The 14-3-3 proteins constitute a family of highly conserved and broadly expressed multifunctional polypeptides that are involved in a variety of important cellular processes that include cell cycle ...progression, growth, differentiation, and apoptosis. Although the exact cellular function(s) of 14-3-3 proteins is not fully elucidated, as a rule these proteins act by binding to protein ligands, thus regulating their activity; so far more than 300 cellular proteins have been reported to interact with 14-3-3 proteins. Binding to cognate interacting partners is isoform-specific, but redundancy also exists as several binding peptides can be recognized by all isoforms, and some functions can be carried out by any isoform indistinctly. Moreover by interacting with different ligands in a spatially and temporally regulated fashion the same isoform can play multiple possibly even opposing roles where the resultant cellular outcome will be determined by the integration of the various effects. Although there is a large body of literature on specific aspects of 14-3-3 biology, not much is known on the coordinated aspects of 14-3-3 isoform expression, post-translational modifications, and subcellular localization. To address the question of isoform-specific differences, we carried out a comparative analysis of the patterns of expression, phosphorylation, and subcellular localization of the 14-3-3 β, ɛ, σ, τ, and ζ protein isoforms in transformed human amnion (AMA) cells. To validate as well as broaden our observations we analyzed the occurrence of the various isoforms in a large number of established cell lines and mammary and urothelial tissue specimens. Given the systematic approach we undertook and our application of isoform-discriminating technologies to the analysis of various cellular systems, we expect the data presented in this study to serve as an enabling resource for researchers working with 14-3-3 proteins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Invasive apocrine carcinomas (IACs), as defined by morphological features, correspond to 0.3–4% of all invasive ductal carcinomas (IDC), and despite the fact that they are histologically distinct ...from other breast lesions there are currently no standard molecular criteria available for their diagnosis and no unequivocal information as to their prognosis. In an effort to address these concerns we have been using protein expression profiling technologies in combination with mass spectrometry and immunohistochemistry (IHC) to discover specific biomarkers that could allow us to molecularly characterize these lesions as well as to dissect some of the steps in the processes underlying breast apocrine metaplasia and development of precancerous apocrine lesions. Establishing these apocrine-specific markers as best practice for the routine pathology evaluation of breast cancer, however, will require their validation in large cohorts of patients. Towards this goal we have composed a panel of antibodies against components of an apocrine protein signature that includes probes against the apocrine-specific markers 15-prostaglandin dehydrogenase (15-PGDH), and acyl-CoA synthetase medium-chain family member 1 (ACSM1), in addition to a set of categorizing markers that are consistently expressed (AR, CD24) or not expressed (ERα, PgR, Bcl-2, and GATA-3) by apocrine metaplasia in benign breast lesions and apocrine sweat glands. This panel was used to analyze a well-defined cohort consisting of 14 apocrine ductal carcinoma
in situ (ADCIS), and 33 IACs diagnosed at the Cancer Institute Hospital, Tokyo between 1997 and 2001. Samples were originally classified on the basis of cellular morphology with all cases having more than 90% of the tumour cells exhibiting cytological features typical of apocrine cells. Using the expression of 15-PGDH and/or ACSM1 as the main criterion, but taking into account the expression of other markers, we were able to identify unambiguously 13 out of 14 ADCIS (92.9%) and 20 out of 33 (60.6%) IAC samples, respectively, as being of apocrine origin. Our results demonstrate that IACs correspond to a distinct, even if heterogeneous, molecular subgroup of breast carcinomas that can be readily identified in an unbiased way using a combination of markers that recapitulate the phenotype of apocrine sweat glands (15-PGDH
+, ACSM1
+, AR
+, CD24
+, ERα
−, PgR
−, Bcl-2
−, and GATA-3
−). These results pave the way for addressing issues such as prognosis of IACs, patient stratification for targeted therapeutics, as well as research strategies for identifying novel therapeutic targets for developing new cancer therapies.
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FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Breast cancer is a heterogeneous disease that encompasses a wide range of histopathological types including: invasive ductal carcinoma, lobular carcinoma, medullary carcinoma, mucinous carcinoma, ...tubular carcinoma, and apocrine carcinoma among others. Pure apocrine carcinomas represent about 0.5% of all invasive breast cancers according to the Danish Breast Cancer Cooperative Group Registry, and despite the fact that they are morphologically distinct from other breast lesions, there are at present no standard molecular criteria available for their diagnosis. In addition, the relationship between benign apocrine changes and breast carcinoma is unclear and has been a matter of discussion for many years. Recent proteome expression profiling studies of breast apocrine macrocysts, normal breast tissue, and breast tumours have identified specific apocrine biomarkers 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase) present in early and advanced apocrine lesions. These biomarkers in combination with proteins found to be characteristically upregulated in pure apocrine carcinomas (psoriasin, S100A9, and p53) provide a protein expression signature distinctive for benign apocrine metaplasias and apocrine cystic lesions. These studies have also presented compelling evidence for a direct link, through the expression of the prostaglandin degrading enzyme 15-PGDH, between early apocrine lesions and pure apocrine carcinomas. Moreover, specific antibodies against the components of the expression signature have identified precursor lesions in the linear histological progression to apocrine carcinoma. Finally, the identification of proteins that characterize the early stages of mammary apocrine differentiation such as 15-PGDH, HMG-CoA reductase, and cyclooxygenase 2 (COX-2) has opened a window of opportunity for pharmacological intervention, not only in a therapeutic manner but also in a chemopreventive setting. Here we review published and recent results in the context of the current state of research on breast apocrine cancer.
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BFBNIB, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Our limited understanding of the biological impact of the whole spectrum of early breast lesions together with a lack of accurate molecular-based risk criteria for the diagnosis and assignment of ...prognostic significance to biopsy findings presents an important problem in the clinical management of patients harboring precancerous breast lesions. As a result, there is a need to identify biomarkers that can better determine the outcome of early breast lesions by identifying subpopulations of cells in breast premalignant disease that are at high-risk of progression to invasive disease. A first step towards achieving this goal will be to define the molecular phenotypes of the various cell types and precursors – generated by the stem cell hierarchy – that are present in normal and benign conditions of the breast. To date there have been very few systematic proteomic studies aimed at characterizing the phenotypes of the different cell subpopulations present in normal human mammary tissue, partly due to the formidable heterogeneity of mammary tissue, but also due to limitations of the current proteomic technologies. Work in our laboratories has attempted to address in a systematic fashion some of these limitations and here we present our efforts to search for biomarkers using normal fresh tissue from non-neoplastic breast samples. From the data generated by the 2D gel-based proteomic profiling we were able to compile a protein database of normal human breast epithelial tissue that was used to support the biomarker discovery program. We review and present new data on the putative cell-progenitor marker cytokeratin 15 (CK15), and describe a novel marker, dihydropyriminidase-related protein 3 (DRP3) that in combination with CK15 and other well known proteins were used to define molecular phenotypes of normal human breast epithelial cells and their progenitors in resting acini, lactating alveoli, and large collecting ducts of the nipple. Preliminary results are also presented concerning DRP3 positive usual ductal hyperplasias (UDHs) and on single cell layer columnar cells (CCCs). At least two
bona fide biomarkers of undifferentiated ERα/PgR negative luminal cells emerged from these studies, CK15 and c-KIT, which in combination with transformation markers may lead to the establishment of a protein signature able to identify breast precancerous at risk of progressing to invasive disease.
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FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Introduction: Tumor-associated proteins released by cancer cells and by tumor stroma cells, referred as 'cancer secretome', represent a valuable resource for discovery of potential cancer biomarkers. ...The last decade was marked by a great increase in number of studies focused on various aspects of cancer secretome including, composition and identification of components externalized by malignant cells and by the components of tumor microenvironment.
Areas covered: Here, we provide an overview of achievements in the proteomic analysis of the cancer secretome, elicited through the tumor-associated interstitial fluid recovered from malignant tissues ex vivo or the protein component of conditioned media obtained from cultured cancer cells in vitro. We summarize various bioinformatic tools and approaches and critically appraise their outcomes, focusing on problems and challenges that arise when applied for the analysis of cancer secretomic databases.
Expert commentary: Recent achievements in the omics- analysis of structural and metabolic aspects of altered cancer secretome contribute greatly to the various hallmarks of cancer including the identification of clinically significant biomarkers and potential targets for therapeutic intervention.
Discovery‐driven translational research in breast cancer is moving steadily from the study of cell lines to the analysis of clinically relevant samples that, together with the ever increasing number ...of novel and powerful technologies available within genomics, proteomics and functional genomics, promise to have a major impact on the way breast cancer will be diagnosed, treated and monitored in the future. Here we present a brief report on long‐term ongoing strategies at the Danish Centre for Translational Breast Cancer Research to search for markers for early detection and targets for therapeutic intervention, to identify signalling pathways affected in individual tumours, as well as to integrate multiplatform ‘omic’ data sets collected from tissue samples obtained from individual patients. The ultimate goal of this initiative is to coalesce knowledge‐based complementary procedures into a systems biology approach to fight breast cancer.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Proteomics
1
The term proteome was coined by Wilkins et al., Biotech. Genet. Eng. Rev. 13 (1996) 19–50.
1
is an emerging area of research of the post-genomic era that deals with the global analysis ...of gene expression using a plethora of techniques to resolve (high resolution two-dimensional polyacrylamide gel electrophoresis, 2D PAGE), identify (peptide sequencing by Edman degradation, mass spectrometry, Western immunoblotting, etc.), quantitate and characterize proteins, as well as to store (comprehensive 2D PAGE databases), communicate and interlink protein and DNA sequence and mapping information from genome projects. Here we review the current status as well as applications of human and mouse proteomic 2D PAGE databases that are being systematically constructed for the global analysis of gene expression in both health and disease (http://biobase.dk/cgi-bin/celis). Furthermore, we discuss the problems one faces when using powerful proteomic technology to study heterogeneous tissue and tumor biopsies, and emphasize the importance of building comprehensive databases that contain a critical mass of information for both known and novel proteins in normal and disease conditions.
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BFBNIB, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Proteomics and immunohistochemistry were used to reveal tumor heterogeneity among urothelial papillomas (UPs) with the long
term goal of predicting their biological potential in terms of outcome. ...First, we identified proteins that were deregulated
in invasive fresh lesions as compared with normal urothelium, and thereafter we immunostained UPs with a panel of antibodies
against some of the markers. Twenty-two major proteins showing variations of 2-fold or more in at least one-third of the invasive
lesions were selected. Specific antibodies against several of the proteins were obtained, but only a few reacted positively
in immunostaining. A panel consisting of antibodies against keratinocytes (CKs) 5, 13, 18, and 20 and markers of squamous
metaplasia (CKs 7, 8, and 14) was used to probe normal urothelium and 30 UPs collected during a period of five years. Four
UPs showed a normal phenotype, whereas the rest could be grouped in five major types that shared aberrant staining with the
CK20 antibody. Type 1 heterogeneity ( n = 4) showed preferred staining of the umbrella cells with the CK8 antibody. Type 2 ( n = 11) was typified by the staining of the basal and intermediate layers with the CK20 antibody. Type 3 ( n = 7) was characterized by the predominant staining of the basal cell layer with the CK5 antibody. Type 4 ( n = 1) showed areas of CK7 negative cells, whereas type 5 ( n = 3) showed loss of staining of the basal cells with the CK20. 29% of the patients experienced recurrences, but none progressed
to invasive disease. Patients harboring phenotypic alterations in the basal cell compartment (types 3 and 5) showed the highest
number of recurrences (4/7 and 2/3, respectively), and all type 3 lesions progressed to a higher degree of dedifferentiation . Even though a long term prospective study involving a larger sample size is required to assess the biological potential of
these lesions, we believe that this approach will prove instrumental for revealing early phenotypic changes in different types
of cancer.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Decoding of the genome information in terms of regulation and function will be the next great challenge in the life sciences in this millennium and indeed, today we are experiencing a rapid explosion ...of technology for the high throughput expression analysis of genes and their products (functional genomics). In particular, the field of proteomics is booming as proteins are often the functional molecules and represent important targets for the pharmaceutical industry. The proteomic technology is complex, and comprises a plethora of state-of-the-art techniques to resolve, identify and detect their interacting partners, as well as to store and communicate protein information in comprehensive two-dimensional polyacrylamide gel electrophoresis (2D PAGE) databases. Besides annotating the genome, these databases will offer a global approach to the study of gene expression both in health and disease. Here, we review the current status of human 2D PAGE databases that we are systematically constructing for the study of bladder cancer and skin ageing.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Molecular cDNA cloning, two-dimensional gel immunoblotting, and amino acid microsequencing identified three sequence-unique and distinct proteins that constitute a subfamily of ubiquitously expressed ...heterogeneous nuclear ribonucleoproteins corresponding to hnRNPs H, H′, and F. These proteins share epitopes and sequence identity with two other proteins, isoelectric focusing sample spot numbers 2222 (37.6 kDa; pI 6.5) and 2326 (39.5 kDa; pI 6.6), indicating that the subfamily may contain additional members. The identity between hnRNPs H and H′ is 96%, between H and F 78%, and between H′ and F 75%, respectively. The three proteins contain three repeats, which we denote quasi-RRMs (qRRMs) since they have a remote similarity to the RNA recognition motif (RRM). The three qRRMs of hnRNP H, with a few additional NH2-terminal amino acids, were constructed by polymerase chain reaction amplification and used for ribohomopolymer binding studies. Each qRRM repeat bound poly(rG), while only the NH2-terminal qRRM bound poly(rC) and poly(rU). None of the repeats bound detectable amounts of poly(rA). The expression levels of hnRNPs H and F were differentially regulated in pairs of normal and transformed fibroblasts and keratinocytes. In normal human keratinocytes, the expression level of H was unaffected by treatment with several substances tested including two second messengers and seven cytokines. Likewise the expression level of F was independent of these substances, although it was strikingly down-regulated by long term treatment with 4β-phorbol 12-myristate 13-acetate, indicating that the protein kinase C signaling pathway regulates its expression. No effect of 4β-phorbol 12-myristate 13-acetate was observed on the expression of hnRNP H. The genes coding for hnRNPs H, H′, and F were chromosome-mapped to 5q35.3 (HNRPH1), 6q25.3-q26, and/or Xq22 (HNRPH2) and 10q11.21-q11.22 (HNRPF), respectively.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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