Urine screening by Seldi-Tof, followed by biomarker identification, in a Brazilian cohort of patients with renal cell carcinoma (RCC).

PURPOSE: To screen proteins/peptides in urine of Renal Cell Carcinoma (RCC) patients by SELDI-TOF (Surface Enhanced Laser Desorption Ionization - Time of Flight) in search of possible biomarkers. MATERIAL AND METHODS: Sixty-one urines samples from Clear Cell RCC and Papillary RCC were compared to 29 samples of control urine on CM10 chip. Mass analysis was performed in a ProteinChip Reader PCS 4,000 (Ciphergen Biosystems, Fremont, CA) with the software Ciphergen Express 3.0. All chips were read at low and at high laser energy. For statistical analysis the urine samples were clustered according to the histological classification (Clear Cell and Papillary Carcinoma). For identification urine was loaded on a SDS PAGE gel and bands of most interest were excised, trypsinized and identified by MS/MS. Databank searches were performed in Swiss-Prot database using the MASCOT search algorithm and in Profound. RESULTS: Proteins that were identified from urine of controls included immunoglobulin light chains, albumin, secreted and transmembrane 1 precursor (protein K12), mannan-binding lectin-associated serine protease-2 (MASP-2) and vitelline membrane outer layer 1 isoform 1. Identification of immunoglobulins and isoforms of albumin are quite common by proteomics and therefore cannot be considered as possible molecular markers. K12 and MASP-2 play important physiological roles, while vitellite membrane outer layer 1 role is unknown since it was never purified in humans. CONCLUSIONS: The down expression of Protein K-12 and MASP-2 make them good candidates for RCC urine marker and should be validated in a bigger cohort including the other less common histological RCC subtypes.

Alien inhibits E2F1 gene expression and cell proliferation.

Recently, using a proteomic approach we have identified the corepressor Alien as a novel interacting factor of the cell cycle regulator E2F1. Unclear was whether this interaction influences cell proliferation and endogenous E2F1 target gene expression. Here, we show by chromatin immunoprecipitation (ChIP) that Alien is recruited in vivo to the E2F binding sites present in the E2F1 gene promoter, inhibits the transactivation of E2F1 and represses endogenous E2F1 gene expression. Interestingly, using synchronized cells to assess the expression of Alien profile during cell cycle the levels of endogenous Alien are increased during G1, G1/S and G2 phase. Furthermore, stable transfection of Alien leads to reduction of cell proliferation. Thus, the data suggest that Alien acts as a corepressor for E2F1 and is involved in cell cycle regulation.

Identification of HNP3 as a tumour marker in CD4+ and CD4- lymphocytes of patients with cutaneous T-cell lymphoma.

Cutaneous T-cell lymphomas (CTCL) are characterized by malignant proliferation of skin homing T-cells. Although prognosis is generally good, reliable markers are needed to identify patients at risk for a more aggressive course. ProteinChip (SELDI) technology was used as a tool for the discovery of protein patterns in lymphocytes from patients with CTCL (n=25) and unaffected controls (n=25). Lymphocytes were separated in CD4+ and CD4- fractions by magnetic cell sorting (MACS). Each whole protein extract was analysed by ProteinChip technology. The resulting protein profiles were submitted for bioinformatic analysis including a clustering algorithm, a rule extraction, a rating and a rule-base construction step. For the generated combined rule base for the CD4- cell fraction, both the sensitivity and specificity for the prediction of CTCL reached 96%, while for the CD4+ fraction they were 92% and 84%, respectively, for sensitivity and specificity. The most significant peak at 3489Da could be identified as HNP3, an alpha-defensin, by immunocapturing. These results open up both the possibility for the use of this protein signature, especially HNP3, to more effectively monitor and screen CTCL, and the avenue to identify the other relevant peaks for a better understanding of the development of this tumour.

The corepressor activity of Alien is controlled by CREB-binding protein/p300.

The regulation of gene repression by corepressors is a controlled process. Surface-enhanced laser desorption ionization MS proteomic analysis and a yeast two-hybrid screen showed independently that the corepressor Alien interacts with the CREB-binding protein (CBP) coactivator. This interaction was further confirmed by coimmunoprecipitation and glutathione S-transferase pull-down experiments, suggesting that Alien interacts in vivo and in vitro with the histone acetyltransferase (HAT) coactivators CBP and its paralog p300. Acetylation detection experiments indicated that Alien is acetylated in vivo. Furthermore, Alien interacts with the central region of CBP/p300 containing the HAT domain and becomes acetylated in vitro. When an inhibitor of CBP/p300 HAT activity was employed, the Alien-mediated silencing was enhanced. Thus, these findings suggest crosstalk between corepressors and coactivators, and indicate fine-tuning of corepressor function by post-translational modification through corepressor acetylation. STRUCTURED DIGITAL ABSTRACT: p300 binds to Alien α by pull down (View interaction) Alien α physically interacts with CBP by two hybrid (View interaction) Alien α physically interacts with MLK2 by two hybrid (View interaction) p300 acetylates Alien α by acetylation assay (View interaction) Alien α physically interacts with NAP1 by two hybrid (View interaction) Alien α physically interacts with TAFI68 by two hybrid (View interaction) Alien α physically interacts with CBP by anti bait coimmunoprecipitation (View Interaction: 1, 2, 3) Alien α binds to CBP by pull down (View interaction).

Confirmation of the biological significance of transthyretin as a biomarker for cutaneous T-cell lymphoma by its protein interaction partners.

We previously identified transthyretin (TTR) and its posttranslational modifications as a down-regulated marker in mycosis fungoides (MF), a benign subtype of cutaneous T-cell lymphoma (CTCL). In order to more precisely understand the biological role of TTR in the etiology of MF, it is essential to clarify the pathways of progression by identifying further interacting proteins. This study is the first to combine blue native polyacrylamide gel electrophoresis (BN-PAGE) with surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to detect new TTR interaction partners and to determine whether these TTR interaction partners can themselves be used as biomarkers. By this procedure, apolipoprotein A1, which was additionally found to be down-regulated in the serum of MF patients, apolipoprotein A4, retinol binding protein 4 (RBP-4), and retinoid X receptor ß (RXR-ß) were identified as interaction partners of TTR. The RXR family plays a role in cell differentiation and proliferation and is known to be the target of bexarotene, which is used in the treatment of CTCL. In conclusion, the combination of BN-PAGE and SELDI-TOF-MS used in this study allowed for the detection of protein interaction partners, which, in the case of RBP-4 and RXR, indicated a connection between the common tumor marker TTR and tumor progression in CTCL.

S100A8 cellular distribution in normal epithelium, hyperplasia, dysplasia and squamous cell carcinoma and its concentration in serum.

OBJECTIVE: To investigate the function of S100A8, a member of the calcium-binding S100 protein family, in oral tumorigenesis. We analyzed its cellular distribution and its serum level in patients with squamous cell carcinoma and normal controls. STUDY DESIGN: We investigated the histopathologic features by tissue microarrays (TMAs) including 8 normal, 66 hyperplastic and dysplastic and 26 oral squamous cell carcinoma (OSCC) tissue cores. The serum level of S100A8 was measured by an enzyme-linked immunosorbent assay using 33 healthy volunteers, 20 patients with hyperproliferative lesions and 23 patients with OSCC. RESULTS: The TMA analysis resulted in different findings. The strongest expression of S100A8 was found in severe dysplasias and carcinoma in situ. In tumor tissue an increased expression occurred only focally. In the normal tissue cores the epithelium showed a moderate reaction, but basal and parabasal cells were completely negative. The serum levels of S100A8 were marginally reduced in cancer patients. The expression between healthy controls and patients with hyperproliferative lesions displayed no difference. CONCLUSION: The expression of S100A8 is helpful only in the transition from severe dysplastic tissue to cancer.

Comparative proteomic analysis of normal and tumor stromal cells by tissue on chip based mass spectrometry (toc-MS).

In carcinoma tissues, genetic and metabolic changes not only occur at the tumor cell level, but also in the surrounding stroma. This carcinoma-reactive stromal tissue is heterogeneous and consists e.g. of non-epithelial cells such as fibroblasts or fibrocytes, inflammatory cells and vasculature-related cells, which promote carcinoma growth and progression of carcinomas. Nevertheless, there is just little knowledge about the proteomic changes from normal connective tissue to tumor stroma. In the present study, we acquired and analysed specific protein patterns of small stromal sections surrounding head and neck cell complexes in comparison to normal subepithelial connective tissue. To gain defined stromal areas we used laser-based tissue microdissection. Because these stromal areas are limited in size we established the highly sensitive 'tissue on chip based mass spectrometry' (toc-MS). Therefore, the dissected areas were directly transferred to chromatographic arrays and the proteomic profiles were subsequently analysed with mass spectrometry. At least 100 cells were needed for an adequate spectrum. The locating of differentially expressed proteins enables a precise separation of normal and tumor stroma. The newly described toc-MS technology allows an initial insight into proteomic differences between small numbers of exactly defined cells from normal and tumor stroma.

Urine screening by Seldi-Tof, followed by biomarker identification, in a Brazilian cohort of patients with Renal Cell Carcinoma (RCC)

Purpose To screen proteins/peptides in urine of Renal Cell Carcinoma (RCC) patients by SELDI-TOF (Surface Enhanced Laser Desorption Ionization - Time of Flight) in search of possible biomarkers. Material and Methods Sixty-one urines samples from Clear Cell RCC and Papillary RCC were compared to 29 samples of control urine on CM10 chip. Mass analysis was performed in a ProteinChip Reader PCS 4,000 (Ciphergen Biosystems, Fremont, CA) with the software Ciphergen Express 3.0. All chips were read at low and at high laser energy. For statistical analysis the urine samples were clustered according to the histological classification (Clear Cell and Papillary Carcinoma). For identification urine was loaded on a SDS PAGE gel and bands of most interest were excised, trypsinized and identified by MS/MS. Databank searches were performed in Swiss-Prot database using the MASCOT search algorithm and in Profound. Results Proteins that were identified from urine of controls included immunoglobulin light chains, albumin, secreted and transmembrane 1 precursor (protein K12), mannan-binding lectin-associated serine protease-2 (MASP-2) and vitelline membrane outer layer 1 isoform 1. Identification of immunoglobulins and isoforms of albumin are quite common by proteomics and therefore cannot be considered as possible molecular markers. K12 and MASP-2 play important physiological roles, while vitellite membrane outer layer 1 role is unknown since it was never purified in humans. Conclusions The down expression of Protein K-12 and MASP-2 make them good candidates for RCC urine marker and should be validated in a bigger cohort including the other less common histological RCC subtypes. .

Various members of the E2F transcription factor family interact in vivo with the corepressor alien.

Proteins perform their activities in cells by the cooperation within protein complexes. For this reason, it is important to investigate protein-protein interactions to receive insights in physiological processes. A multitude of proteins are involved in the regulation of the cell cycle. Specific key factors participating here are members of the E2F transcription factors. Using an in vivo protein-protein complex detection assay, which comprises mass spectrometric and immunological techniques, we detected a number of known as well as new protein-protein interactions. We describe here for the first time protein complexes containing the corepressor Alien and members of the E2F transcription factor family. Furthermore, we assessed the functional relevance and show a repression of the transcriptional activity of E2F by Alien. Additionally, we detected new interactions that link endogenously expressed Alien with the tumor suppressor retinoblastoma protein (pRB) and with proteins involved in cell cycle regulation.

Posttranslational modifications of transthyretin are serum markers in patients with mycosis fungoides.

Cutaneous T-cell lymphomas (CTCLs) are characterized by the recruitment of malignant T-cell clones, predominantly of the CD4(+) T-helper subpopulation, into the skin. Mycosis fungoides (MF) is the most common type of CTCL and accounts for almost 50% of all primary cutaneous lymphomas. The ProteinChip technology surface-enhanced laser desorption/ionization time of flight/mass spectrometry (SELDI-TOF-MS) was used to detect biomarkers in sera from MF patients (n = 25) and healthy controls (n = 26). Therefore, diluted sera were applied to IMAC30 ProteinChip arrays, and the resulting protein profiles were bioinformatically analyzed. A protein set that distinguishes MF patients from healthy controls with a sensitivity of 82.6% and a specificity of 100% was identified. Four significant peaks were identified by two-dimensional gel electrophoresis, immunodepletion, and SELDI-TOF-MS as transthyretin (TTR) and three TTR modifications. A subsequent enzyme-linked immunosorbent assay confirmed these findings. The ability to detect and identify proteins and protein modifications using SELDI-TOF-MS might reveal a better insight on this kind of disease and may lead to a better understanding and earlier detection of MF patients.

Detection and identification of transcription factors as interaction partners of alien in vivo.

Interacting proteins are often involved in the same cellular processes, and thus the identification of interacting partners of a given protein with unknown function may give insight into the physiological role of this protein. For the detection of protein-protein interactions of the corepressor Alien we used a proteomic approach comprising mass spectrometry and immunological techniques. We assessed solely endogenously expressed proteins. In this study we present for the first time that Alien is interacting within a network of proteins involved in transcriptional regulation, DNA repair, and cell cycle in vivo. In this way we detected protein interactions of Alien involving nucleophosmin, ERCC3, TRIP11, as well as CRSP3.

Protein profiling of oral brush biopsies: S100A8 and S100A9 can differentiate between normal, premalignant, and tumor cells.

In oral mucosa lesions it is frequently difficult to differentiate between precursor lesions and already manifest oral squamous cell carcinoma. Therefore, multiple scalpel biopsies are necessary to detect tumor cells already in early stages and to guarantee an accurate follow-up. We analyzed oral brush biopsies (n = 49) of normal mucosa, inflammatory and hyperproliferative lesions, and oral squamous cell carcinoma with ProteinChip Arrays (SELDI) as a non-invasive method to characterize putative tumor cells. Three proteins were found that differentiated between these three stages. These three proteins are able to distinguish between normal cells and tumor cells with a sensitivity of 100% and specificity of 91% and can distinguish inflammatory/hyperproliferative lesions from tumor cells with a sensitivity of up to 91% and specificity of up to 90%. Two of these proteins have been identified by immunodepletion as S100A8 and S100A9 and this identification was confirmed by immunocytochemistry. For the first time, brush biopsies have been successfully used for proteomic biomarker discovery. The identified protein markers are highly specific for the distinction of the three analyzed stages and therewith reflect the progression from normal to premalignant non-dysplastic and finally to tumor tissue. This knowledge could be used as a first diagnostic step in the monitoring of mucosal lesions.

The tumor suppressors p33ING1 and p33ING2 interact with alien in vivo and enhance alien-mediated gene silencing.

The tumor suppressor p33ING1 is involved in DNA repair and cell cycle regulation. Furthermore, p33ING1 is a transcriptional silencer that recognizes the histone mark for trimethylated lysine 4 at histone H3. Interestingly, expression of p33ING1 and p33ING2 is able to induce premature senescence in primary human fibroblasts. The corepressor Alien is involved in gene silencing mediated by selected members of nuclear hormone receptors. In addition, Alien acts as a corepressor for E2F1, a member of the E2F cell cycle regulatory family. Furthermore, recent findings suggest that Alien is complexed with transcription factors participating in DNA repair and chromatin. Here, using a proteomic approach by surface-enhanced laser desorption ionization and mass spectrometry (SELDI-MS) combined with immunological techniques, we show that Alien interacts in vivo with the tumor suppressor p33ING1 as well as with the related tumor suppressor candidate p33ING2. The interaction of Alien with p33ING1 and p33ING2 was confirmed in vitro with GST-pull-down, suggesting a direct binding of Alien to these factors. The binding domain was mapped to a central region of Alien. Functionally, the expression of p33ING1 or p33ING2 enhances the Alien-mediated silencing, suggesting that the interaction plays a role in transcriptional regulation. Thus, the findings suggest that the identified interaction between Alien and the tumor suppressors p33ING1 and p33ING2 reveals a novel cellular protein network.

Protein profiling of microdissected pancreas carcinoma and identification of HSP27 as a potential serum marker.

BACKGROUND: Patients with pancreatic adenocarcinomas have a poor prognosis because of late clinical manifestation and the tumor's aggressive nature. We used proteomic techniques to search for markers of pancreatic carcinoma. METHODS: We performed protein profiling of microdissected cryostat sections of 9 pancreatic adenocarcinomas and 10 healthy pancreatic tissue samples using ProteinChip technology (surface-enhanced laser desorption/ionization). We identified proteins by use of 2-dimensional gel electrophoresis, peptide fingerprint mapping, and immunodepletion and used immunohistochemistry for in situ localization of the proteins found. We used ELISA to quantify these proteins in preoperative serum samples from 35 patients with pancreatic cancer and 37 healthy individuals. RESULTS: From among the differentially expressed signals that were detected by ProteinChip technology, we identified 2 proteins, DJ-1 and heat shock protein 27 (HSP27). We then detected HSP27 in sera of patients by use of ELISA, indicating a sensitivity of 100% and a specificity of 84% for the recognition of pancreatic cancer. CONCLUSIONS: The detection of DJ-1 and HSP27 in pure defined tissue and the retrieval of HSP27 in serum by antibody-based methods identifies a potential marker for pancreatic cancer.

Detection and identification of protein interactions of S100 proteins by ProteinChip technology.

The aim of this work was to establish an approach for identification of protein interactions. This assay used an anti-S100A8 antibody coupled on beads and incubated with cell extract. The bead eluates were analyzed using ProteinChip technology and subsequently subjected to an appropriate digestion. Molecular masses of digestion fragments were determined by SELDI-MS, and database analysis revealed S100A10 as interacting protein. This result was confirmed by co-immunoprecipitation and immunocapturing. Using S100A10 as new bait, a specific interaction with S100A7 was detectable.