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  • 1.
    Ahmadian, Afshin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ren, Z P
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Pontén, F
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Pontén, J
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Genetic instability in the 9q22.3 region is a late event in the development of squamous cell carcinoma.1998In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 17, no 14Article in journal (Refereed)
    Abstract [en]

    Squamous cell carcinoma (SCC) of the skin represents a group of neoplasms which is associated with exposure to UV light. Recently, we obtained data suggesting that invasive skin cancer and its precursors derive from one original neoplastic clone. Here, the analysis were extended by loss of heterozygosity (LOH) analysis in the chromosome 9q22.3 region. A total of 85 samples, taken from twenty-two sections of sun-exposed sites, corresponding to normal epidermis, morphological normal cells with positive immuno-staining for the p53 protein (p53 patches), dysplasias, cancer in situ (CIS) and squamous cell carcinomas (SCC) of the skin were analysed. Overall, about 70% of p53 patches had mutations in the p53 gene but not LOH in the p53 gene or 9q22.3 region. Approximately 70% of the dysplasias showed p53 mutations of which about 40% had LOH in the p53 region but not in the 9q22.3 region. In contrast, about 65% of SCC and CIS displayed LOH in the 9q22.3 region, as well as frequent (80%) mutations and/or LOH in the p53 gene. These findings strongly suggest that alterations in the p53 gene is an early event in the progression towards SCC, whereas malignant development involves LOH and alterations in at least one (or several) tumor suppressor genes located in chromosome 9q22.3.

  • 2. Alvarez-Baron, Claudia P
    et al.
    Jonsson, Philip
    Thomas, Christoforos
    Dryer, Stuart E
    Williams, Cecilia
    University of Houston.
    The two-pore domain potassium channel KCNK5: induction by estrogen receptor alpha and role in proliferation of breast cancer cells.2011In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 25, no 8, 1326-36 p.Article in journal (Refereed)
    Abstract [en]

    The growth of many human breast tumors requires the proliferative effect of estrogen acting via the estrogen receptor α (ERα). ERα signaling is therefore a clinically important target for breast cancer prevention and therapeutics. Although extensively studied, the mechanism by which ERα promotes proliferation remains to be fully established. We observed an up-regulation of transcript encoding the pH-sensitive two-pore domain potassium channel KCNK5 in a screen for genes stimulated by 17β-estradiol (E2) in the ERα(+) breast cancer cell lines MCF-7 and T47D. KCNK5 mRNA increased starting 1 h after the onset of E2 treatment, and protein levels followed after 12 h. Estrogen-responsive elements are found in the enhancer region of KCNK5, and chromatin immunoprecipitation assays revealed binding of ERα to the KCNK5 enhancer in E2-treated MCF-7 cells. Cells treated with E2 also showed increases in the amplitude of pH-sensitive potassium currents, as assessed by whole-cell recordings. These currents are blocked by clofilium. Although confocal microscopy suggested that most of the channels are located in intracellular compartments, the increase in macroscopic currents suggests that E2 treatment increases the number of active channels at the cell surface. Application of small interfering RNA specific for KCNK5 decreased pH-sensitive potassium currents and also reduced the estrogen-induced proliferation of T47D cells. We conclude that E2 induces the expression of KCNK5 via ERα(+) in breast cancer cells, and this channel plays a role in regulating proliferation in these cell lines. KCNK5 may therefore represent a useful target for treatment, for example, of tamoxifen-resistant breast cancer.

  • 3. Arabi, A.
    et al.
    Ullah, K.
    Branca, R. M. M.
    Johansson, J.
    Bandarra, D.
    Haneklaus, M.
    Fu, J.
    Ariës, I.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Den Boer, M. L.
    Pokrovskaja, K.
    Grandér, D.
    Xiao, G.
    Rocha, S.
    Lehtiö, J.
    Sangfelt, O.
    Proteomic screen reveals Fbw7 as a modulator of the NF-kappa B pathway2012In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 3, 976- p.Article in journal (Refereed)
    Abstract [en]

    Fbw7 is a ubiquitin-ligase that targets several oncoproteins for proteolysis, but the full range of Fbw7 substrates is not known. Here we show that by performing quantitative proteomics combined with degron motif searches, we effectively screened for a more complete set of Fbw7 targets. We identify 89 putative Fbw7 substrates, including several disease-associated proteins. The transcription factor NF-κB2 (p100/p52) is one of the candidate Fbw7 substrates. We show that Fbw7 interacts with p100 via a conserved degron and that it promotes degradation of p100 in a GSK3 2 phosphorylation-dependent manner. Fbw7 inactivation increases p100 levels, which in the presence of NF-κB pathway stimuli, leads to increased p52 levels and activity. Accordingly, the apoptotic threshold can be increased by loss of Fbw7 in a p100-dependent manner. In conclusion, Fbw7-mediated destruction of p100 is a regulatory component restricting the response to NF-κB2 pathway stimulation.

  • 4. Aydoğdu, Eylem
    et al.
    Katchy, Anne
    Tsouko, Efrosini
    Lin, Chin-Yo
    Haldosén, Lars-Arne
    Helguero, Luisa
    Williams, Cecilia
    University of Houston.
    MicroRNA-regulated gene networks during mammary cell differentiation are associated with breast cancer.2012In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 33, no 8, 1502-11 p.Article in journal (Refereed)
    Abstract [en]

    MicroRNAs (miRNAs) play pivotal roles in stem cell biology, differentiation and oncogenesis and are of high interest as potential breast cancer therapeutics. However, their expression and function during normal mammary differentiation and in breast cancer remain to be elucidated. In order to identify which miRNAs are involved in mammary differentiation, we thoroughly investigated miRNA expression during functional differentiation of undifferentiated, stem cell-like, murine mammary cells using two different large-scale approaches followed by qPCR. Significant changes in expression of 21 miRNAs were observed in repeated rounds of mammary cell differentiation. The majority, including the miR-200 family and known tumor suppressor miRNAs, was upregulated during differentiation. Only four miRNAs, including oncomiR miR-17, were downregulated. Pathway analysis indicated complex interactions between regulated miRNA clusters and major pathways involved in differentiation, proliferation and stem cell maintenance. Comparisons with human breast cancer tumors showed the gene profile from the undifferentiated, stem-like stage clustered with that of poor-prognosis breast cancer. A common nominator in these groups was the E2F pathway, which was overrepresented among genes targeted by the differentiation-induced miRNAs. A subset of miRNAs could further discriminate between human non-cancer and breast cancer cell lines, and miR-200a/miR-200b, miR-146b and miR-148a were specifically downregulated in triple-negative breast cancer cells. We show that miR-200a/miR-200b can inhibit epithelial-mesenchymal transition (EMT)-characteristic morphological changes in undifferentiated, non-tumorigenic mammary cells. Our studies propose EphA2 as a novel and important target gene for miR-200a. In conclusion, we present evidentiary data on how miRNAs are involved in mammary cell differentiation and indicate their related roles in breast cancer.

  • 5.
    Ayoglu, Burcu
    et al.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Häggmark, Anna
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Neiman, Maja
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Igel, Ulrika
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Schwenk, Jochen
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Systematic antibody and antigen-based proteomic profiling with microarrays2011In: EXPERT REVIEW OF MOLECULAR DIAGNOSTICS, ISSN 1473-7159, Vol. 11, no 2, 219-234 p.Article, review/survey (Refereed)
    Abstract [en]

    Current approaches within affinity-based proteomics are driven both by the accessibility and availability of antigens and capture reagents, and by suitable multiplexed technologies onto which these are implemented. By combining planar microarrays and other multiparallel systems with sets of reagents, possibilities to discover new and unpredicted protein disease associations, either via directed hypothesis-driven or via undirected hypothesis-generating target selection, can be created. In the following stages, the discoveries made during these screening phases have to be verified for potential clinical relevance based on both technical and biological aspects. The use of affinity tools throughout discovery and verification has the potential to streamline the introduction of new markers, as transition into clinically required assay formats appears straightforward. In this article, we summarize some of the current building blocks within array-and affinity-based proteomic profiling with a focus on body fluids, by giving a perspective on how current and upcoming developments in this bioscience could enable a path of pursuit for biomarker discovery.

  • 6. Bidon, Tobias
    et al.
    Janke, Axel
    Fain, Steven R.
    Eiken, Hans Geir
    Hagen, Snorre B.
    Saarma, Urmas
    Hallström, Björn M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lecomte, Nicolas
    Hailer, Frank
    Brown and Polar Bear Y Chromosomes Reveal Extensive Male-Biased Gene Flow within Brother Lineages2014In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 31, no 6, 1353-1363 p.Article in journal (Refereed)
    Abstract [en]

    Brown and polar bears have become prominent examples in phylogeography, but previous phylogeographic studies relied largely on maternally inherited mitochondrial DNA (mtDNA) or were geographically restricted. The male-specific Y chromosome, a natural counterpart to mtDNA, has remained underexplored. Although this paternally inherited chromosome is indispensable for comprehensive analyses of phylogeographic patterns, technical difficulties and low variability have hampered its application in most mammals. We developed 13 novel Y-chromosomal sequence and microsatellite markers from the polar bear genome and screened these in a broad geographic sample of 130 brown and polar bears. We also analyzed a 390-kb-long Y-chromosomal scaffold using sequencing data from published male ursine genomes. Y chromosome evidence support the emerging understanding that brown and polar bears started to diverge no later than the Middle Pleistocene. Contrary to mtDNA patterns, we found 1) brown and polar bears to be reciprocally monophyletic sister (or rather brother) lineages, without signals of introgression, 2) male-biased gene flow across continents and on phylogeographic time scales, and 3) male dispersal that links the Alaskan ABC islands population to mainland brown bears. Due to female philopatry, mtDNA provides a highly structured estimate of population differentiation, while male-biased gene flow is a homogenizing force for nuclear genetic variation. Our findings highlight the importance of analyzing both maternally and paternally inherited loci for a comprehensive view of phylogeographic history, and that mtDNA-based phylogeographic studies of many mammals should be reevaluated. Recent advances in sequencing technology render the analysis of Y-chromosomal variation feasible, even in nonmodel organisms.

  • 7. Biscaia, S. M. P.
    et al.
    Carbonero, E. R.
    Bellan, D. L.
    Borges, B. S.
    Costa, C. R.
    Rossi, G. R.
    Goncalves, J. P.
    Melo, C. M.
    Livero, F. A. R.
    Ruthes, Andrea C.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Zotz, R.
    Silva, E. V.
    Oliveira, C. C.
    Acco, A.
    Nader, H. B.
    Chammas, R.
    Iacomini, M.
    Franco, C. R. C.
    Trindade, E. S.
    Safe therapeutics of murine melanoma model using a novel antineoplasic, the partially methylated mannogalactan from Pleurotus eryngii2017In: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 178, 95-104 p.Article in journal (Refereed)
    Abstract [en]

    A heteropolysaccharide was isolated by cold aqueous extraction from edible mushroom Pleurotus eryngii ("King Oyster") basidiocarps and its biological properties were evaluated. Structural assignments were carried out using mono-and bidimensional NMR spectroscopy, monosaccharide composition, and methylation analyses. A man-nogalactan having a main chain of (1 -> 6)-linked alpha-D-galactopyranosyl and 3-O-methyl-alpha-D-galactopyranosyl residues, both partially substituted at OH-2 by beta-D-Manp (MG-Pe) single-unit was found. Biological effects of mannogalactan from P. eryngii (MG-Pe) were tested against murine melanoma cells. MG-Pe was non-cytotoxic, but reduced in vitro melanoma cells invasion. Also, 50 mg/kg MG-Pe administration to melanoma-bearing C57BL/6 mice up to 10 days decreased in 60% the tumor volume compared to control. Additionally, no changes were observed when biochemical profile, complete blood cells count (CBC), organs, and body weight were analyzed. Mg-Pe was shown to be a promising anti-melanoma molecule capable of switching melanoma cells to a non-invasive phenotype with no toxicity to melanoma-bearing mice.

  • 8.
    Blomfeldt, Thomas O. J.
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Kuktaite, Ramune
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Johansson, Eva
    Hedenqvist, Mikael S.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Mechanical Properties and Network Structure of Wheat Gluten Foams2011In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 12, no 5, 1707-1715 p.Article in journal (Refereed)
    Abstract [en]

    This Article reports the influence of the protein network structure on the mechanical properties of foams produced from commercial wheat gluten using freeze-drying. Foams were produced from alkaline aqueous solutions at various gluten concentrations with or without glycerol, modified with bacterial cellulose nanosized fibers, or both. The results showed that 20 wt % glycerol was sufficient for plasticization, yielding foams with low modulus and high strain recovery. It was found that when fibers were mixed into the foams, a small but insignificant increase in elastic modulus was achieved, and the foam structure became more homogeneous. SEM indicated that the compatibility between the fibers and the matrix was good, with fibers acting as bridges in the cell walls. IR spectroscopy and SE-HPLC revealed a relatively low degree of aggregation, which was highest in the presence of glycerol. Confocal laser scanning microscopy revealed distinct differences in HMW-glutenin subunits and gliadin distributions for all of the different samples.

  • 9. Bondesson, Maria
    et al.
    Hao, Ruixin
    Lin, Chin-Yo
    Williams, Cecilia
    University of Houston, United States.
    Gustafsson, Jan-Åke
    Estrogen receptor signaling during vertebrate development2015In: Biochimica et Biophysica Acta. Gene Regulatory Mechanisms, ISSN 1874-9399, E-ISSN 1876-4320, Vol. 1849, no 2, 142-151 p.Article, review/survey (Refereed)
    Abstract [en]

    Estrogen receptors are expressed and their cognate ligands produced in all vertebrates, indicative of important and conserved functions. Through evolution estrogen has been involved in controlling reproduction, affecting both the development of reproductive organs and reproductive behavior. This review broadly describes the synthesis of estrogens and the expression patterns of aromatase and the estrogen receptors, in relation to estrogen functions in the developing fetus and child. We focus on the role of estrogens for the development of reproductive tissues, as well as non-reproductive effects on the developing brain. We collate data from human, rodent, bird and fish studies and highlight common and species-specific effects of estrogen signaling on fetal development. Morphological malformations originating from perturbed estrogen signaling in estrogen receptor and aromatase knockout mice are discussed, as well as the clinical manifestations of rare estrogen receptor alpha and aromatase gene mutations in humans.

  • 10. Buus, S.
    et al.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Forsström, Björn
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schafer-Nielsen, C.
    High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays2012In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 12, 1790-1800 p.Article in journal (Refereed)
    Abstract [en]

    Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against linear epitopes of the human proteome and obtained very detailed descriptions of the involved specificities. The epitopes identified ranged from 4 to 12 amino acids in size. In general, the antibodies were of exquisite specificity, frequently disallowing even single conservative substitutions. In several cases, multiple distinct epitopes could be identified for the same target protein, suggesting an efficient approach to the generation of paired antibodies. Two alternative epitope mapping approaches identified similar, although not necessarily identical, epitopes. These results show that ultrahigh-density peptide microarrays can be used for linear epitope mapping. With an upper theoretical limit of 2,000,000 individual peptides per array, these peptide microarrays may even be used for a systematic validation of antibodies at the proteomic level.

  • 11.
    Christakou, Athanasia E.
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Solid tumor spheroid formation by temperature-controlled high voltage ultrasound in a multi-well microdevice2014In: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, Chemical and Biological Microsystems SocietyChemical and Biological Microsystems Society , 2014, 573-575 p.Conference paper (Refereed)
    Abstract [en]

    In the present work we demonstrate effective 3D growth of human hepatocellular carcinoma (HCC) HepG2 cell spheroids in parallel in a multi-well microdevice actuated with high voltage ultrasound in a temperature-controlled system. We compare the spheroid formation during continuous ultrasound exposure for one week where we formed spheroids in 59% of the wells, with the spheroid formation without ultrasound actuation, where we obtained 0% spheroids. Furthermore, we present an application of the tumor spheroids for investigating natural killer (NK) cells behavior against solid tumors.

  • 12.
    Dahlberg, Carl
    Department of Solid Mechanics, KTH.
    The Functional Response of Mesenchymal Stem Cells to Electron-Beam Patterend Elastomeric Surfaces Presenting Micrometer to Nanoscale Heterogeneous Rigidity2017In: Advanced Materials, ISSN 0935-9648, E-ISSN 1521-4095Article in journal (Refereed)
  • 13. Dahlman-Wright, Karin
    et al.
    Qiao, Yichun
    Jonsson, Philip
    Gustafsson, Jan-Åke
    Williams, Cecilia
    University of Houston.
    Zhao, Chunyan
    Interplay between AP-1 and estrogen receptor α in regulating gene expression and proliferation networks in breast cancer cells2012In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 33, no 9, 1684-91 p.Article in journal (Refereed)
    Abstract [en]

    Estrogen receptor α (ERα) is a ligand-dependent transcription factor that plays an important role in breast cancer. Estrogen-dependent gene regulation by ERα can be mediated by interaction with other DNA-binding proteins, such as activator protein-1 (AP-1). The nature of such interactions in mediating the estrogen response in breast cancer cells remains unclear. Here we show that knockdown of c-Fos, a component of the transcription factor AP-1, attenuates the expression of 37% of all estrogen-regulated genes, suggesting that c-Fos is a fundamental factor for ERα-mediated transcription. Additionally, knockdown of c-Fos affected the expression of a number of genes that were not regulated by estrogen. Pathway analysis reveals that silencing of c-Fos downregulates an E2F1-dependent proproliferative gene network. Thus, modulation of the E2F1 pathway by c-Fos represents a novel mechanism by which c-Fos enhances breast cancer cell proliferation. Furthermore, we show that c-Fos and ERα can cooperate in regulating E2F1 gene expression by binding to regulatory elements in the E2F1 promoter. To start to dissect the molecular details of the cross talk between AP-1 and estrogen signaling, we identify a novel ERα/AP-1 target, PKIB (cAMP-dependent protein kinase inhibitor-β), which is overexpressed in ERα-positive breast cancer tissues. Knockdown of PKIB results in robust growth suppression of breast cancer cells. Collectively, our findings support c-Fos as a critical factor that governs estrogen-dependent gene expression and breast cancer proliferation programs.

  • 14. Daub, Jennifer
    et al.
    Gardner, Paul P.
    Tate, John
    Ramsköld, Daniel
    KTH.
    Manske, Magnus
    Scott, William G.
    Weinberg, Zasha
    Griffiths-Jones, Sam
    Bateman, Alex
    The RNA WikiProject: Community annotation of RNA families2008In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 14, no 12, 2462-2464 p.Article in journal (Refereed)
    Abstract [en]

    The online encyclopedia Wikipedia has become one of the most important online references in the world and has a substantial and growing scientific content. A search of Google with many RNA-related keywords identifies a Wikipedia article as the top hit. We believe that the RNA community has an important and timely opportunity to maximize the content and quality of RNA information in Wikipedia. To this end, we have formed the RNA WikiProject (http://en.wikipedia.org/wiki/Wikipedia: WikiProject_RNA) as part of the larger Molecular and Cellular Biology WikiProject. We have created over 600 new Wikipedia articles describing families of noncoding RNAs based on the Rfam database, and invite the community to update, edit, and correct these articles. The Rfam database now redistributes this Wikipedia content as the primary textual annotation of its RNA families. Users can, therefore, for the first time, directly edit the content of one of the major RNA databases. We believe that this Wikipedia/Rfam link acts as a functioning model for incorporating community annotation into molecular biology databases.

  • 15.
    Dekki Shalaly, Nancy
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ria, Massimiliano
    Johansson, Ulrika
    KTH, School of Biotechnology (BIO), Protein Technology.
    Avall, Karin
    Berggren, Per-Olof
    Hedhammar, My
    KTH, School of Biotechnology (BIO), Protein Technology.
    Silk matrices promote formation of insulin-secreting islet-like clusters2016In: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 90, 50-61 p.Article in journal (Refereed)
    Abstract [en]

    Ex vivo expansion of endocrine cells constitutes an interesting alternative to be able to match the unmet need of transplantable pancreatic islets. However, endocrine cells become fragile once removed from their extracellular matrix (ECM) and typically become senescent and loose insulin expression during conventional 2D culture. Herein we develop a protocol where 3D silk matrices functionalized with ECM derived motifs are used for generation of insulin-secreting islet-like clusters from mouse and human primary cells. The obtained clusters were shown to attain an islet-like spheroid shape and to maintain functional insulin release upon glucose stimulation in vitro. Furthermore, in vivo imaging of transplanted murine clusters showed engraftment with increasing vessel formation during time. There was no sign of cell death and the clusters maintained or increased in size throughout the period, thus suggesting a suitable cluster size for transplantation.

  • 16. Dey, Prasenjit
    et al.
    Jonsson, Philip
    Hartman, Johan
    Williams, Cecilia
    Ström, Anders
    Gustafsson, Jan-Åke
    Estrogen receptors β1 and β2 have opposing roles in regulating proliferation and bone metastasis genes in the prostate cancer cell line PC32012In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 26, no 12, 1991-2003 p.Article in journal (Refereed)
    Abstract [en]

    The estrogen receptor (ER)β1 is successively lost during cancer progression, whereas its splice variant, ERβ2, is expressed in advanced prostate cancer. The latter form of cancer often metastasizes to bone, and we wanted to investigate whether the loss of ERβ1 and/or the expression of ERβ2 affect such signaling pathways in prostate cancer. Using PC3 and 22Rv1 prostate cancer cell lines that stably express ERβ1 or ERβ2, we found that the ERβ variants differentially regulate genes known to affect tumor behavior. We found that ERβ1 repressed the expression of the bone metastasis regulator Runx2 in PC3 cells. By contrast, RUNX2 expression was up-regulated at the mRNA level by ERβ2 in PC3 cells, whereas Slug was up-regulated by ERβ2 in both PC3 and 22Rv1 cells. In addition, the expression of Twist1, a factor whose expression strongly correlates with high Gleason grade prostate carcinoma, was increased by ERβ2. In agreement with the increased Twist1 expression, we found increased expression of Dickkopf homolog 1; Dickkopf homolog 1 is a factor that has been shown to increase the RANK ligand/osteoprotegerin ratio and enhance osteoclastogenesis, indicating that the expression of ERβ2 can cause osteolytic cancer. Furthermore, we found that only ERβ1 inhibited proliferation, whereas ERβ2 increased proliferation. The expression of the proliferation markers Cyclin E, c-Myc, and p45(Skp2) was differentially affected by ERβ1 and ERβ2 expression. In addition, nuclear β-catenin protein and its mRNA levels were reduced by ERβ1 expression. In conclusion, we found that ERβ1 inhibited proliferation and factors known to be involved in bone metastasis, whereas ERβ2 increased proliferation and up-regulated factors involved in bone metastasis. Thus, in prostate cancer cells, ERβ2 has oncogenic abilities that are in strong contrast to the tumor-suppressing effects of ERβ1.

  • 17. Dória, M Luisa
    et al.
    Ribeiro, Ana S
    Wang, Jun
    Cotrim, Cândida Z
    Domingues, Pedro
    Williams, Cecilia
    University of Houston.
    Domingues, M Rosário
    Helguero, Luisa A
    Fatty acid and phospholipid biosynthetic pathways are regulated throughout mammary epithelial cell differentiation and correlate to breast cancer survival2014In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 28, no 10, 4247-64 p.Article in journal (Refereed)
    Abstract [en]

    This work combined gene and protein expression, gas chromatography-flame ionization detector, and hydrophilic interaction liquid chromatography-tandem mass spectrometry to compare lipid metabolism changes in undifferentiated/proliferating vs. functionally differentiated mammary epithelial cells (MECs) and to study their correlation to breast cancer survival. Sixty-eight genes involved in lipid metabolism were changed in MEC differentiation. Differentiated cells showed induction of Elovl6 (2-fold), Scd1 (4-fold), and Fads2 (2-fold), which correlated with increased levels of C16:1 n-7 and C18:1 n-9 (1.5-fold), C20:3 n-6 (2.5-fold), and C20:4 n-6 (6-fold) fatty acids (FAs) and more phospholipids (PLs) containing these species. Further, increased expression (2- to 3-fold) of genes in phosphatidylethanolamine (PE) de novo biosynthesis resulted in a 20% PE increase. Proliferating/undifferentiated cells showed higher C16:0 (1.7-fold) and C18:2 n-6 (4.2-fold) levels and more PLs containing C16:0 FAs [PC(16:0/16:1), PG(16:0/18:2), PG(16:0/18:1), and SM(16:0/18:0)]. Kaplan-Meier analysis of data from 3455 patients with breast cancer disclosed a positive correlation for 59% of genes expressed in differentiated MECs with better survival. PE biosynthesis and FA oxidation correlated with better prognosis in patients with breast cancer, including the basal-like subtype. Therefore, genes involved in mammary gland FA and PL metabolism and their resulting molecular species reflect the cellular proliferative ability and differentiation state and deserve further studies as potential markers of breast cancer progression

  • 18. Edvardsson, Karin
    et al.
    Nguyen-Vu, Trang
    Kalasekar, Sharanya M
    Pontén, Fredrik
    Gustafsson, Jan-Åke
    Williams, Cecilia
    University of Houston.
    Estrogen receptor β expression induces changes in the microRNA pool in human colon cancer cells2013In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 34, no 7, 1431-41 p.Article in journal (Refereed)
    Abstract [en]

    There is epidemiological, animal and in vitro evidence that estrogen receptor β (ERβ) can mediate protective effects against colon cancer, but the mechanism is not completely understood. Previous research has indicated critical pathways whereby ERβ acts in an antitumorigenic fashion. In this study, we investigate ERβ's impact on the microRNA (miRNA) pool in colon cancer cells using large-scale genomic approaches, bioinformatics and focused functional studies. We detect and confirm 27 miRNAs to be significantly changed following ERβ expression in SW480 colon cancer cells. Among these, the oncogenic miR-17-92 cluster and miR-200a/b are strongly downregulated. Using target prediction and anticorrelation to gene expression data followed by focused mechanistic studies, we demonstrate that repression of miR-17 is a secondary event following ERβ's downregulatory effect on MYC. We show that re-introduction of miR-17 can reverse the antiproliferative effects of ERβ. The repression of miR-17 also influences cell death upon DNA damage and mediates regulation of NCOA3 (SRC-3) and CLU in colon cancer cells. We further determine that the downregulation of miR-200a/b mediates increased ZEB1 while decreasing E-cadherin levels in ERβ-expressing colon cancer cells. Changes in these genes correspond to significant alterations in morphology and migration. Our work contributes novel data of ERβ and miRNA in the colon. Elucidating the mechanism of ERβ and biomarkers of its activity has significant potential to impact colon cancer prevention and treatment.

  • 19. Edvardsson, Karin
    et al.
    Ström, Anders
    Jonsson, Philip
    Gustafsson, Jan-Åke
    Williams, Cecilia
    University of Houston.
    Estrogen receptor β induces antiinflammatory and antitumorigenic networks in colon cancer cells.2011In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 25, no 6, 969-79 p.Article in journal (Refereed)
    Abstract [en]

    Several studies suggest estrogen to be protective against the development of colon cancer. Estrogen receptor β (ERβ) is the predominant estrogen receptor expressed in colorectal epithelium and is the main candidate to mediate the protective effects. We have previously shown that expression of ERβ reduces growth of colorectal cancer in xenografts. Little is known of the actions of ERβ and its effect on gene transcription in colon cancers. To dissect the processes that ERβ mediates and to investigate cell-specific mechanisms, we reexpressed ERβ in three colorectal cancer cell lines (SW480, HT29, and HCT-116) and conducted genome-wide expression studies in combination with gene-pathway analyses and cross-correlation to ERβ-chromatin-binding sites. Although induced gene regulation was cell specific, overrepresentation analysis of functional classes indicated that the same biological themes, including apoptosis, cell differentiation, and regulation of the cell cycle, were affected in all three cell lines. Novel findings include a strong ERβ-mediated down-regulation of IL-6 and downstream networks with significant implications for inflammatory mechanisms involved in colon carcinogenesis. We also discovered cross talk between the suggested nuclear receptor coregulator PROX1 and ERβ, demonstrating that ERβ both regulates and shares target genes with PROX1. The influence of ERβ on apoptosis was further explored using functional studies, which suggested an increased DNA-repair capacity. We conclude that reexpression of ERβ induces transcriptome changes that, through several parallel pathways, converge into antitumorigenic capabilities in all three cell lines. We propose that enhancing ERβ action has potential as a novel therapeutic approach for prevention and/or treatment of colon cancer.

  • 20. Ehrlund, Anna
    et al.
    Jonsson, Philip
    Vedin, Lise-Lotte
    Williams, Cecilia
    University of Houston.
    Gustafsson, Jan-Åke
    Treuter, Eckardt
    Knockdown of SF-1 and RNF31 affects components of steroidogenesis, TGFβ, and Wnt/β-catenin signaling in adrenocortical carcinoma cells.2012In: PloS one, ISSN 1932-6203, Vol. 7, no 3, e32080- p.Article in journal (Refereed)
    Abstract [en]

    The orphan nuclear receptor Steroidogenic Factor-1 (SF-1, NR5A1) is a critical regulator of development and homeostasis of the adrenal cortex and gonads. We recently showed that a complex containing E3 ubiquitin ligase RNF31 and the known SF-1 corepressor DAX-1 (NR0B1) interacts with SF-1 on target promoters and represses transcription of steroidogenic acute regulatory protein (StAR) and aromatase (CYP19) genes. To further evaluate the role of SF-1 in the adrenal cortex and the involvement of RNF31 in SF-1-dependent pathways, we performed genome-wide gene-expression analysis of adrenocortical NCI-H295R cells where SF-1 or RNF31 had been knocked down using RNA interference. We find RNF31 to be deeply connected to cholesterol metabolism and steroid hormone synthesis, strengthening its role as an SF-1 coregulator. We also find intriguing evidence of negative crosstalk between SF-1 and both transforming growth factor (TGF) β and Wnt/β-catenin signaling. This crosstalk could be of importance for adrenogonadal development, maintenance of adrenocortical progenitor cells and the development of adrenocortical carcinoma. Finally, the SF-1 gene profile can be used to distinguish malignant from benign adrenocortical tumors, a finding that implicates SF-1 in the development of malignant adrenocortical carcinoma.

  • 21. Feliu, Neus
    et al.
    Walter, Marie Valérie
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Montañez, Maria I.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Kunzmann, Andrea
    Hult, Anders
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Nyström, Andreas
    Malkoch, Michael
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Fadeel, Bengt
    Stability and biocompatibility of a library of polyester dendrimers in comparison to polyamidoamine dendrimers2012In: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 33, no 7, 1970-1981 p.Article in journal (Refereed)
    Abstract [en]

    Dendrimers can be designed for several biomedical applications due to their well-defined structure, functionality and dimensions. The present study focused on the in vitro biocompatibility evaluation of a library of aliphatic polyester dendrimers based on 2,2-bis(methylol)propionic acid (bis-MPA) with an overall diameter of 0.5-2 nm. In addition, dendrimers with two different chemical surfaces (neutral with hydroxyl end group and anionic with carboxylic end group) and dendrons corresponding to the structural fragments of the dendrimers were evaluated. Commercial polyamidoamine dendrimers (PAMAM) with cationic (amine) or neutral (hydroxyl) end group were also included for comparison. Cell viability studies were conducted in human cervical cancer (HeLa) and acute monocytic leukemia cells (THP.1) differentiated into macrophage-like cells as well as in primary human monocyte-derived macrophages. Excellent biocompatibility was observed for the entire hydroxyl functional bis-MPA dendrimer library, whereas the cationic, but not the neutral PAMAM exerted dose-dependent cytotoxicity in cell lines and primary macrophages. Studies to evaluate material stability as a function of pH, temperature, and time, demonstrated that the stability of the 4th generation hydroxyl functional bis-MPA dendrimer increased at acidic pH. Taken together, bis-MPA dendrimers are degradable and non-cytotoxic to human cell lines and primary cells.

  • 22.
    Fontana, Jacopo
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fritz, Nicolas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aperia, Anita
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Apoptosis caused by excessive mitochondrial albumin uptake in renal cells is initiated by increased mitochondrial calcium concentration2015In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 29Article in journal (Other academic)
  • 23.
    Fritz, Nicolas
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Akkuratov, Evgeny
    KTH.
    Liebmann, Thomas
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lindskog, Maria
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aperia, Anita
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Functional consequences of the disease-causing T613M mutation of NKAalpha3 for hippocampal neurons2015In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 29Article in journal (Other academic)
  • 24.
    Gantelius, Jesper
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Bass, Tarek
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Sjöberg, Ronald
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays2011In: International Journal of Molecular Sciences, ISSN 1661-6596, Vol. 12, no 11, 7748-7759 p.Article in journal (Refereed)
    Abstract [en]

    Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<30 ng/mL) determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.

  • 25. Grunewald, J.
    et al.
    Kaiser, Y.
    Ostadkarampour, M.
    Rivera, N. V.
    Vezzi, F.
    Lötstedt, B.
    Olsen, R. -A
    Sylwan, L.
    Lundin, Sverker
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sandalova, T.
    Ahlgren, K. M.
    Wahlström, J.
    Achour, A.
    Ronninger, M.
    Eklund, A.
    T-cell receptor-HLA-DRB1 associations suggest specific antigens in pulmonary sarcoidosis2016In: European Respiratory Journal, ISSN 0903-1936, E-ISSN 1399-3003, Vol. 47, no 3, 898-909 p.Article in journal (Refereed)
    Abstract [en]

    In pulmonary sarcoidosis, CD4+; T-cells expressing T-cell receptor Vα2.3 accumulate in the lungs of HLA-DRB1∗03+; patients. To investigate T-cell receptor-HLA-DRB1∗03 interactions underlying recognition of hitherto unknown antigens, we performed detailed analyses of T-cell receptor expression on bronchoalveolar lavage fluid CD4+; T-cells from sarcoidosis patients. Pulmonary sarcoidosis patients (n=43) underwent bronchoscopy with bronchoalveolar lavage. T-cell receptor α and β chains of CD4+; T-cells were analysed by flow cytometry, DNA-sequenced, and threedimensional molecular models of T-cell receptor-HLA-DRB1∗03 complexes generated. Simultaneous expression of Vα2.3 with the Vβ22 chain was identified in the lungs of all HLADRB1∗ 03+; patients. Accumulated Vα2.3/Vβ22-expressing T-cells were highly clonal, with identical or near-identical Vα2.3 chain sequences and inter-patient similarities in Vβ22 chain amino acid distribution. Molecular modelling revealed specific T-cell receptor-HLA-DRB1∗03-peptide interactions, with a previously identified, sarcoidosis-associated vimentin peptide, (Vim)429-443 DSLPLVDTHSKRTLL, matching both the HLA peptide-binding cleft and distinct T-cell receptor features perfectly. We demonstrate, for the first time, the accumulation of large clonal populations of specific Vα2.3/Vβ22 T-cell receptor-expressing CD4+; T-cells in the lungs of HLA-DRB1∗03+; sarcoidosis patients. Several distinct contact points between Vα2.3/Vβ22 receptors and HLA-DRB1∗03 molecules suggest presentation of prototypic vimentin-derived peptides.

  • 26. Gunnarson, E.
    et al.
    Axehult, G.
    Baturina, G.
    Zelenin, S.
    Zelenina, Marina
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Aperia, A.
    Lead induces increased water permeability in astrocytes expressing aquaporin 42005In: Neuroscience, ISSN 0306-4522, E-ISSN 1873-7544, Vol. 136, no 1, 105-114 p.Article in journal (Refereed)
    Abstract [en]

    The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes. There is now compelling evidence that AQP4 may contribute to an unfavorable course in brain edema. Acute lead intoxication is a condition that causes brain damage preceded by brain edema. Here we report that lead increases AQP4 water permeability (P-f) in astrocytes. A rat astrocyte cell line that does not express aquaporin 4 was transiently transfected with aquaporin 4 tagged with green fluorescent protein (GFP). Using confocal laser scanning microscopy we measured water permeability in these cells and in AQP4-negative cells located on the same plate. AQP4-expressing astrocytes had a three-fold higher water permeability than astrocytes not expressing AQP4. Lead exposure induced a significant, 40%, increase in water permeability in astrocytes expressing AQP4, but had no effect on Pf in astrocytes not expressing AQP4. The increase in water permeability persisted after lead washout, while treatment with a lead chelator, meso-2,3-dimercaptosuccinic acid, abolished the lead-induced increase in Pf. The effect of lead was attenuated in the presence of a calcium (Ca2+)/ calmodulin-dependent protein kinase 11 (CaMKII) inhibitor, but not in the presence of a protein kinase C inhibitor. In cells expressing AQP4 where the consensus site for CaMKII phosphorylation was mutated, lead failed to increase water permeability. Lead exposure also increased Pf in rat astroglial cells in primary culture, which express endogenous AQP4. Lead had no effect on Pf in astrocytes transfected with aquaporin 3. In situ hybridization studies on rat brain after oral lead intake for three days showed no change in distribution of AQP4 mRNA. It is suggested that lead-triggered stimulation of water transport in AQP4-expressing astrocytes may contribute to the pathology of acute lead intoxication.

  • 27. Gunnarson, Eli
    et al.
    Song, Yutong
    Kowalewski, Jacob M
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Brines, Michael
    Cerami, Anthony
    Andersson, Ulf
    Zelenina, Marina
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Aperia, Anita
    Erythropoietin modulation of astrocyte water permeability as a component of neuroprotection.2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 1091-6490, Vol. 106, no 5, 1602-7 p.Article in journal (Refereed)
    Abstract [en]

    Disturbed brain water homeostasis with swelling of astroglial cells is a common complication in stroke, trauma, and meningitis and is considered to be a major cause of permanent brain damage. Astroglial cells possess the water channel aquaporin 4 (AQP4). Recent studies from our laboratory have shown that glutamate, acting on group I metabotropic glutamate receptors (mGluRs), increases the permeability of astrocyte AQP4, which, in situations of hypoxia-ischemia, will increase astrocyte water uptake. Here we report that erythropoietin (EPO), which in recent years has emerged as a potent neuro-protective agent, antagonizes the effect of a group I mGluR agonist on astrocyte water permeability. Activation of group I mGluRs triggers fast and highly regular intracellular calcium oscillations and we show that EPO interferes with this signaling event by altering the frequency of the oscillations. These effects of EPO are immediate, in contrast to the neuroprotective effects of EPO that are known to depend upon gene activation. Our findings indicate that EPO may directly reduce the risk of astrocyte swelling in stroke and other brain insults. In support of this conclusion we found that EPO reduced the neurological symptoms in a mouse model of primary brain edema known to depend upon AQP4 water transport.

  • 28. Gunnarson, Eli
    et al.
    Zelenina, Marina
    Axehult, Gustav
    Song, Yutong
    Bondar, Alexander
    Krieger, Patrik
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Zelenin, Sergey
    Aperia, Anita
    Identification of a molecular target for glutamate regulation of astrocyte water permeability2008In: Glia, ISSN 0894-1491, E-ISSN 1098-1136, Vol. 56, no 6, 587-596 p.Article in journal (Refereed)
    Abstract [en]

    Astrocytes play a key role for maintenance of brain water homeostasis, but little is known about mechanisms of short-term regulation of astrocyte water permeability. Here, we report that glutamate increases astrocyte water permeability and that the molecular target for this effect is the aquaporin-4 (AQP4) serine 111 residue, which is in a strategic position for control of the water channel gating. The glutamate effect involves activation of group I metabotropic glutamate receptors (mGluR), intracellular calcium release, and activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and nitric oxide synthase (NOS). The physiological impact of our results is underlined by the finding that mGluR activation increases the rate of hypoosmotic tissue swelling in acute rat hippocampal slices. Cerebral ischemia is associated with an excessive release of glutamate, and in postischemic cerebral edema ablation of AQP4 attenuates the degree of damage. Thus, we have identified AQP4 as the molecular target for drugs that may attenuate the development of brain edema.

  • 29. Gustafsson, JK
    et al.
    Ermund, Anna
    Ambort, Daniel
    Johansson, MEV
    Nilsson, HE
    Thorell, K
    Hebert, Hans
    Karolinska Institutet, Sweden .
    Sjövall, H
    Hansson, Gunnar
    Bicarbonate and functional CFTR channel are required for proper mucin secretion and link cystic fibrosis with its mucus phenotype2012In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 209, no 7, 1263-1272 p.Article in journal (Refereed)
    Abstract [en]

    Cystic fibrosis (CF) is caused by a nonfunctional chloride and bicarbonate ion channel (CF transmembrane regulator [CFTR]), but the link to the phenomenon of stagnant mucus is not well understood. Mice lacking functional CFTR (CftrΔ508) have no lung phenotype but show similar ileal problems to humans. We show that the ileal mucosa in CF have a mucus that adhered to the epithelium, was denser, and was less penetrable than that of wild-type mice. The properties of the ileal mucus of CF mice were normalized by secretion into a high concentration sodium bicarbonate buffer (~100 mM). In addition, bicarbonate added to already formed CF mucus almost completely restored the mucus properties. This knowledge may provide novel therapeutic options for CF.

  • 30. Hartman, Johan
    et al.
    Edvardsson, Karin
    Lindberg, Karolina
    Zhao, Chunyan
    Williams, Cecilia
    Karolinska Institutet.
    Ström, Anders
    Gustafsson, Jan-Åke
    Tumor repressive functions of estrogen receptor beta in SW480 colon cancer cells2009In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 69, no 15, 6100-6 p.Article in journal (Refereed)
    Abstract [en]

    Estrogen receptor beta (ERbeta) is the predominant ER in the colorectal epithelium. Compared with normal colon tissue, ERbeta expression is reduced in colorectal cancer. Our hypothesis is that ERbeta inhibits proliferation of colon cancer cells. Hence, the aim of this study has been to investigate the molecular function of ERbeta in colon cancer cells, focusing on cell cycle regulation. SW480 colon cancer cells have been lentivirus transduced with ERbeta expression construct with or without mutated DNA-binding domain or an empty control vector. Expression of ERbeta resulted in inhibition of proliferation and G(1) phase cell cycle arrest and this effect was dependent on a functional DNA-binding region. c-Myc is overexpressed in an overwhelming majority of colorectal tumors. By Western blot and real-time PCR, we found c-Myc to be down-regulated in the ERbeta-expressing cells. Furthermore, the c-Myc target gene p21((Waf1/Cip1)) was induced and Cdc25A was reduced by ERbeta at the transcriptional level. The second cdk2-inhibitor, p27(Kip1), was induced by ERbeta, but this regulation occurred at the posttranscriptional level, probably through ERbeta-mediated repression of the F-box protein p45(Skp2). Expression of the ERbeta-variant with mutated DNA binding domain resulted in completely different cell cycle gene regulation. We performed in vivo studies with SW480 cells +/- ERbeta transplanted into severe combined immunodeficient/beige mice; after three weeks of ERbeta-expression, a 70% reduction of tumor volume was seen. Our results show that ERbeta inhibits proliferation as well as colon cancer xenograft growth, probably as a consequence of ERbeta-mediated inhibition of cell-cycle pathways. Furthermore, this ERbeta-mediated cell cycle repression is dependent on functional ERE binding.

  • 31.
    Hedhammar, My
    KTH, School of Biotechnology (BIO), Proteomics.
    Strategies for facilitated protein recovery after recombinant production in Escherichia coli2005Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The successful genomic era has resulted in a great demand for efficient production and purification of proteins. The main objective of the work described in this thesis was to develop methods to facilitate recovery of target proteins after recombinant production in Escherichia coli.

    A positively charged purification tag, Zbasic, has previously been constructed by protein design of a compact three-helix bundle domain, Z. The charged domain was investigated for general use as a fusion partner. All target proteins investigated could be selectively captured by ion-exchange chromatography under conditions excluding adsorption of the majority of Escherichia coli host proteins. A single cation-exchange chromatography step at physiological pH was sufficient to provide Zbasic fusion proteins of high purity close to homogeneity. Moreover, efficient isolation directly from unclarified Escherichia coli homogenates could also be accomplished using an expanded bed mode. Since the intended use of a recombinant protein sometimes requires removal of the purification tag, a strategy for efficient release of the Zbasic moiety using an immobilised protease was developed. The protease columns were reusable without any measurable decrease in activity. Moreover, subsequent removal of the released tag, Zbasic, was effected by adsorption to a second cation-exchanger.

    Using a similar strategy, a purification tag with a negatively charged surface, denoted Zacid, was constructed and thoroughly characterised. Contrary to Zbasic, the negatively charged Zacid was highly unstructured in a low conductivity environment. Despite this, all Zacid fusion proteins investigated could be efficiently purified from whole cell lysates using anion-exchange chromatography

    Synthesis of polypeptides occurs readily in Escherichia coli providing large amounts of protein in cells of this type, albeit often one finds the recombinant proteins sequestered in inclusion bodies. Therefore, a high throughput method for screening of protein expression was developed. Levels of both soluble and precipitated protein could simultaneously be assessed in vivo by the use of a flow cytometer.

    The positively charged domain, Zbasic, was shown also to be selective under denaturing conditions, providing the possibility to purify proteins solubilised from inclusion bodies. Finally, a flexible process for solid-phase refolding was developed, using Zbasic as a reversible linker to the cation-exchanger resin.

  • 32. Jakobsen, Lis
    et al.
    Vanselow, Katja
    Skogs, Marie
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Toyoda, Yusuke
    Lundberg, Emma
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Poser, Ina
    Falkenby, Lasse G.
    Bennetzen, Martin
    Westendorf, Jens
    Nigg, Erich A.
    Uhlen, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Hyman, Anthony A.
    Andersen, Jens S.
    Novel asymmetrically localizing components of human centrosomes identified by complementary proteomics methods2011In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 30, no 8, 1520-1535 p.Article in journal (Refereed)
    Abstract [en]

    Centrosomes in animal cells are dynamic organelles with a proteinaceous matrix of pericentriolar material assembled around a pair of centrioles. They organize the microtubule cytoskeleton and the mitotic spindle apparatus. Mature centrioles are essential for biogenesis of primary cilia that mediate key signalling events. Despite recent advances, the molecular basis for the plethora of processes coordinated by centrosomes is not fully understood. We have combined protein identification and localization, using PCP-SILAC mass spectrometry, BAC transgeneOmics, and antibodies to define the constituents of human centrosomes. From a background of non-specific proteins, we distinguished 126 known and 40 candidate centrosomal proteins, of which 22 were confirmed as novel components. An antibody screen covering 4000 genes revealed an additional 113 candidates. We illustrate the power of our methods by identifying a novel set of five proteins preferentially associated with mother or daughter centrioles, comprising genes implicated in cell polarity. Pulsed labelling demonstrates a remarkable variation in the stability of centrosomal protein complexes. These spatiotemporal proteomics data provide leads to the further functional characterization of centrosomal proteins.

  • 33. Jonsson, Philip
    et al.
    Coarfa, Cristian
    Mesmar, Fahmi
    Raz, Tal
    Rajapakshe, Kimal
    Thompson, John F
    Gunaratne, Preethi H
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Single-Molecule Sequencing Reveals Estrogen-Regulated Clinically Relevant lncRNAs in Breast Cancer2015In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 29, no 11Article in journal (Refereed)
    Abstract [en]

    Estrogen receptor (ER)α-positive tumors are commonly treated with ERα antagonists or inhibitors of estrogen synthesis, but most tumors develop resistance, and we need to better understand the pathways that underlie the proliferative and tumorigenic role of this estrogen-activated transcription factor. We here present the first single-molecule sequencing of the estradiol-induced ERα transcriptome in the luminal A-type human breast cancer cell lines MCF7 and T47D. Sequencing libraries were prepared from the polyadenylated RNA fraction after 8 hours of estrogen or vehicle treatment. Single-molecule sequencing was carried out in biological and technical replicates and differentially expressed genes were defined and analyzed for enriched processes. Correlation analysis with clinical expression and survival were performed, and follow-up experiments carried out using time series, chromatin immunoprecipitation and quantitative real-time PCR. We uncovered that ERα in addition to regulating approximately 2000 protein-coding genes, also regulated up to 1000 long noncoding RNAs (lncRNAs). Most of these were up-regulated, and 178 lncRNAs were regulated in both cell lines. We demonstrate that Long Intergenic Non-protein Coding RNA 1016 (LINC01016) and LINC00160 are direct transcriptional targets of ERα, correlate with ERα expression in clinical samples, and show prognostic significance in relation to breast cancer survival. We show that silencing of LINC00160 results in reduced proliferation, demonstrating that lncRNA expression have functional consequences. Our findings suggest that ERα regulation of lncRNAs is clinically relevant and that their functions and potential use as biomarkers for endocrine response are important to explore.

  • 34. Jonsson, Philip
    et al.
    Katchy, Anne
    Williams, Cecilia
    Support of a bi-faceted role of estrogen receptor β (ERβ) in ERα-positive breast cancer cells.2014In: Endocrine-Related Cancer, ISSN 1351-0088, E-ISSN 1479-6821, Vol. 21, no 2, 143-160 p.Article in journal (Refereed)
    Abstract [en]

    The expression of estrogen receptor α (ERα) in breast cancer identifies patients most likely to respond to endocrine treatment. The second ER, ERβ, is also expressed in breast tumors, but its function and therapeutic potential need further study. Although in vitro studies have established that ERβ opposes transcriptional and proliferative functions of ERα, several clinical studies report its correlation with proliferative markers and poorer prognosis. The data demonstrate that ERβ opposes ERα are primarily based on transient expression of ERβ. Here, we explored the functions of constitutively expressed ERβ in ERα-positive breast cancer lines MCF7 and T47D. We found that ERβ, under these conditions heterodimerized with ERα in the presence and absence of 17β-estradiol, and induced genome-wide transcriptional changes. Widespread anti-ERα signaling was, however, not observed and ERβ was not antiproliferative. Tamoxifen antagonized proliferation and ER-mediated gene regulation both in the presence and absence of ERβ. In conclusion, ERβ's role in cells adapted to its expression appears to differ from its role in cells with transient expression. Our study is important because it provides a deeper understanding of ERβ's role in breast tumors that coexpress both receptors and supports an emerging bi-faceted role of ERβ.

  • 35. Katchy, Anne
    et al.
    Edvardsson, Karin
    Aydogdu, Eylem
    Williams, Cecilia
    University of Houston, United States .
    Estradiol-activated estrogen receptor α does not regulate mature microRNAs in T47D breast cancer cells.2012In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 128, no 3-5, 145-53 p.Article in journal (Refereed)
    Abstract [en]

    Breast cancers are sensitive to hormones such as estrogen, which binds to and activates estrogen receptors (ER) leading to significant changes in gene expression. microRNAs (miRNA) have emerged as a major player in gene regulation, thus identification of miRNAs associated with normal or disrupted estrogen signaling is critical to enhancing our understanding of the diagnosis and prognosis of breast cancer. We have previously shown that 17β-estradiol (E2) induced activation of ERα in T47D cells results in significant changes in the expression of protein-coding genes involved in cell cycle, proliferation, and apoptosis. To identify miRNAs regulated by E2-activated ERα, we analysed their expression in T47D cells following E2-activation using both dual-color microarrays and TaqMan Low Density Arrays, and validations were carried out by real-time PCR. Although estrogen treatment results in altered expression of up to 900 protein-coding transcripts, no significant changes in mature miRNA expression levels could be confirmed. Whereas previous studies aiming to elucidate the role of miRNA in ER-positive breast cancers cell lines have yielded conflicting results, the work presented here represents a thorough investigation of and significant step forward in our understanding of ERα mediated miRNA regulation.

  • 36. Katchy, Anne
    et al.
    Pinto, Caroline
    Jonsson, Philip
    Nguyen-Vu, Trang
    Pandelova, Marchela
    Riu, Anne
    Schramm, Karl-Werner
    Samarov, Daniel
    Gustafsson, Jan-Åke
    Bondesson, Maria
    Williams, Cecilia
    University of Houston, United States .
    Coexposure to phytoestrogens and bisphenol a mimics estrogenic effects in an additive manner2014In: Toxicological Sciences, ISSN 1096-6080, E-ISSN 1096-0929, Vol. 138, no 1, 21-35 p.Article in journal (Refereed)
    Abstract [en]

    Endocrine-disrupting chemicals (EDC) are abundant in our environment. A number of EDCs, including bisphenol A (BPA) can bind to the estrogen receptors (ER), ERα and ERβ, and may contribute to estrogen-linked diseases such as breast cancer. Early exposure is of particular concern; many EDCs cross the placenta and infants have measurable levels of, eg, BPA. In addition, infants are frequently fed soy-based formula (SF) that contains phytoestrogens. Effects of combined exposure to xeno- and phytoestrogens are poorly studied. Here, we extensively compared to what extent BPA, genistein, and an extract of infant SF mimic estrogen-induced gene transcription and cell proliferation. We investigated ligand-specific effects on ER activation in HeLa-ERα and ERβ reporter cells; on proliferation, genome-wide gene regulation and non-ER-mediated effects in MCF7 breast cancer cells; and how coexposure influenced these effects. The biological relevance was explored using enrichment analyses of differentially regulated genes and clustering with clinical breast cancer profiles. We demonstrate that coexposure to BPA and genistein, or SF, results in increased functional and transcriptional estrogenic effects. Using statistical modeling, we determine that BPA and phytoestrogens act in an additive manner. The proliferative and transcriptional effects of the tested compounds mimic those of 17β-estradiol, and are abolished by cotreatment with an ER antagonist. Gene expression profiles induced by each compound clustered with poor prognosis breast cancer, indicating that exposure may adversely affect breast cancer prognosis. This study accentuates that coexposure to BPA and soy-based phytoestrogens results in additive estrogenic effects, and may contribute to estrogen-linked diseases, including breast cancer.

  • 37. Katchy, Anne
    et al.
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Department of Biology and Biochemistry, University of Houston, Houston.
    Expression Profiles of Estrogen-Regulated MicroRNAs in Breast Cancer Cells2016In: Methods in Molecular Biology: The Estrogen Receptors / [ed] Kathleen M. Eyster, Springer, 2016, Vol. 1366Chapter in book (Other academic)
    Abstract [en]

    Molecular signaling through both estrogen and microRNAs are critical for breast cancer development and growth. The activity of estrogen is mediated by transcription factors, the estrogen receptors. Here we describe a method for robust characterization of estrogen-regulated microRNA profiles. The method details how to prepare cells for optimal estrogen response, directions for estrogen treatment, RNA extraction, microRNA large-scale profiling, and subsequent confirmations.

  • 38. Kaucka, Marketa
    et al.
    Ivashkin, Evgeny
    Gyllborg, Daniel
    Zikmund, Tomas
    Tesarova, Marketa
    Kaiser, Jozef
    Xie, Meng
    Petersen, Julian
    Pachnis, Vassilis
    Nicolis, Silvia K.
    Yu, Tian
    Sharpe, Paul
    Arenas, Ernest
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Clevers, Hans
    Suter, Ueli
    Chagin, Andrei S.
    Fried, Kaj
    Hellander, Andreas
    Adameyko, Igor
    Analysis of neural crest-derived clones reveals novel aspects of facial development2016In: Science Advances, ISSN 2375-2548, Vol. 2, no 8, e1600060Article in journal (Refereed)
    Abstract [en]

    Cranial neural crest cells populate the future facial region and produce ectomesenchyme-derived tissues, such as cartilage, bone, dermis, smooth muscle, adipocytes, and many others. However, the contribution of individual neural crest cells to certain facial locations and the general spatial clonal organization of the ectomesenchyme have not been determined. We investigated how neural crest cells give rise to clonally organized ectomesenchyme and how this early ectomesenchyme behaves during the developmental processes that shape the face. Using a combination of mouse and zebrafish models, we analyzed individual migration, cell crowd movement, oriented cell division, clonal spatial overlapping, and multilineage differentiation. The early face appears to be built from multiple spatially defined overlapping ectomesenchymal clones. During early face development, these clones remain oligopotent and generate various tissues in a given location. By combining clonal analysis, computer simulations, mouse mutants, and live imaging, we show that facial shaping results from an array of local cellular activities in the ectomesenchyme. These activities mostly involve oriented divisions and crowd movements of cells during morphogenetic events. Cellular behavior that can be recognized as individual cell migration is very limited and short-ranged and likely results from cellular mixing due to the proliferation activity of the tissue. These cellular mechanisms resemble the strategy behind limb bud morphogenesis, suggesting the possibility of common principles and deep homology between facial and limb outgrowth.

  • 39.
    Khorshidi, Mohammad Ali
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Kowalewski, Jacob M.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Garrod, Kym R.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Cahalan, Michael D.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Analysis of transient migration behavior of natural killer cells imaged in situ and in vitro2011In: Integrative Biology, ISSN 1757-9694, E-ISSN 1757-9708, Vol. 3, no 7, 770-778 p.Article in journal (Refereed)
    Abstract [en]

    We present a simple method for rapid and automatic characterization of lymphocyte migration from time-lapse fluorescence microscopy data. Time-lapse imaging of natural killer (NK) cells in vitro and in situ, both showed that individual cells transiently alter their migration behavior. Typically, NK cells showed periods of high motility, interrupted by transient periods of slow migration or almost complete arrests. Analysis of in vitro data showed that these periods frequently coincided with contacts with target cells, sometimes leading to target cell lysis. However, NK cells were also commonly observed to stop independently of contact with other cells. In order to objectively characterize the migration of NK cells, we implemented a simple method to discriminate when NK cells stop or have low motilities, have periods of directed migration or undergo random movement. This was achieved using a sliding window approach and evaluating the mean squared displacement (MSD) to assess the migration coefficient and MSD curvature along trajectories from individual NK cells over time. The method presented here can be used to quickly and quantitatively assess the dynamics of individual cells as well as heterogeneity within ensembles. Furthermore, it may also be used as a tool to automatically detect transient stops due to the formation of immune synapses, cell division or cell death. We show that this could be particularly useful for analysis of in situ time-lapse fluorescence imaging data where most cells, as well as the extracellular matrix, are usually unlabelled and thus invisible.

  • 40.
    Klevebring, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology.
    On Transcriptome Sequencing2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci.

  • 41.
    Kodura, Magdalena
    et al.
    KTH, School of Biotechnology (BIO). Neurocentrum, Karolinska University Hospital.
    Souchelnytskyi, S.
    Breast carcinoma metastasis suppressor gene 1 (BRMS1): update on its role as the suppressor of cancer metastases2015In: Cancer Metastasis Review, ISSN 0167-7659, E-ISSN 1573-7233, Vol. 34, no 4, 611-618 p.Article in journal (Refereed)
    Abstract [en]

    BRMS1 was discovered over a decade ago as a potential tumor suppressor gene. In this review, we summarize the recent findings about the structure of BRMS1, mechanisms of its action and a role of BRMS1 in the cancer progression. As a suppressor of metastasis, BRMS1 has demonstrated a variety of ways to act on the cell functions, such as cell migration, invasiveness, angiogenesis, cell survival, cytoskeleton rearrangements, cell adhesion, and immune recognition. This variety of effects is a likely reason behind the robustness of anti-metastatic influence of BRMS1. Intracellular signaling mechanisms employed by BRMS1 include regulation of transcription, EGF/HER2 signaling, and expression of NF-kB, fascin, osteopontin, and IL-6. Recently reported clinical studies confirm that BRMS1 can indeed be used as a prognostic marker. Approaches to employ BRMS1 in a development of anti-cancer treatment have also been made. The studies reviewed here with respect to BRMS1 structure, cellular effects, intracellular signaling, and clinical value consolidate the importance of BRMS1 in the development of metastasis.

  • 42. Krementsov, Dimitry N
    et al.
    Katchy, Anne
    Case, Laure K
    Carr, Frances E
    Davis, Barbara
    Williams, Cecilia
    University of Houston, United States .
    Teuscher, Cory
    Studies in experimental autoimmune encephalomyelitis do not support developmental bisphenol a exposure as an environmental factor in increasing multiple sclerosis risk2013In: Toxicological Sciences, ISSN 1096-6080, E-ISSN 1096-0929, Vol. 135, no 1, 91-102 p.Article in journal (Refereed)
    Abstract [en]

    Multiple sclerosis (MS), a demyelinating immune-mediated central nervous system disease characterized by increasing female penetrance, is the leading cause of disability in young adults in the developed world. Epidemiological data strongly implicate an environmental factor, acting at the population level during gestation, in the increasing incidence of female MS observed over the last 50 years, yet the identity of this factor remains unknown. Gestational exposure to bisphenol A (BPA), an endocrine disruptor used in the manufacture of polycarbonate plastics since the 1950s, has been reported to alter a variety of physiological processes in adulthood. BPA has estrogenic activity, and we hypothesized that increased gestational exposure to environmental BPA may therefore contribute to the increasing female MS risk. To test this hypothesis, we utilized two different mouse models of MS, experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice (chronic progressive) and in SJL/J mice (relapsing-remitting). Dams were exposed to physiologically relevant levels of BPA in drinking water starting 2 weeks prior to mating and continuing until weaning of offspring. EAE was induced in adult offspring. No significant changes in EAE incidence, progression, or severity were observed with BPA exposure, despite changes in cytokine production by autoreactive T cells. However, endocrine disruption was evidenced by changes in testes development, and transcriptomic profiling revealed that BPA exposure altered the expression of several genes important for testes development, including Pdgfa, which was downregulated. Overall, our results do not support gestational BPA exposure as a significant contributor to the increasing female MS risk.

  • 43.
    Laurell, Cecilia
    KTH, School of Biotechnology (BIO).
    Microarray Based Gene Expression Analysis in Cancer Research2006Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Biotechnological inventions during the 20th century have resulted in a wide range of approaches for explorations in the functional genomics field. Microarray technology is one of the recent advances which have provided us with snapshots of which genes are expressed in cells of various tissues and diseases. Methods to obtain reliable microarray data are continuously being developed and improved to meet the demands of biological researchers.

    In this thesis microarrays have been used to investigate gene expression patterns in cancer research. Four studies in three different areas were carried out covering adrenocortical tumors, p53 target genes and a comparison of RNA amplification methods.

    Adrenocortical tumours are among the most common tumours with an incidence of 7-9%. Malignancy of these tumors is rare. Distinction between malignant and benign tumours is often difficult to establish which makes an improvement of diagnostic approaches important. To elucidate biological processes in adrenocortical tumour development and to examine if there is a molecular signature associated with malignancy, microarray analysis was performed on 29 adrenocortical tumors and four normal specimens. It was possible to classify malignant and benign samples based on the entire expression profile. A number of potential biomarkers was identified which will be further evaluated.

    P53 is a gene which is mutated in 50% of all cancers. Functional p53 is a transcription factor which is activated upon cellular stress and DNA damage. Target genes are mainly involved in cell cycle arrest and apoptosis. In solid tumors cells are stressed by hypoxia. To examine which target genes p53 activate under hypoxic conditions a microarray study of the cell lines HCT116p53+/+ and HCT116p53-/- was performed. A set of novel potential p53 target genes was identified while many known target genes were found to be not transcriptionally activated during hypoxia. Follow up which was focused on how p53 affected hypoxia induced apoptosis showed that the death receptor Fas was critical.

    When small amounts of tissue are available, amplification of the transcript population is necessary for microarray analysis. A new strategy for amplification based on PCR was evaluated and compared to a commercial in vitro transcription protocol. Both protocols produced reliable results. Advantages with the PCR based method are a lower cost and a high flexibility due to compatibility with both sense and antisense strand microarrays.

    Keywords: adrenocortical tumour, apoptosis, cancer, classification, gene expression, microarray, p53, RNA amplification

  • 44. Li, J.
    et al.
    Newberg, J. Y.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Murphy, R. F.
    Automated Analysis and Reannotation of Subcellular Locations in Confocal Images from the Human Protein Atlas2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, e50514- p.Article in journal (Refereed)
    Abstract [en]

    The Human Protein Atlas contains immunofluorescence images showing subcellular locations for thousands of proteins. These are currently annotated by visual inspection. In this paper, we describe automated approaches to analyze the images and their use to improve annotation. We began by training classifiers to recognize the annotated patterns. By ranking proteins according to the confidence of the classifier, we generated a list of proteins that were strong candidates for reexamination. In parallel, we applied hierarchical clustering to group proteins and identified proteins whose annotations were inconsistent with the remainder of the proteins in their cluster. These proteins were reexamined by the original annotators, and a significant fraction had their annotations changed. The results demonstrate that automated approaches can provide an important complement to visual annotation.

  • 45. Li, J.
    et al.
    Shariff, A.
    Wiking, Mikaela
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rohde, G. K.
    Murphy, R. F.
    Estimating Microtubule Distributions from 2D Immunofluorescence Microscopy Images Reveals Differences among Human Cultured Cell Lines2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, e50292- p.Article in journal (Refereed)
    Abstract [en]

    Microtubules are filamentous structures that are involved in several important cellular processes, including cell division, cellular structure and mechanics, and intracellular transportation. Little is known about potential differences in microtubule distributions within and across cell lines. Here we describe a method to estimate information pertaining to 3D microtubule distributions from 2D fluorescence images. Our method allows for quantitative comparisons of microtubule distribution parameters (number of microtubules, mean length) between different cell lines. Among eleven cell lines compared, some showed differences that could be accounted for by differences in the total amount of tubulin per cell while others showed statistically significant differences in the balance between number and length of microtubules. We also observed that some cell lines that visually appear different in their microtubule distributions are quite similar when the model parameters are considered. The method is expected to be generally useful for comparing microtubule distributions between cell lines and for a given cell line after various perturbations. The results are also expected to enable analysis of the differences in gene expression underlying the observed differences in microtubule distributions among cell types.

  • 46. Li, Yanhong
    et al.
    Marcoux, Marie-Odile
    Gineste, Martine
    Vanpee, Mireille
    Zelenina, Marina
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Casper, Charlotte
    Expression of water and ion transporters in tracheal aspirates from neonates with respiratory distress2009In: Acta Paediatrica, ISSN 0803-5253, E-ISSN 1651-2227, Vol. 98, no 11, 1729-1737 p.Article in journal (Refereed)
    Abstract [en]

    Aim: The aim of the study was to determine whether neonatal respiratory distress is related to changes in water and ion transporter expression in lung epithelium. Methods: The study included 32 neonates on mechanical ventilation: 6 patients with normal lung X-rays (control group), eight with respiratory distress syndrome (RDS), eight with transient tachypnea of the newborn (TTN), 10 with abnormal lung X-rays (mixed group). The protein abundance of water channel AQP5, epithelial sodium channel (ENaC; alpha-, beta- and gamma-ENaC) and Na+, K+-ATPase alpha 1 were examined in tracheal aspirates using semiquantitative immunoblotting. Results: beta-ENaC level was significantly lower in RDS group compared with infants with TTN and infants in the control group. AQP5 expression was significantly higher in TTN compared with the infants with RDS and all other infants with abnormal lung X-rays. Conclusion: Neonatal respiratory distress is associated with changes in beta-ENaC and AQP5 expression. The lower beta-ENaC expression may be one of the factors that predispose to the development of RDS. The higher AQP5 expression may provide the possibility for reabsorption of postnatal lung liquid, which contributes to quick recovery of infants with TTN.

  • 47. Li, Yanhong
    et al.
    Zelenina, Marina
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. Karolinska Institutet, Sweden.
    Plat-Willson, Genevieve
    Marcoux, Marie-Odile
    Aperia, Anita
    Casper, Charlotte
    Urinary aquaporin-2 excretion during ibuprofen or indomethacin treatment in preterm infants with patent ductus arteriosus2011In: Acta Paediatrica, ISSN 0803-5253, E-ISSN 1651-2227, Vol. 100, no 1, 59-66 p.Article in journal (Refereed)
    Abstract [en]

    Aim: Water channel AQP2 is the target for vasopressin (AVP) and a major determinant of urinary concentrating capacity. In mature kidneys, prostaglandins counteract the effect of AVP on AQP2 expression at functional sites. We investigated whether disturbances in water homeostasis in infants with patent ductus arteriosus (PDA) treated with prostaglandin inhibitors can be attributed to activation of AQP2. Methods: In 53 infants with symptomatic PDA (gestational age 24-33 weeks), 30 receiving ibuprofen and 23 indomethacin starting at 2-15 days of life, clinical and biochemical data were collected before treatment and after each dose of the drugs. Urinary AQP2 was determined by dot immunoblotting. Results: Urinary AQP2 level and osmolality were decreased in both groups. Urinary osmolality was overall low and correlated inversely with fluid uptake. In ibuprofen group, there was no correlation of AQP2 level with urinary osmolality. Conclusion: There was no AQP2 upregulation in the infants. The low urinary osmolality and dissociation between urinary osmolality and urinary AQP2 level indicate that the fluid retention sometimes observed in PDA infants treated with prostaglandin inhibitors is not caused by increased levels of functional AQP2. Thus, knowledge about the renal physiology of the adult cannot always be transferred to the infant kidney.

  • 48. Ling, G
    et al.
    Ahmadian, A
    Persson, A
    Undén, A B
    Afink, G
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Uhlén, M
    Toftgård, R
    Lundeberg, J
    Pontén, F
    PATCHED and p53 gene alterations in sporadic and hereditary basal cell cancer.2001In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 20, no 53Article in journal (Refereed)
    Abstract [en]

    It is widely accepted that disruption of the hedgehog-patched pathway is a key event in development of basal cell cancer. In addition to patched gene alterations, p53 gene mutations are also frequent in basal cell cancer. We determined loss of heterozygosity in the patched and p53 loci as well as sequencing the p53 gene in tumors both from sporadic and hereditary cases. A total of 70 microdissected samples from tumor and adjacent skin were subjected to PCR followed by fragment analysis and DNA sequencing. We found allelic loss in the patched locus in 6/8 sporadic basal cell cancer and 17/19 hereditary tumors. All sporadic and 7/20 hereditary tumors showed p53 gene mutations. Loss of heterozygosity in the p53 locus was rare in both groups. The p53 mutations detected in hereditary tumors included rare single nucleotide deletions and unusual double-base substitutions compared to the typical ultraviolet light induced missense mutations found in sporadic tumors. Careful microdissection of individual tumors revealed genetically linked subclones with different p53 and/or patched genotype providing an insight on time sequence of genetic events. The high frequency and co-existence of genetic alterations in the patched and p53 genes suggest that both these genes are important in the development of basal cell cancer.

  • 49. Nadadhur, A. G.
    et al.
    Melero, J. E.
    Meijer, M.
    Schut, D.
    Jacobs, G.
    Wan Li, K.
    Hjorth, Johannes
    KTH, School of Computer Science and Communication (CSC), Computational Science and Technology (CST). KTH, Centres, Science for Life Laboratory, SciLifeLab. Vrije Universiteit, Netherlands.
    Meredith, R. M.
    Toonen, R. F.
    Van Kesteren, R. E.
    Smit, A. B.
    Verhage, M.
    Heine, V. M.
    Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 6, 0178533Article in journal (Refereed)
    Abstract [en]

    Generation of neuronal cultures from induced pluripotent stem cells (hiPSCs) serve the studies of human brain disorders. However we lack neuronal networks with balanced excitatory- inhibitory activities, which are suitable for single cell analysis. We generated low-density networks of hPSC-derived GABAergic and glutamatergic cortical neurons. We used two different co-culture models with astrocytes. We show that these cultures have balanced excitatory-inhibitory synaptic identities using confocal microscopy, electrophysiological recordings, calcium imaging and mRNA analysis. These simple and robust protocols offer the opportunity for single-cell to multi-level analysis of patient hiPSC-derived cortical excitatory- inhibitory networks; thereby creating advanced tools to study disease mechanisms underlying neurodevelopmental disorders.

  • 50. Nguyen-Vu, Trang
    et al.
    Vedin, Lise-Lotte
    Liu, Ka
    Jonsson, Philip
    Lin, Jean Z
    Candelaria, Nicholes R
    Candelaria, Lindsay P
    Addanki, Sridevi
    Williams, Cecilia
    University of Houston.
    Gustafsson, Jan-Åke
    Steffensen, Knut R
    Lin, Chin-Yo
    Liver × receptor ligands disrupt breast cancer cell proliferation through an E2F-mediated mechanism2013In: Breast Cancer Research, ISSN 1465-5411, E-ISSN 1465-542X, Vol. 15, no 3, R51Article in journal (Refereed)
    Abstract [en]

    INTRODUCTION: Liver × receptors (LXRs) are members of the nuclear receptor family of ligand-dependent transcription factors and have established functions as regulators of cholesterol, glucose, and fatty acid metabolism and inflammatory responses. Published reports of anti-proliferative effects of synthetic LXR ligands on breast, prostate, ovarian, lung, skin, and colorectal cancer cells suggest that LXRs are potential targets in cancer prevention and treatment.

    METHODS: To further determine the effects of LXR ligands and identify their potential mechanisms of action in breast cancer cells, we carried out microarray analysis of gene expression in four breast cancer cell lines following treatments with the synthetic LXR ligand GW3965. Differentially expressed genes were further subjected to gene ontology and pathway analyses, and their expression profiles and associations with disease parameters and outcomes were examined in clinical samples. Response of E2F target genes were validated by real-time PCR, and the posited role of E2F2 in breast cancer cell proliferation was tested by RNA interference experiments.

    RESULTS: We observed cell line-specific transcriptional responses as well as a set of common responsive genes. In the common responsive gene set, upregulated genes tend to function in the known metabolic effects of LXR ligands and LXRs whereas the downregulated genes mostly include those which function in cell cycle regulation, DNA replication, and other cell proliferation-related processes. Transcription factor binding site analysis of the downregulated genes revealed an enrichment of E2F binding site sequence motifs. Correspondingly, E2F2 transcript levels are downregulated following LXR ligand treatment. Knockdown of E2F2 expression, similar to LXR ligand treatment, resulted in a significant disruption of estrogen receptor positive breast cancer cell proliferation. Ligand treatment also decreased E2F2 binding to cis-regulatory regions of target genes. Hierarchical clustering of breast cancer patients based on the expression profiles of the commonly downregulated LXR ligand-responsive genes showed a strong association of these genes with patient survival.

    CONCLUSIONS: Taken together, these results indicate that LXR ligands target gene networks, including those regulated by E2F family members, are critical for tumor biology and disease progression and merit further consideration as potential agents in the prevention and treatment of breast cancers.

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