogenase 2)、S100A10、prefoldin subunit 2和核内转运蛋白,这些蛋白与细胞代谢、凋亡、增殖、解毒和信号转导等有关.Real-time PCR、蛋白质印迹法和免疫细胞化学法验证结果与蛋白质组学研究结果一致.结论:鉴定得到的A549和A549/DDP细胞中的差异蛋白为研究肺癌细胞DDP耐药提供新依据.     

Exploration of metastasis-related proteins based upon proteomic analysis

Omics analysis is defined as a comprehensive analysis of various biological aspects from gene sequence to metabolite pattern via protein expression, and it might revolutionarily alter the design of scientific experiments as well as the process of biological investigation. Furthermore, omics technologies allow the generation of huge amounts of data at high-throughput. Particularly, proteomics is one of the omics technologies, which is a comprehensive analysis of protein, and it is hoped that proteomics accelerates translational research in the field of malignant neoplasm. Given the present status of proteomics technologies, we performed differential analysis between two categories such as a metastasis-positive group and a negative group using the clinical materials of a single kind of malignant neoplasm, and detected various kinds of protein molecules associated with metastasis. However, we could not obtain any evidence that these molecules were directly related with metastasis. The close relationship between clinical doctors, molecular pathologists and technical experts concerning proteomics is essential, because an experimental design, which they would generate, is extremely important for the detection of the targeted molecules. Additionally, in this paper some recent reports concerning the exploration of metastasis-related molecules using proteomic technology proteomics are introduced.     

An approach to proteomic analysis of human tumors

A strategy for proteomic analysis of microdissected cells derived from human tumor specimens is described and demonstrated by using esophageal cancer as an example. Normal squamous epithelium and corresponding tumor cells from two patients were procured by laser-capture microdissection and studied by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Fifty thousand cells resolved approximately 675 distinct proteins (or isoforms) with molecular weights ranging between 10 and 200 kDa and isoelectric points of pH 3-10. Comparison of the microdissected protein profiles showed a high degree of similarity between the matched normal-tumor samples (98% identical). However, 17 proteins showed tumor-specific alterations, including 10 that were uniquely present in the tumors and seven that were observed only in the normal epithelium. Two of the altered proteins were characterized by mass spectrometry and immunoblot analysis and were identified as cytokeratin 1 and annexin I. Acquisition of 2D-PAGE protein profiles, visualization of disregulated proteins, and subsequent determination of the identity of selected proteins through high-sensitivity MS-MS microsequencing are possible from microdissected cell populations. These separation and analytical techniques are uniquely capable of detecting tumor-specific alterations. Continued refinement of techniques and methodologies to determine the abundance and status of proteins in vivo holds great promise for future study of normal cells and associated neoplasms. Mol.     

Reduction in protein tyrosine phosphorylation during differentiation of human leukemia cell line K-562

Human chronic myelogenous leukemia cell line K-562 expresses the bcr/c-abl fusion protein which is an active protein tyrosine kinase. Multiple tyrosine-phosphorylated proteins were detected in K-562 cells by immunoblotting with a high-affinity anti-phosphotyrosine antibody. When K-562 cells were induced with hemin to progress through the erythroid differentiation pathway, reduction in the levels of these tyrosine-phosphorylated proteins was observed. This reduction in tyrosine-phosphorylated proteins was not found in another chronic myelogenous leukemia cell line which could not be induced to differentiate by hemin. This and other observations established that the reduction in protein tyrosine phosphorylation is a specific differentiation response. The bcr/c-abl protein synthesis was reduced in hemin-treated K-562 cells. Thus, erythroid differentiation of K-562 cells reduces the level of the bcr/c-abl tyrosine kinase and the phosphotyrosine content of its substrate proteins.     

A single lysis solution for the analysis of tissue samples by different proteomic technologies

Cancer, being a major healthcare concern worldwide, is one of the main targets for the application of emerging proteomic technologies and these tools promise to revolutionize the way cancer will be diagnosed and treated in the near future. Today, as a result of the unprecedented advances that have taken place in molecular biology, cell biology and genomics there is a pressing need to accelerate the translation of basic discoveries into clinical applications. This need, compounded by mounting evidence that cellular model systems are unable to fully recapitulate all biological aspects of human dissease, is driving scientists to increasingly use clinically relevant samples for biomarker and target discovery. Tissues are heterogeneous and as a result optimization of sample preparation is critical for generating accurate, representative, and highly reproducible quantitative data. Although a large number of protocols for preparation of tissue lysates has been published, so far no single recipe is able to provide a “one-size fits all” solubilization procedure that can be used to analyse the same lysate using different proteomics technologies. Here we present evidence showing that cell lysis buffer 1 (CLB1), a lysis solution commercialized by Zeptosens [a division of Bayer (Schweiz) AG], provides excellent sample solubilization and very high 2D PAGE protein resolution both when using carrier ampholytes and immobilized pH gradient strips. Moreover, this buffer can also be used for array-based proteomics (reverse-phase lysate arrays or direct antibody arrays), allowing the direct comparison of qualitative and quantitative data yielded by these technologies when applied to the same samples. The usefulness of the CLB1 solution for gel-based proteomics was further established by 2D PAGE analysis of a number of technically demanding specimens such as breast carcinoma core needle biopsies and problematic tissues such as brain cortex, cerebellum, skeletal muscle, kidney cortex and tongue. This solution when combined with a specific sample preparation technique – cryostat sectioning of frozen specimens – simplifies tissue sample preparation and solves most of the difficulties associated with the integration of data generated by different proteomic technologies.     

Lung cancer diagnosis from proteomic analysis of preinvasive lesions

Early detection may help improve survival from lung cancer. In this study, our goal was to derive and validate a signature from the proteomic analysis of bronchial lesions that could predict the diagnosis of lung cancer. Using previously published studies of bronchial tissues, we selected a signature of nine matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) mass-to-charge ratio features to build a prediction model diagnostic of lung cancer. The model was based on MALDI MS signal intensity (MALDI score) from bronchial tissue specimens from our 2005 published cohort of 51 patients. The performance of the prediction model in identifying lung cancer was tested in an independent cohort of bronchial specimens from 60 patients. The probability of having lung cancer based on the proteomic analysis of the bronchial specimens was characterized by an area under the receiver operating characteristic curve of 0.77 (95% CI 0.66-0.88) in this validation cohort. Eight of the nine features were identified and validated by Western blotting and immunohistochemistry. These results show that proteomic analysis of endobronchial lesions may facilitate the diagnosis of lung cancer and the monitoring of high-risk individuals for lung cancer in surveillance and chemoprevention trials.     

蛋白质组学研究在确定新的肿瘤标志物中的意义

随着蛋白质组学技术、高通量技术及生物信息学技术的发展,越来越多的肿瘤标志物被发现,并将逐步应用于临床,这些进展必将为肿瘤的早期检测和风险评价提供可靠的依据.现综述蛋白质组学研究策略在肿瘤标志物的筛选和鉴定中的应用.     

Regulation of biosynthesis and phosphorylation of P210 protein during differentiation induction of K 562 cells

The changes of P210^bcr/abl and other tyrosine phosphorylated proteins in K562 cells during growth and differentiation were studied by metabolic labeling with ³²PO₄ and immunoprecipitation with anti-phosphotyrosine sera. The anti-phosphotyrosine sera recognized P210^bcr/abl and other phosphoproteins with mol wt of 150, 115, 100, 70 and 64 kD. The 2-day incubation of K562 cells with inducers for differentiation, hemin, sodium butyrate and TPA, decreased the level of P210^bcr/abl protein and other phosphoproteins. Inhibitors of tyrosine protein kinase, amiloride and genistein, also reduced the phosphorylation of P210^bcr/abl protein and other substrates, but did not induce differentiation of the cells. Although most of these additives inhibited the cell growth, cytotoxic agents such as adriamycin, vincristine and Ara-C did not affect the level of P210^bcr/abl protein. The experiments using ³⁵S-methionine labeled cells and the immunoprecipitation with anti-abl sera suggested that reduced biosynthesis but not dephosphorylation of P210^bcr/abl protein mainly accounted for the reduction of P210^bcr/abl protein reacting to anti-phosphotyrosine sera in differentiation-induced cells. These results indicate that the reduction of P210^bcr/abl protein synthesis plays some roles in cellular differentiation of K562 cells.     

血清蛋白质谱诊断模型在乳腺癌辅助诊断中的价值

目的 建立乳腺癌的血清蛋白质谱诊断模型,评价其在乳腺癌辅助诊断中的价值.方法 应用表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术检测113例乳腺癌患者、103例乳腺良性肿瘤患者及92例健康女性的血清蛋白质谱,采用Biomarker Pattern(BPS)软件分析蛋白质谱,建立分类树模型,然后对模型进行盲筛验证.结果 比较乳腺癌患者与健康女性的血清蛋白质谱,筛选出12个差异蛋白质峰.经验证,所建立的分类树模型Ⅰ诊断乳腺癌的灵敏度为91.9%,特异度为81.2%.比较乳腺良性肿瘤患者与健康女性的血清蛋白质谱,筛选出11个差异蛋白质峰.经验证,所建立的分类树模型Ⅱ诊断乳腺良性肿瘤的灵敏度为87.9%,特异度为81.2%.比较乳腺癌与乳腺良性肿瘤患者的血清蛋白质谱,筛选出2个差异蛋白质峰.经验证,所建立的分类树模型Ⅲ诊断乳腺癌的灵敏度为81.8%,特异度为78.3%.应用这些差异蛋白及分类树模型,分别对93例CA15-3阴性乳腺癌患者与36例乳腺良性疾病患者的血清、20例CA15-3阳性乳腺癌患者与36例乳腺良性疾病患者的血清进行盲筛,诊断乳腺癌的敏感度和特异度分别为80.6%和91.7%、75.0%和91.7%,明显高于传统的乳腺癌标志物CA15-3,而CA15-3阴性与CA15-3阳性乳腺癌未见明显的差异蛋白质峰.结论 应用SELDI-TOF-MS技术可筛选出乳腺癌、乳腺良性肿瘤和健康女性血清蛋白质谱存在的差异蛋白质峰,据此建立的诊断模型可用于乳腺癌的辅助诊断.     

Identification of serum biomarkers for pancreatic adenocarcinoma by proteomic analysis

Diagnosis of pancreatic adenocarcinoma (PaCa) at an early stage is important for successful treatment and improving the prognosis of patients. Serum samples were applied to strong anionic exchange chromatography (SAX) protein chips for protein profiling by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to distinguish PaCa from noncancer. The Wilcoxon rank-sum test, decision tree algorithm, and logistic regression were used to statistically analyze the multiple protein peaks. Sixty-one protein peaks between 2000 and 30 000  m/z ratios were detected to establish multiple decision classification trees for differentiating the known disease states. A sensitivity of 0.833 and a specificity of 1.000 were obtained in distinguishing PaCa from healthy controls and benign pancreatic diseases. Six protein biomarkers related to different PaCa TNM stages were detected ( P  < 0.01). One protein biomarker ( m/z 4016) rich in PaCa had a down-regulated trend when preoperative and postoperative samples ( P  < 0.05) were compared. Three protein biomarkers ( m/z 4155, 4791, and 28 068) were detected in the differential diagnosis of the three test groups ( P  < 0.05). A peak m/z 28 068 was identified as C14orf16 using ProteinChip immunoassay. C14orf166 levels were significantly higher in the serum of patients with PaCa compared with the control group using a sandwich immunoenzymatic system. Immunolabeling of tissue sections revealed that the C14orf166 protein was strongly expressed in tumor cells. The results suggest that SELDI-TOF-MS serum profiling is helpful for the diagnostic, prognostic or therapeutic effects of PaCa, which is superior to CA 19-9. The identified protein biomarker C14orf166 is a potential biomarker of PaCa. ( Cancer Sci 2009; 100: 2292–2301)     

Identification of serum biomarkers for nasopharyngeal carcinoma by proteomic analysis

BACKGROUND: Early diagnosis of nasopharyngeal carcinoma (NPC) remains a challenge. Serum protein profiling is a promising approach for the classification of cancer versus noncancer samples. The objective of the current study was to assess the feasibility of mass spectrometry-based protein profiling and a classification tree algorithm for discriminating between patients with NPC and noncancer controls. METHODS: Serum samples from patients with NPC and noncancer controls were analyzed by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). The study was divided into a preliminary training set and a blind test set: A preliminary training set and a classification tree of spectra derived from 55 patients with NPC and a group of 60 noncancer controls were used to develop a proteomic model that discriminated cancer from noncancer effectively. Then, the validity of the classification tree was challenged with a blind test set, which included another 25 patients with NPC and 28 noncancer controls. RESULTS: Four protein peaks at 4097 daltons (Da), 4180 Da, 5912 Da, and 8295 Da were chosen automatically as a biomarker pattern in the training set that discriminated cancer from noncancer with sensitivity of 94.5% and specificity of 96.7%. When the SELDI marker pattern was tested with the blinded test set, it yielded a sensitivity of 92%, a specificity of 92.9%, and an accuracy rate of 92.5%. The accuracy of 2 protein peaks (4581 Da and 7802 Da) was 80% for predicting stage I and II NPC and 86% for predicting stage III and IV NPC. CONCLUSIONS: The high sensitivity and specificity obtained by the serum protein profiling approach demonstrated that SELDI-TOF-MS combined with a tree analysis model both can facilitate discriminating between NPC and noncancer controls and can provide an innovative clinical diagnostic platform to improve the detection of NPC.