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Results for “Protein Array Analysis”

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PubMed2005

Effect of retinoic acid and interferon alpha-2a on transitional cell carcinoma of bladder.

PURPOSE: Retinoids modulate the growth and differentiation of normal and malignant epithelial cells in vitro and in vivo. Retinoids and their analogues have been used in animal models and clinical trials of chemoprevention and superficial bladder cancer treatment. Interferons are cytokines that have antiviral, antiproliferative and immunomodulatory function. They are used in many clinical trials for the treatment of different cancers. To identify new effective agents and develop novel approaches for the chemoprevention and treatment of superficial bladder cancer we investigated the effects of a combination of retinoids and interferon alpha-2a (IFN) on growth and apoptosis in bladder cancer cell lines. MATERIALS AND METHODS: The 4 bladder cancer cell lines UM-UC-6, UM-UC-9, UM-UC-10 and UM-UC-13 were treated with 2 retinoids, namely all-trans-retinoic acid (ATRA) and 9-cis retinoic acid (9cRA), as well as with IFN or with combinations of retinoids and IFN. The ability of these agents used alone and in combination to inhibit growth, induce apoptosis and modulate gene expression was investigated. The effects of retinoids on an INF related gene were also examined. RESULTS: Most bladder cancer cell lines were resistant to growth inhibition and apoptosis induction by ATRA and 9cRA, even at a high concentration. The effects of these retinoids on cell growth and apoptosis were enhanced by IFN. The combination of ATRA and IFN induced retinoic acid receptor beta, and signal transducer and activator of transcription 1 expression in 3 bladder cancer cell lines, as detected by reverse transcriptase-polymerase chain reaction and Western blot analysis. Retinoids increased IFN-related gene expression detected by microarray analysis and real-time reverse transcriptase-polymerase chain reaction. CONCLUSIONS: The results demonstrate that IFN acts synergistically with ATRA and 9cRA in the growth and apoptosis of bladder cancer cells in vitro and suggest that this combination has a potential for the treatment of transitional cell carcinoma of the bladder.

Antineoplastic Agents↗

PubMed2005

Development of a protein microarray library and a system for rheumatoid-arthritis disease marker screening.

Early detection and intervention can delay the onset of symptoms of RA (rheumatoid arthritis), but current diagnostic tests suffer from low sensitivity and specificity, making such early disease detection difficult. Analysis of body fluids for the identification of multiple molecular markers and the subsequent determination of modifications in their associated protein structures emerges as a possibility for early detection. The structural modifications of these markers are thought to play a pivotal role in defining the pathological state of diseased joints. Therefore a proteome library and system for RA disease screening has been developed and investigated in the present study. The large (39 S) mammalian ribosomal subunit has been identified as a potential RA marker. This protein is known as L 35, and antibodies to this protein have been found to be expressed in patients suffering from RA.

Amino Acid Sequence↗

PubMed2005

Hyaluronan oligosaccharide-induced activation of transcription factors in bovine articular chondrocytes.

OBJECTIVE: To document the activity profile of transcription factors following chondrocyte stimulation with hyaluronan (HA) hexasaccharides (HA(6)) and to determine the expression of genes whose transcriptional activation is tightly associated with the transcription factors. METHODS: Nuclear extracts from bovine articular chondrocytes treated with HA(6) were subjected to transcription factor protein-DNA array analysis. Electrophoretic mobility shift assay (EMSA) analyses were performed to confirm the results of protein-DNA array. The gene expressions of matrix metalloproteinase 3 (MMP-3), type II collagen, and cartilage oligomeric matrix protein (COMP) were examined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), and protease activity was assessed by casein zymography. RESULTS: In the protein-DNA array analysis, 12 transcription factors were up-regulated and 2 transcription factors were down-regulated in the chondrocytes treated with HA(6). The transcription factors retinoic acid receptor (RAR), retinoid X receptor (RXR), and Sp-1 exhibited >2-fold increased activity by HA(6) treatment, as confirmed by EMSA. RT-PCR analysis showed that the expression levels of MMP-3, type II collagen, and COMP messenger RNA, which are tightly associated with the activation of RAR, RXR, or Sp-1, were up-regulated by treatment with HA(6). Addition of high molecular mass HA after HA(6) treatment resulted in abrogation of the MMP-3 induction. CONCLUSION: These results suggest that HA(6) increase the activity of multiple transcription factors in chondrocytes and signal the enhanced expression of key genes involved in cartilage-matrix remodeling and turnover. The data also demonstrate that high molecular mass HA has a potential to suppress the signaling activated by HA(6).

Adjuvants, Immunologic↗

PubMed2006

Development of recombinant-based mass spectrometric immunoassay with application to resistin expression profiling.

This report addresses the need for additional assays for human resistin (hRES) by developing a rational progression of the mass spectrometric immunoassay to incorporate recombinant proteins. The recombinant-based hRES mass spectrometric immunoassay (RES-MSIA) was initially developed for the qualitative analysis of the human resistin homodimer from normal (healthy) plasma samples. The method involved selective extraction and detection of both endogenous and recombinant resistant proteins. RES-MSIA was then applied to the rigorous quantification of resistin. The resistin standard addition curve was constructed from serially diluted concentrations of rhRES using endogenous hRES, inherent in the human plasma, as the internal reference standard (IRS). The roles of endogenous and recombinant resistin were subsequently reversed, using rhRES as the IRS during RES-MSIA quantification. Concurrently, the relative ratio of hRES to rhRES was used as an ancillary technique to rapidly determine the relative concentration of hRES in each of plasma samples. Overall, normal hRES levels determined by RES-MSIA were found to be comparable to those selected and determined by ELISA. With regard to gender, female donor samples were slightly elevated over males. Four single cardiac samples were analyzed and found to have hRES concentrations approximately three times that of the normal. The recombinant-based RES-MSIA is rapid and is amendable to parallel high-throughput robotic processing of resistin related disease cohorts.

Gene Expression Profiling↗

PubMed2005

Differential expression of cyclin D3 in ALK+ and ALK- anaplastic large cell lymphoma.

As defined in the World Health Organization classification, anaplastic large cell lymphoma (ALCL) is a distinct type of non-Hodgkin lymphoma of T/null cell lineage, a subset of which is associated with translocations involving 2p23 resulting in expression of anaplastic lymphoma kinase (ALK). The most common translocation, the t(2;5)(p23;q35), results in expression of nucleophosmin (NPM)-ALK. NPM-ALK has been shown to activate signal transducer and activator of transcription (STAT) 3, a transcriptional regulator of cyclin D3. In this study, we assessed cyclin D3 expression in 2 ALK+ ALCL cell lines (Karpas 299 and SU-DHL1) and 1 ALK- ALCL cell line (Mac2A) by Western blot analysis. We also assessed cyclin D3 expression in 52 ALCL tumors (32 ALK+, 20 ALK-) by immunohistochemistry using tissue microarrays. These results were compared with phosphorylated (activated) STAT3 (pSTAT3) expression. Both ALK+ ALCL cell lines, but not the ALK- ALCL cell line, expressed cyclin D3 and pSTAT3. Cyclin D3 was expressed in 25 (78%) of 32 ALK+ ALCL tumors and in 4 (20%) of 20 ALK- ALCL tumors (P < .001, Fisher exact test ). In ALK+ ALCL tumors, the mean percentage of cyclin D3-positive tumor cells was 40.6% compared with 5.1% in ALK- ALCL tumors (P < .001, Mann-Whitney U test). The percentages of cyclin D3-positive and pSTAT3-positive tumor cells were positively correlated (Spearman R = 0.35, P = .036). We conclude that cyclin D3 is differentially expressed in ALK+ and ALK- ALCL and that high expression levels of cyclin D3 in ALK+ ALCL may be attributable to STAT3 activation.

Anaplastic Lymphoma Kinase↗

PubMed2005

Proteomic analysis of nipple aspirate fluid using SELDI-TOF-MS.

Proteomic analysis of body fluids, including breast nipple aspirate fluid (NAF), holds promise to aid in early cancer detection. We conducted a prospective trial that collected NAF from women scheduled for diagnostic breast surgery to determine 1) the consistency of proteomic results, 2) protein masses associated with breast cancer, 3) subsets of women with a unique proteomic profile and 4) a breast cancer predictive model. NAF was collected preoperatively in 114 women and analyzed by SELDI-TOF mass spectrometry over a 3-50 kDa range using H4, NP and SAX ProteinChips. For all 3 chips, the same protein peaks were detected over 90% of the time in duplicate samples. The overall coefficient of variation was < or = 0.17% for each chip for the internal standard and < or = 0.29% for the unknown proteins. Seven candidate protein ion masses frequently expressed in NAF were identified. Three (5,200-H4, p=.04, 11,880-H4, p=.07 and 13,880 Da-SAX, p=.03) were differentially expressed in women with/without breast cancer. Protein expression differed between women with/without pathologic nipple discharge (PND), but the 5,200, 11,880 and 13,880 proteins remained associated with breast cancer even if PND samples were excluded. Subset analysis identified differences in expression between benign disease and DCIS and between DCIS and invasive cancer for the 5,200 and 33,400 Da proteins. The best cancer detection model included age, parity and the 11,880 Da protein, and excluded women with PND. 1) NAF proteomic analysis using SELDI-TOF is reproducible with the same sample set across different platforms, 2) differential proteomic expression exists between women/without breast cancer and 3) combining proteomic and clinical information that are available before surgery optimizes the prediction of which women have breast cancer.

Adult↗

PubMed2005

Growth-related oncogene produced in human breast cancer cells and regulated by Syk protein-tyrosine kinase.

Syk, a nonreceptor type of protein tyrosine kinase widely expressed in hematopoietic cells, is a candidate suppressor gene in human breast cancer. Reduced expression of Syk protein is correlated with poor prognosis, while its overexpression can reduce the malignant phenotype of breast cancer cells. The mechanism of action of Syk remains unclear. In this study, we utilized low Syk-expressing, highly invasive MDA-MB-231 and high Syk-expressing, less invasive MCF-7 breast cancer cells to investigate the possibility that part of the functional effects of Syk are mediated by cytokines known to play roles in cell migration, invasion or metastasis. Using protein array technology, we determined that MDA-MB-231 cells secrete a number of cytokines known to regulate cellular growth and motility. One such cytokine, growth-related oncogene (GRO), has previously not been described in breast cancer. Of the compounds detected in the culture supernatant of MDA-MB-231, GRO was the only one that was significantly altered by modulation of Syk expression; overexpression of Syk caused a marked reduction in secreted levels of GRO. Conversely, downregulation of the relatively high levels of Syk produced in MCF-7 cells upregulated GRO secretion. At the mRNA level, overexpression of Syk in MDA-MB-231 differentially regulated the 3 GRO isotypes such that message levels of GROalpha and gamma were downregulated while that of GRObeta was not affected. Matrigel invasion assays demonstrated a link between Syk expression and resulting GRO activity in mediating the invasive potential of MDA-MB-231 cells. In summary, our findings provide evidence that human breast cancer cells express and secrete GRO and implicate this cytokine as an essential mediator of the antiinvasive properties of Syk tyrosine kinase.

Blotting, Western↗

PubMed2003

BCL-2 family proteins in peripheral T-cell lymphomas: correlation with tumour apoptosis and proliferation.

The present study investigated expression levels of the anti-apoptotic proteins BCL-2, BCL-XL and MCL-1 and the pro-apoptotic proteins BAX and BCL-XS in a series of 112 peripheral T-cell lymphomas (PTCLs) classified according to the WHO classification. Using immunohistochemical methods and a 10% cut-off, each protein was detected in a subset of PTCLs: BCL-2 in 46%, BCL-XS in 49%, BAX in 57%, BCL-XL in 57%, and MCL-1 in 65%. The mean percentage of positive cells for these proteins varied significantly among the PTCL types. Only two types of PTCL, ALK-positive anaplastic large cell lymphoma (ALCL) and enteropathy-type T-cell lymphoma, had a distinctive pattern of expression; all were BCL-2-negative and MCL-1-positive. The mean percentage of BAX-positive and BCL-XS-positive tumour cells was higher in ALK-positive ALCL than in ALK-negative ALCL or other types of PTCL (p = 0.06 and p = 0.01, respectively, Kruskal-Wallis test). MCL-1 was detected significantly more frequently (p = 0.01, chi-square test) and at higher levels (p = 0.0001, Kruskal-Wallis test) in ALK-positive ALCL and ALK-negative ALCL than in other PTCL types. The apoptotic rate, evaluated by the TUNEL assay, correlated inversely with BCL-2 expression (p = 0.035). The proliferation index, assessed by the MIB-1 antibody, correlated with expression levels of MCL-1 (R = 0.42, p = 0.003), BCL-2 (R = 0.32, p = 0.027), BAX (R = 0.33, p = 0.014), and BCL-XL (R = 0.34, p = 0.015) (Spearman rank). In conclusion, BCL-2 family proteins are expressed by a subset of PTCLs and their levels correlate with some histological types, apoptotic rate, and proliferation index. Expression of these proteins may explain the poor response of many types of PTCL to standard chemotherapy. These proteins may also provide novel targets for experimental therapy.

Anaplastic Lymphoma Kinase↗

PubMed2006

Profiling humoral autoimmune repertoire of dilated cardiomyopathy (DCM) patients and development of a disease-associated protein chip.

Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37,200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.

Amino Acid Sequence↗

PubMed2005

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of 4-sulfophenyl isothiocyanate-derivatized peptides on AnchorChip sample supports using the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone and diammonium citrate.

The reagent 4-sulfophenyl isothiocyanate (SPITC) is an effective, stable, and inexpensive alternative to commercially available reagents used in the N-terminal sulfonation of peptides for enhanced postsource decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analyses. However, suppression of ionization of sulfonated peptides due to sample and matrix contaminants such as sodium can be a problem when using prestructured MALDI target sample supports, such as the Bruker Daltonics AnchorChip. We show that use of the salt-tolerant matrix 2,4,6-trihydroxyacetophenone containing diammonium citrate (THAP/DAC) as an alternative to alpha-cyanohydroxycinnamic acid (HCCA) reduces the need for extensive washing of ZipTip-bound peptides or additional on-target sample clean-up steps. Use of the THAP/DAC matrix results in selective ionization of sulfonated peptides with greater peptide coverage, as well as detection of higher mass derivatized peptides, than was observed for HCCA or THAP alone. The THAP/DAC matrix is quite tolerant of sodium contamination, with SPITC-peptides detectable in preparations containing up to 50 mM NaCl. In addition, THAP/DAC matrix was found to promote efficient PSD fragmentation of sulfonated peptides. We demonstrated the utility of using the THAP/DAC MALDI matrix for peptide sequencing with DNA polymerase beta tryptic peptide mixture, as well as tryptic peptides derived from Xiphophorus maculatus brain extract proteins previously separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).

Acetophenones↗

PubMed2006

Characterization of matrix metalloproteinase expressed by human embryonic kidney cells.

There is little information available on the proteases expressed by human embryonic kidney (HEK) cells, which are often used for expression of recombinant proteins and production of adenovirus vector. The expression profile of proteases in HEK cell line was investigated using zymography, mRNA analysis, western blotting and protein array. The major protease was gelatinase A [or matrix metalloproteinase (MMP)-2]. Beside, other MMPs, such as MMP-1, -2, -3, -8, -9, -10, -13 and membrane type (MT) 1- and 3-MMP, as well as tissue inhibitors of metalloproteinase (TIMP)-1, -2 and -3, were also expressed by HEK cells. Characterization of MMP and TIMP profiles expressed by HEK cells provides the basis for degradation control of recombinant protein and adenovirus vector during culture and purification processes.

Cells, Cultured↗

PubMed2006

Application of complement 1q for the site-selective recognition of immune complex in protein chip.

Complement 1q (C1q) was applied for the specific recognition of antibody-antigen complex in antibody-based protein chip. The specific binding of C1q to antibody-antigen complex was investigated by surface plasmon resonance (SPR) with respect to Yersinia entericolitica, Salmonella typimurium, insulin, and bovine serum albumin. The protein chip was fabricated with two different kinds of antibodies a zigzag configuration. When one of antigens and fluorescein-isothiocyanate (FITC)-labeled C1q was applied on the protein chip, the specific binding event of C1q to immune complexes formed on protein chip was observed by fluorescence microscopy. These results implicate that the C1q can be used as an alternative to many antibodies that may be utilized individually on each spot of the protein chip.

Antigen-Antibody Complex↗

PubMed2004

Facile synthesis of metal-chelating peptides on chip for protein array.

A unique peptide sequence of HGGHHG screening from a combinatorial synthetic peptide library showed a good chelating ability to bind a transition metal on a chip better than hexa-His peptide. It was directly conjugated with a His-Tagged proteins onto a chip in a mild aqueous solution and can be used this chip as a high throughput technique for protein array in order to detect and purify the His-Tagged proteins.

Chelating Agents↗

PubMed2005

Protein microarray using alpha-amino acids as metal tags on chips.

Procedures for synthesizing alpha-amino acids on a chip for coordination with transitional metal ions and a His-Tagged protein have been successfully developed as a stable protein microarray. Using the recombinant His-Tagged 3CL-protease (3CLpro) as a model for attachment to chips containing D-/L-Glu, Asp, Orn, Ser via different transitional metal ions, it was found that the Orn chip was the best of affinity binding and stability by which Zn2+ was the best metal ion for affinity while Co2+ was the best metal ion for stability. Thus, this protein microarray can be alternatively used as a high throughput screening method for rapid detection against SARS CoV 3CLpro and/or efficient purification of other Tagged proteins.

Amino Acids↗

PubMed2007

Functional glycosylation of human podoplanin: glycan structure of platelet aggregation-inducing factor.

Podoplanin (Aggrus) is a mucin-type sialoglycoprotein that plays a key role in tumor cell-induced platelet aggregation. Podoplanin possesses a platelet aggregation-stimulating (PLAG) domain, and Thr52 in the PLAG domain of human podoplanin is important for its activity. Endogenous or recombinant human podoplanin were purified, and total glycosylation profiles were surveyed by lectin microarray. Analyses of glycopeptides produced by Edman degradation and mass spectrometry revealed that the disialyl-corel (NeuAc alpha2-3Gal beta l-3(NeuAc alpha2-6)GalNAc alpha l-O-Thr) structure was primarily attached to a glycosylation site at residue Thr52. Sialic acid-deficient podoplanin recovered its activity after additional sialylation. These results indicated that the sialylated Corel at Thr52 is critical for podoplanin-induced platelet aggregation.

Amino Acid Sequence↗

PubMed2003

Biochip as a potential platform of serological interferon alpha2b antibody assay.

The formation of antibodies against cytokines may play a major role in the generation of the immune response and may affect treatment protocols with recombinant cytokines. Interferon (IFN) is one of the effective therapeutic agents with anti-viral and anti-tumor specific effects. The appearance of IFN antibodies in patients may limit the natural and the therapeutic effect by IFNs. In contrast to conventional ELISA techniques, we here report a simple biochip methodology that enables identification of antibodies against cytokines and peptides. The method takes advantage of a functionalized self-assembled monolayer modified by N-hydroxysuccinimide (NHS). To validate this surface, four human proteins: IFNalpha2b, leptin, growth hormone and human IgG, with molecular sizes ranging between 14 and 150 kDa, were used. A number of other parameters for protein assay conditions by array technology were evaluated concomitantly. Finally, 56 serum samples from patients treated with recombinant human IFNalpha2b were simultaneously tested on single chip. In these patients, 16.1% (9 of 56 cases) were positive for IFNalpha2b antibodies. All results were confirmed in an ELISA, specific for the identification of IFNalpha specific antibodies in human samples. The potential application of this protein biochip can be amplified rapidly and reliably to test not only IFNalpha2b, but also other cytokine specific antibodies. The clinical relevance of such assays for investigations in autoimmune disorders is expected.

Adsorption↗

PubMed2006

Robust increase of cutaneous sensitivity, cytokine production and sympathetic sprouting in rats with localized inflammatory irritation of the spinal ganglia.

We investigated the role and mechanisms of inflammatory responses within the dorsal root ganglion (DRG) in the development of chemogenic pathological pain. DRG inflammation was induced by a single deposit of the immune activator zymosan in incomplete Freund's adjuvant in the epidural space near the L5 DRG via a small hole drilled through the transverse process. After a single zymosan injection, rats developed bilateral mechanical hyperalgesia and allodynia which began by day 1 after surgery, peaked at days 3-7, and lasted up to 28 days. The number of macrophages in ipsilateral and contralateral DRGs increased significantly, lasting over 14 days. Robust glial activation was observed in inflamed ganglia. Cytokine profile analysis using a multiplexing protein array system showed that, in normal DRG, all but interleukin (IL)-5, IL-10 and granulocyte-macrophage colony stimulating factor (GM-CSF) were detectable with concentrations of up to 180 pg/mg protein. Local inflammatory irritation selectively increased IL-1beta, IL-6, IL-18, monocyte chemoattractant protein-1 (MCP-1), and growth-related oncogene (GRO/KC) up to 17-fold, and decreased IL-2 and IL-12 (p70) up to threefold. Inflaming the DRG also remarkably increased the incidence of spontaneous activity of A- and C-fibers recorded in the dorsal root. Many of the spontaneously active A-fibers exhibited a short-bursting discharge pattern. Changes in cytokines and spontaneous activity correlated with the time course of pain behaviors, especially light stroke-evoked tactile allodynia. Finally, local inflammation induced extensive sprouting of sympathetic fibers, extending from vascular processes within the inflamed DRG. These results demonstrate the feasibility of inducing chronic localized inflammatory responses in the DRG in the absence of traumatic nerve damage, and highlight the possible contribution of several inflammatory cytokines/chemokines to the generation of spontaneous activity and development and persistence of chemogenic pathologic pain.

Action Potentials↗

PubMed2005

Artificial polypeptide scaffold for protein immobilization.

An artificial polypeptide scaffold composed of surface anchor and protein capture domains was designed and expressed in vivo. By using a mutant E. coli phenylalanyl-tRNA synthetase, the photoreactive amino acid para-azidophenylalanine was incorporated into the surface anchor domain. Octyltrichlorosilane-treated surfaces were functionalized with this polypeptide by spin coating and photocrosslinking. The resulting protein films were shown to immobilize recombinant proteins through association of coiled coil heterodimer.

Amino Acid Sequence↗