Gaining insights into the Bcr-Abl activity-independent mechanisms of resistance to imatinib mesylate in KCL22 cells: A comparative proteomic approach

Imatinib mesylate is a potent inhibitor of Bcr-Abl tyrosine kinase, an oncoprotein that plays a key role in the development of chronic myeloid leukemia. Consequently, imatinib is used as front-line therapy for this disease. A major concern in imatinib treatment is the emergence of resistance to the drug. Here we used the imatinib-resistant KCL22R and imatinib-sensitive KCL22S cells in which none of the known resistance mechanisms has been detected and hence novel Bcr-Abl activity-independent mechanisms could be envisaged. We characterized proteins that were differentially expressed between the KCL22R and KCL22S cells. Using two-dimensional differential gel electrophoresis coupled with mass spectrometry and Western blot analysis we identified 51 differentially expressed proteins: 27 were over-expressed and 24 were under-expressed in KCL22R versus KCL22S cells. Several of these proteins are likely to be involved in such survival mechanisms as modulation of redox balance and activation of anti-apoptotic pathways mediated by NF-κB and Ras-MAPK signaling. The data reported may be useful for further studies on mechanisms of imatinib resistance and for the screening of biomarkers to develop new combinatorial therapeutic approaches.     

Molecular changes associated with induction of cell death in a human T-cell leukaemia line: putative nucleases identified as histones

Following treatment of the human T-cell leukaemia line, CEM-C7, with the glucocorticoid, dexamethasone, a rapid decrease in viability occurred after 40 h which coincided with fragmentation of DNA in these cells. A similar pattern of DNA fragmentation was observed when these cells were gamma-irradiated or treated with cycloheximide. Distinct morphological changes occurred after treatment, indicating a form of cell death, regulated from within, termed apoptosis. A set of nuclear proteins ranging in size from 10-18 kDa appeared by 40 h following treatment with dexamethasone. Treatment of cells with gamma-irradiation or cycloheximide also produced the same protein pattern. This set of proteins, and a doublet approximately 55 kDa in size, had apparent nuclease activity which was not observed in untreated cells. However, protein microsequencing of these bands in the 10-18 kDa region revealed that they were histone proteins. These results cast doubt on a recent report which provided evidence that these proteins were induced nucleases.     

A multidrug-resistant MCF-7 human breast cancer cell line which exhibits cross-resistance to antiestrogens and hormone-independent tumor growth in vivo

MCF-7 human breast cancer cells provide a useful in vitro model system to study hormone-responsive breast cancer as they contain receptors for estrogen and progesterone, and estrogen both induces the synthesis of specific proteins in these cells and increases their rate of proliferation. An MCF-7 cell line which was selected for resistance to adriamycin (MCF-7/AdrR) exhibits the phenotype of multidrug resistance (MDR), and displays multiple biochemical changes. MDR in MCF-7/AdrR is also associated with a loss of mitogenic response to estrogen and the development of cross-resistance to the antiestrogen 4-hydroxytamoxifen. In addition, while the parental MCF-7 cell line responds to estrogen with increased levels of progesterone receptors and the secretion of specific proteins, these estrogen responses are lost in MCF-7/AdrR. Furthermore, while the formation of tumors in nude mice by wild-type MCF-7 cells is dependent upon the presence of estrogen, MCF-7/AdrR cells form tumors in the absence of exogenous estrogen administration. These changes in hormonal sensitivity and estrogen-independent tumorigenicity of the multidrug-resistant MCF-7 cell line are associated with a loss of the estrogen receptor and a concomitant increase in the level of receptors for epidermal growth factor. Thus, in MCF-7/AdrR cells, the development of MDR is associated with alterations in the expression of both cytosolic and membrane receptors, resulting in resistance to hormonal agents and the expression of hormone-independent tumor formation.     

A proteomic approach to cisplatin resistance in the cervix squamous cell carcinoma cell line A431

Since drug resistance is a complex and multifactorial event involving activation/repression of multiple biochemical pathways, we used a proteomic approach to study cisplatin resistance and drug response in human tumor cell lines. The cervix squamous cell carcinoma cell line A431 and its cisplatin-resistant subline, A431/Pt, were used as a model system. The experimental set-up involved not just a two-way comparison of the control vs. the drug-resistant cell line, but also an acute cisplatin treatment of both cell lines, leading to a four-way comparison, as follows: 1) A431 vs. A431/Pt cells; 2) A431 vs. A431 cisplatin exposed cells; 3) A431/Pt vs. A431/Pt cisplatin exposed cells; 4) A431 cisplatin exposed cells vs. A431/Pt cisplatin exposed cells. We found modulation of proteins, which could be classified under various categories, such as molecular chaperones (e.g. heat-shock proteins HSP60, HSP90, HSC71, heat-shock cognate 71 kDa protein), Ca2+-binding proteins (e.g. calmodulin, calumenin), proteins involved in drug detoxification (such as peroxiredoxins PRX 2 and PRX 6, and glutathione-S-transferase, GST), anti-apoptotic proteins (such as 14-3-3 switched on in cisplatin-exposed cells) and ion channels (such as VDAC-1, voltage-dependent anion-selective channel). In particular, the basal levels of HSC71 and HSP60 were increased in A431/Pt cells as compared to A431 cells, and cisplatin exposure resulted in up-regulation of HSP60 and HSP90 only in A431 cells. Moreover, cisplatin exposure up-regulated the anti-apoptotic 14-3-3 protein in both cell lines, GST in sensitive cells and PRX6 in A431/Pt cells. These findings are consistent with a constitutive expression of defence factors by resistant cells and with activation by cisplatin of mechanisms acting to protect cells from drug-induced damage. This pattern of response, also observed in parental cells, could reflect an intrinsic resistance of this tumor type.     

A proteomic screen identified stress-induced chaperone proteins as targets of Akt phosphorylation in mesangial cells

The serine-threonine kinase Akt regulates mesangial cell apoptosis, proliferation, and hypertrophy. To define Akt signaling pathways in mesangial cells, we performed a functional proteomic screen for rat mesangial cell proteins phosphorylated by Akt. A group of chaperone proteins, heat shock protein (Hsp) 70, Hsp90alpha, Hsp90beta, Glucose-regulated protein (Grp) Grp78, Grp94, and protein disulfide isomerase (PDI) were identified as potential Akt substrates by two techniques: (a) in vitro phosphorylation of mesangial cell lysate by recombinant active Akt followed by protein separation by SDS-PAGE or 2-DE and phosphoprotein identification by peptide mass fingerprinting using MALDI-MS, or (b) immunoblot analysis of proteins from PDGF-stimulated mesangial cells using an anti-Akt phospho-motif antibody. In vitro kinase reactions using recombinant proteins confirmed that Akt phosphorylates Hsp70, Hsp90alpha and beta, Grp94, and PDI. Immunoprecipitation of Akt from mesangial cell lysate coprecipitated Grp78 and Hsp70. PDGF stimulation of mesangial cells caused an acidic shift in the isoelectric point of Hsp70, Hsp90, and PDI that was dependent on PI-3K activity for Hsp70 and Hsp90. The data suggest that Akt-mediated phosphorylation of stress-induced chaperones represents a mechanism for regulation of chaperone function during mesangial cell responses to physiologic and pathologic stimuli.     

Metabolic and proteomic study of NS0 myeloma cell line following the adaptation to protein-free medium

Proteomics and metabolomics technologies are potentially useful tool for the study of the very complex process of cell adaptation to protein-free medium. In this work, we used the iTRAQ technology to analyze different protein levels in adapted and non-adapted NS0 myeloma cell line. Several proteins with differential expression profile were characterized and quantified. Carbohydrate metabolism, protein synthesis and membrane transport were the principal pathways that change after the adaptation. Changes in lactate production rate with respect to glucose consumption rate were observed according to the changes observed by proteomic.     

Aeromonas hydrophila vacuolating activity in the Caco-2 human enterocyte cell line as a putative virulence factor

A non-enterotoxigenic strain of Aeromonas hydrophila isolated from diarrheic stools of an 8-month-old child was found to cause vacuolation in Caco-2 cells. The vacuoles became prominent 60 min after addition of the bacterial culture to the cell monolayers and, after 120 min, a complete disruption of the monolayers was observed. Cell vacuolation was not detected when Caco-2 monolayers were tested with sterile filtrates of overnight cultures of the A. hydrophila vacuolating strain AH14846. This strain produced a diffuse adherence pattern in Caco-2 cell monolayers, but did not produce detectable cytotonic enterotoxin in the suckling mouse test and only produced small quantities of aerolysin. By demonstrating the ability to induce vacuolation in mammalian cells of enterocytic lineage, the current study raises the possibility that such activity might contribute to gastrointestinal symptoms in infections involving Aeromonas strains which do not express well-established enterotoxins.     

人食管癌EC9706单克隆的分离培养及其生物学特性的研究

用限制性稀释法对食管癌EC9706细胞系进行单细胞克隆分离,建立了3个亚系,分别命名为EC1、EC2和EC3。细胞遗传学分析显示,EC1仍为混合克隆,EC2和EC3的染色体众数分别为117条和62条。对EC2和EC3的生物学特性进行比较,生长曲线、平皿集落形成能力实验、流式细胞分析和细胞运动能力实验均表明,两者的恶性程度有显著差别,EC2较低,EC3较高。成功分离的2个食管癌单克隆细胞亚系,可为食管癌发生发展的分子机制研究、基因治疗研究、耐药机制研究以及抗肿瘤药物筛选提供实验材料。     

Blood-based lung cancer biomarkers identified through proteomic discovery in cancer tissues,cell lines and conditioned medium

Background

Support for early detection of lung cancer has emerged from the National Lung Screening Trial (NLST), in which low-dose computed tomography (LDCT) screening reduced lung cancer mortality by 20 % relative to chest x-ray. The US Preventive Services Task Force (USPSTF) recently recommended annual screening for the high-risk population, concluding that the benefits (life years gained) outweighed harms (false positive findings, abortive biopsy/surgery, radiation exposure). In making their recommendation, the USPSTF noted that the moderate net benefit of screening was dependent on the resolution of most false-positive results without invasive procedures. Circulating biomarkers may serve as a valuable adjunctive tool to imaging.

Results

We developed a broad-based proteomics discovery program, integrating liquid chromatography/mass spectrometry (LC/MS) analyses of freshly resected lung tumor specimens (n = 13), lung cancer cell lines (n = 17), and conditioned media collected from tumor cell lines (n = 7). To enrich for biomarkers likely to be found at elevated levels in the peripheral circulation of lung cancer patients, proteins were prioritized based on predicted subcellular localization (secreted, cell-membrane associated) and differential expression in disease samples. 179 candidate biomarkers were identified. Several markers selected for further validation showed elevated levels in serum collected from subjects with stage I NSCLC (n = 94), relative to healthy smoker controls (n = 189). An 8-marker model was developed (TFPI, MDK, OPN, MMP2, TIMP1, CEA, CYFRA 21–1, SCC) which accurately distinguished subjects with lung cancer (n = 50) from high risk smokers (n = 50) in an independent validation study (AUC = 0.775).

Conclusions

Integrating biomarker discovery from multiple sample types (fresh tissue, cell lines and conditioned medium) has resulted in a diverse repertoire of candidate biomarkers. This unique collection of biomarkers may have clinical utility in lung cancer detection and diagnoses.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9090-9) contains supplementary material, which is available to authorized users.     

Vanadium mediated apoptosis and cell cycle arrest in MCF7 cell line

Vanadium is a metal widely distributed in the environment. It is also a dietary micronutrient. It has shown insulin mimetic and chemopreventive properties and has been considered as an important pharmacological agent. In this study, we evaluated the apoptogenic role of vanadium on human breast cancer cell line MCF7. Exposure of MCF7 cells to vanadium led to the induction of apoptosis in a dose-dependent manner. Percentage of apoptosis was maximum (42.5%) at the highest non-toxic dose (250 microM). It was found that vanadium treatment brought about a prominent chromatin condensation, cell cycle arrest leading to apoptosis. These apoptosis based assays demonstrate that vanadium has the potential to be developed into an anti-cancer drug in the near future.     

A comparative morphological study of lateral line systems in larvae and pupae of Trichoptera

The abdominal lateral line system in Trichoptera, consisting of filaments and lamellae with associated primary and secondary setae, was studied in larvae and pupae of 70 genera representing 26 families. Filaments are slender, generally bifid, tubular extensions of the cuticle that together form a fringe on the lateral abdominal surface of larvae and pupae of many but not all families of case-making Trichoptera (Integripalpia). Larvae and pupae of the Annulipalpia and Spicipalpia lack a lateral fringe of bifid filaments, but single filaments occur in larvae of a few unrelated genera. A lateral fringe may help in regulating the flow of water which is maintained through the case by abdominal undulation. Lamellae occur only in larvae of the Integripalpia, where they are variously distributed among families and genera, and probably have a sensory function. Three types of lamellae are recognized: forked, serrate and single. Filaments and lamellae have significant taxonomic value, and their patterns of occurrence raise questions about the phylogeny of Trichoptera.