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  • 1.
    Blokzijl, Andries
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Friedman, Mikaela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular and Morphological Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Profiling protein expression and interactions: proximity ligation as a tool for personalized medicine2010In: Journal of Internal Medicine, ISSN 0954-6820, E-ISSN 1365-2796, Vol. 268, no 3, p. 232-245Article, review/survey (Refereed)
    Abstract [en]

    Blokzijl A, Friedman M, Ponten F, Landegren U (From the Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden). Profiling protein expression and interactions: proximity ligation as a tool for personalized medicine. (Review) J Intern Med 2010; 268: 232-245. The ability to detect very low levels of expressed proteins has enormous potential for early diagnostics and intervention at curable stages of disease. An extended range of targets such as interacting or post-translationally modified proteins can further improve the potential for diagnostics and patient stratification, and for monitoring response to treatment. These are critical building blocks for personalized treatment strategies to manage disease. The past few decades have seen a remarkably improved understanding of the molecular basis of disease in general, and of tumour formation and progression in particular. This accumulated knowledge creates opportunities to develop drugs that specifically target molecules or molecular complexes critical for survival and expansion of tumour cells. However, tumours are highly variable between patients, necessitating the development of diagnostic tools to individualize treatment through parallel analysis of sets of biomarkers. The proximity ligation assay (PLA) can address many of the requirements for advanced molecular analysis. The method builds on the principle that recognition of target proteins by two, three or more antibodies can bring in proximity DNA strands attached to the antibodies. The DNA strands can then participate in ligation reactions, giving rise to molecules that are amplified for highly sensitive detection. PLA is particularly well suited for sensitive, specific and multiplexed analysis of protein expression, post-translational modifications and protein-protein interactions. The analysis of this extended range of biomarkers will prove critical for the development and implementation of personalized medicine.

  • 2.
    Conze, Tim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Ligation-mediated Molecular Analysis of Influenza Subtypes, Splicing and Protein Glycosylation2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Binder-based assays are employed throughout the life sciences. Powerful signal amplification techniques have enabled detection of very rare molecule species diluted in simple buffers. Unspecific binding of primary binders leads to increased background in more complex samples. By requiring two recognition events, ligation-based molecular analyses provide highly specific detection of biomolecules in complex samples.

    We developed a highly multiplexed padlock-ligation assay targeting signature sequences in the hemagglutinin and neuraminidase genes. From a panel of 77 avian influenza isolates of all major serotypes, 97% were genotyped correctly in accordance with previous classifications by classical diagnostic methods (Paper I).

    Alternative splicing is an important mechanism expanding the proteome. Current analysis techniques fail to provide sequences of complete transcripts beyond the read length of sequencing instruments. We devised and implemented a strategy to compress the sequence information contained in the splicing pattern of a transcript into the presence or absence of sequence-blocks. We demonstrate that this assay yields information about the splicing patterns in thousands of transcripts from cellular cDNA (Paper II).

    Expression changes of mucin proteins and glycosylation structures are frequently observed from the early stages of cancer development. Expression of mucin 2 and sialyl-Tn are common features of intestinal metaplasia and gastric cancer, and are known to co-locate. Here we have developed an in situ proximity ligation assay (PLA) directed against mucin 2 and sialyl-Tn. Our study on intestinal metaplasia and gastric cancer tissue sections identified mucin 2 as a major carrier of sialyl-Tn in these conditions, and demonstrated how conveniently glycosylation of proteins can be studied by in situ PLA (Paper III).

    This thesis shows how the dual recognition requirement of ligation-based assays can be employed to detect target molecules with high specificity, to analyze several sequence features of nucleic acids or to study the proximity of two antigens in situ.

    List of papers
    1. Simultaneous genotyping of all hemagglutinin and neuraminidase subtypes of avian influenza viruses by use of padlock probes
    Open this publication in new window or tab >>Simultaneous genotyping of all hemagglutinin and neuraminidase subtypes of avian influenza viruses by use of padlock probes
    Show others...
    2008 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 46, no 5, p. 1747-1751Article in journal (Refereed) Published
    Abstract [en]

    A subtyping assay for both the hemagglutinin (HA) and neuraminidase (NA) surface antigens of the avian influenza virus (AIV) has been developed. The method uses padlock probe chemistry combined with a microarray output for detection. The outstanding feature of this assay is its capability to designate both the HA and the NA of an AIV sample from a single reaction mixture. A panel of 77 influenza virus strains was tested representing the entire assortment of the two antigens. One hundred percent (77/77) of the samples tested were identified as AIV, and 97% (75/77) were subtyped correctly in accordance with previous examinations performed by classical diagnostic methods. Testing of heterologous pathogens verified the specificity of the assay. This assay is a convenient and practical tool for the study of AIVs, providing important HA and NA data more rapidly than conventional methods.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-16412 (URN)10.1128/JCM.02292-07 (DOI)000255678200027 ()18353937 (PubMedID)
    Available from: 2008-05-22 Created: 2008-05-22 Last updated: 2017-12-08Bibliographically approved
    2. Single molecule analysis of combinatorial splicing
    Open this publication in new window or tab >>Single molecule analysis of combinatorial splicing
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    2010 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 16, p. e163-Article in journal (Refereed) Published
    Abstract [en]

    Alternative splicing forms diverse mRNA isoform populations from a single ancestral pre-mRNA and thereby enhances complexity of transcript structure and of gene function. We describe a method called spliceotyping, which translates combinatorial mRNA splicing patterns into a library of binary strings of nucleic acid tags, encoding the exon composition of transcripts. The transcript abundance is registered by counts of individual molecules and individual exon inclusion patterns are represented as strings of binary data.

    The technique is illustrated by analyzing the splicing patterns of the adenovirus early 1A gene and the beta actin reference transcript. The method permits different genes to be analyzed in parallel and will be valuable for elucidating the complex effects of combinatorial splicing.

    Keywords
    Splicing, Combinatorial, RCA, Single molecule, Amplified Single Molecule Detection
    National Category
    Medical and Health Sciences
    Research subject
    Molecular Medicine
    Identifiers
    urn:nbn:se:uu:diva-96780 (URN)10.1093/nar/gkq581 (DOI)000281720500003 ()20587504 (PubMedID)
    Available from: 2010-03-11 Created: 2008-02-28 Last updated: 2017-12-14Bibliographically approved
    3. MUC2 mucin is a major carrier of the cancer-associated sialyl-Tn antigen in intestinal metaplasia and gastric carcinomas
    Open this publication in new window or tab >>MUC2 mucin is a major carrier of the cancer-associated sialyl-Tn antigen in intestinal metaplasia and gastric carcinomas
    Show others...
    2010 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 2, p. 199-206Article in journal (Refereed) Published
    Abstract [en]

    Changes in mucin protein expression and in glycosylation are common features in pre-neoplastic lesions and cancer and are therefore used as cancer-associated markers. De novo expression of intestinal mucin MUC2 and cancer-associated sialyl-Tn antigen are frequently observed in intestinal metaplasia (IM) and gastric cancer. However, despite that these antigens often co-localize, MUC2 has not been demonstrated to be a carrier of sialyl-Tn. By using the in situ proximity ligation assay (in situ PLA), we herein could show that MUC2 is a major carrier of the sialyl-Tn antigen in all IM cases and in most gastric carcinoma cases. The requirement by in situ PLA for the presence of both antigens in close proximity increases the selectivity compared to measurement of co-localization, as determined by immunohistochemistry. Identification of the mucin which is the carrier of a carbohydrate structure offers unique advantages for future development of more accurate diagnostic and prognostic markers.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-112103 (URN)10.1093/glycob/cwp161 (DOI)000273226500007 ()19815850 (PubMedID)
    Available from: 2010-01-08 Created: 2010-01-08 Last updated: 2017-12-12Bibliographically approved
  • 3.
    Conze, Tim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Carvalho, Ana Sofia
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Almeida, Raquel
    Reis, Celso A.
    David, Leonor
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    MUC2 mucin is a major carrier of the cancer-associated sialyl-Tn antigen in intestinal metaplasia and gastric carcinomas2010In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 2, p. 199-206Article in journal (Refereed)
    Abstract [en]

    Changes in mucin protein expression and in glycosylation are common features in pre-neoplastic lesions and cancer and are therefore used as cancer-associated markers. De novo expression of intestinal mucin MUC2 and cancer-associated sialyl-Tn antigen are frequently observed in intestinal metaplasia (IM) and gastric cancer. However, despite that these antigens often co-localize, MUC2 has not been demonstrated to be a carrier of sialyl-Tn. By using the in situ proximity ligation assay (in situ PLA), we herein could show that MUC2 is a major carrier of the sialyl-Tn antigen in all IM cases and in most gastric carcinoma cases. The requirement by in situ PLA for the presence of both antigens in close proximity increases the selectivity compared to measurement of co-localization, as determined by immunohistochemistry. Identification of the mucin which is the carrier of a carbohydrate structure offers unique advantages for future development of more accurate diagnostic and prognostic markers.

  • 4. Gyarmati, Péter
    et al.
    Conze, Tim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Zohari, Siamak
    LeBlanc, Neil
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Banér, Johan
    Belák, Sándor
    Simultaneous genotyping of all hemagglutinin and neuraminidase subtypes of avian influenza viruses by use of padlock probes2008In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 46, no 5, p. 1747-1751Article in journal (Refereed)
    Abstract [en]

    A subtyping assay for both the hemagglutinin (HA) and neuraminidase (NA) surface antigens of the avian influenza virus (AIV) has been developed. The method uses padlock probe chemistry combined with a microarray output for detection. The outstanding feature of this assay is its capability to designate both the HA and the NA of an AIV sample from a single reaction mixture. A panel of 77 influenza virus strains was tested representing the entire assortment of the two antigens. One hundred percent (77/77) of the samples tested were identified as AIV, and 97% (75/77) were subtyped correctly in accordance with previous examinations performed by classical diagnostic methods. Testing of heterologous pathogens verified the specificity of the assay. This assay is a convenient and practical tool for the study of AIVs, providing important HA and NA data more rapidly than conventional methods.

  • 5.
    Hedborg, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Pediatrics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Ullerås, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Grimelius, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Wassberg, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Thoracic Surgery.
    Maxwell, Patrick H
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Animal Development and Genetics.
    Hero, Barbara
    Berthold, Frank
    Schilling, Freimut
    Harms, Dieter
    Sandstedt, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Franklin, Gary
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Animal Development and Genetics.
    Evidence for hypoxia-induced neuronal-to-chromaffin metaplasia in neuroblastoma2003In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 17, no 6, p. 598-609Article in journal (Refereed)
    Abstract [en]

    We present evidence that in neuroblastoma, a pediatric malignancy of embryonal sympathetic origin, hypoxia, underlies a phenotypic switch from a primitive neuronal to a chromaffin cell type. This conclusion is based on morphological and molecular data on 116 clinical tumors and is supported by data on the phenotypic effects of hypoxia on neuroblastoma cell lines when studied in monolayer culture and as tumor xenografts. In the clinical material, extensive chromaffin features were seen in regions of chronic tumor hypoxia. This was the exclusive form of intra-tumoral maturation of stroma-poor tumors and was also seen in stroma-rich tumors, either exclusively or in combination with ganglion-like cells. In neuroblastoma cell lines, hypoxia induced changes in gene expression associated with the chromaffin features observed in vivo. We therefore propose tumor hypoxia as a major cue determining phenotype in sympathetic tumors of neuroblastic origin. Because it appears to be reversible upon reoxygenation in monolayer culture, we suggest the term metaplasia for the phenomenon.

  • 6.
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Visualization of Protein Activity Status in situ Using Proximity Ligation Assays2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In 2001 the human proteome organization (HUPO) was created with the ambition to identify and characterize all proteins encoded in the human genome according to several criteria; their expression levels in different tissues and under different conditions; the sub-cellular localization; post-translational modifications; interactions, and if possible also the relationship between their structure and function.When the knowledge of different proteins and their potential interactions increases, so does the need for methods able to unravel the nature of molecular processes in cells and organized tissues, and ultimately for clinical use in samples obtained from patients.

    The in situ proximity ligation assay (in situ PLA) was developed to provide localized detection of proteins, post-translational modifications and protein-protein interactions in fixed cells and tissues. Dual recognition of the target or interacting targets is a prerequisite for the creation of a circular reporter DNA molecule, which subsequently is locally amplified for visualization of individual protein molecules in single cells. These features offer the high sensitivity and selectivity required for detection of even rare target molecules.

    Herein in situ PLA was first established and then employed as a tool for detection of both interactions and post-translational modifications in cultured cells and tissue samples. In situ PLA was also adapted to high content screening (HCS) for therapeutic effects, where it was applied for cell-based drug screening of inhibitors influencing post-translational modifications. This was performed using primary cells, paving the way for evaluation of drug effects on cells from patient as a diagnostic tool in personalized medicine.

    In conclusion, this thesis describes the development and applications of in situ PLA as a tool to study proteins, post-translational modifications and protein-protein interactions in genetically unmodified cells and tissues, and for clinical interactomics.

     

    List of papers
    1. Direct observation of individual endogenous protein complexes in situ by proximity ligation
    Open this publication in new window or tab >>Direct observation of individual endogenous protein complexes in situ by proximity ligation
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    2006 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 3, no 12, p. 995-1000Article in journal (Refereed) Published
    Abstract [en]

    Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes-oligonucleotides attached to antibodies against the two target proteins-guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-gamma (IFN-gamma) signaling and low-molecular-weight inhibitors.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-16092 (URN)10.1038/nmeth947 (DOI)000242213900018 ()17072308 (PubMedID)
    Available from: 2008-06-25 Created: 2008-06-25 Last updated: 2017-12-08Bibliographically approved
    2. In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method
    Open this publication in new window or tab >>In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method
    Show others...
    2007 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, no 9, p. 1500-1509Article in journal (Refereed) Published
    Abstract [en]

    Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor β (PDGFRβ) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRβ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRβ in porcine aortic endothelial cells transfected with the β-receptor, but not in cells transfected with the α-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRβ. We furthermore visualized tyrosine phosphorylated PDGFRβ in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-12659 (URN)10.1074/mcp.M700166-MCP200 (DOI)000249237200004 ()17565975 (PubMedID)
    Available from: 2008-05-28 Created: 2008-05-28 Last updated: 2017-12-11Bibliographically approved
    3. Platelet-derived growth factor receptor expression and activation in choroid plexus tumors
    Open this publication in new window or tab >>Platelet-derived growth factor receptor expression and activation in choroid plexus tumors
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    2009 (English)In: American Journal of Pathology, ISSN 0002-9440, E-ISSN 1525-2191, Vol. 175, no 4, p. 1631-1637Article in journal (Refereed) Published
    Abstract [en]

    Choroid plexus tumors are intraventricular neoplasms predominantly affecting young children. In contrast to choroid plexus papillomas, choroid plexus carcinomas progress frequently, necessitating the development of adjuvant treatment concepts. Platelet derived growth factor (PDGF) signaling has been shown to support growth in a variety of tumors. The finding of PDGF receptor expression in choroid plexus tumors prompted us to elucidate PDGF receptor activation state using a novel method, in situ proximity ligation assay, on formalin-fixed, paraffin-embedded, archival samples of 19 choroid plexus tumors. As assessed by in situ proximity ligation assay, the proportion of phosphorylated PDGF receptor alpha was low in choroid plexus papillomas and choroid plexus carcinomas, whereas phosphorylated PDGF receptor beta was found to be significantly higher in choroid plexus carcinomas. In the immortalized choroid plexus epithelial cell line Z310 expressing PDGF receptor beta, PDGF-BB exhibited a time- and dose-dependent proliferative response, which was significantly attenuated by imatinib (gleevec). In conclusion, our findings suggest that PDGF receptor beta is functionally involved in the biology of choroid plexus tumors and may represent a molecular target for therapy. In addition, this study demonstrates the feasibility and usefulness of in situ proximity ligation assay for monitoring receptor tyrosine kinase activation in formalin-fixed, paraffin-embedded, archival tissues.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-112105 (URN)10.2353/ajpath.2009.081022 (DOI)000270503100029 ()19717644 (PubMedID)
    Available from: 2010-01-08 Created: 2010-01-08 Last updated: 2017-12-12Bibliographically approved
    4. High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation
    Open this publication in new window or tab >>High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation
    Show others...
    2010 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 1, p. 178-183Article in journal (Refereed) Published
    Abstract [en]

    The cost of developing new drugs is a major obstacle for pharmaceutical companies and academia with many drugs identified in the drug discovery process failing approval for clinical use due to lack of intended effect or because of severe side effects. Since the early 1990 s, high throughput screening of drug compounds has increased enormously in capacity but has not resulted in a higher success rate of the identified drugs. Thus, there is a need for methods that can identify biologically relevant compounds and more accurately predict in vivo effects early in the drug discovery process. To address this, we developed a proximity ligation-based assay for high content screening of drug effects on signaling pathways. As a proof of concept, we used the assay to screen through a library of previously identified kinase inhibitors, including six clinically used tyrosine kinase inhibitors, to identify compounds that inhibited the platelet-derived growth factor (PDGF) receptor beta signaling pathway in stimulated primary human fibroblasts. Thirteen of the 80 compounds were identified as hits, and the dose responses of these compounds were measured. The assay exhibited a very high Z' factor (0.71) and signal to noise ratio (11.7), demonstrating excellent ability to identify compounds interfering with the specific signaling event. A comparison with regular immunofluorescence detection of phosphorylated PDGF receptor demonstrated a far superior ability by the in situ proximity ligation assay to reveal inhibition of receptor phosphorylation. In addition, inhibitor-induced perturbation of protein-protein interactions of the PDGF signaling pathway could be quantified, further demonstrating the usefulness of the assay in drug discovery.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-112102 (URN)10.1074/mcp.M900331-MCP200 (DOI)000275505900014 ()19864249 (PubMedID)
    Available from: 2010-01-08 Created: 2010-01-08 Last updated: 2017-12-12Bibliographically approved
  • 7.
    Kamali-Moghaddam, Masood
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Ekholm Pettersson, Frida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Wu, Di
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Englund, Hillevi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Darmanis, Spyros
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Lord, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Tavoosidana, Gholamreza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Gustafsdottir, Sigrun
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Nilsson, Lars N. G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Sensitive detection of A beta protofibrils by proximity ligation: relevance for Alzheimer's disease2010In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 11, p. 124-Article in journal (Refereed)
    Abstract [en]

    Background: Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of A beta peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of A beta protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity. Results: For specific detection of A beta protofibrils we have used a monoclonal antibody, mAb158, selective for A beta protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Ab aggregates in solid- phase reactions, allowing detection of just 0.1 pg/ml A beta protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Ab protofibril detection by up to 25- fold. The assay was used to measure soluble Ab aggregates in brain homogenates from mice transgenic for a human allele predisposing to A beta aggregation. Conclusions: The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of A beta aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.

  • 8.
    Kurt, Kevin
    et al.
    Robert Koch Institute, Wernigerode, Germany.
    Alderborn, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Strommenger, Birgit
    Robert Koch Institute, Wernigerode, Germany.
    Witte, Wolfgang
    Robert Koch Institute, Wernigerode, Germany.
    Nübel, Ulrich
    Robert Koch Institute, Wernigerode, Germany.
    Multiplexed genotyping of methicillin-resistant Staphylococcus aureus isolates by use of padlock probes and tag microarrays2009In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 47, no 3, p. 577-585Article in journal (Refereed)
    Abstract [en]

    We developed and tested a ligase-based assay for simultaneous probing of core genome diversity and typing of methicillin resistance determinants in Staphylococcus aureus isolates. This assay uses oligonucleotide padlock probes whose two ends are joined through ligation when they hybridize to matching target DNA. Circularized probes are subsequently amplified by PCR with common primers and analyzed by using a microarray equipped with universal tag probes. Our set of padlock probes includes oligonucleotides targeting diagnostic regions in the mecA, ccrB, and ccrC genes of the SCCmec cassette in methicillin-resistant S. aureus (MRSA). These probes determine the presence and type of SCCmec cassettes (i.e., SCCmec types I to VI). Additional oligonucleotides interrogate a number of highly informative single nucleotide polymorphisms retrieved from a multilocus sequence typing (MLST) database. These latter probes enable the exploration of isolates' phylogenetic affiliation with clonal lineages of MRSA as revealed by MLST. The described assay enables multiplexed genotyping of MRSA based on a single-tube reaction. With a set of clinical isolates of MRSA and methicillin-susceptible S. aureus (n=66), 100% typeability and 100% accuracy were achieved. The assay described here provides valuable genotypic information that may usefully complement existing genotyping procedures. Moreover, the assay is easily extendable by incorporating additional padlock probes and will be valuable for the quick and cost-effective probing of large numbers of polymorphisms at different genomic locations, such as those ascertained through currently ongoing mutation discovery and genome resequencing projects.

  • 9.
    Leuchowius, Karl-Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    High Content Analysis of Proteins and Protein Interactions by Proximity Ligation2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Fundamental to all biological processes is the interplay between biomolecules such as proteins and nucleic acids. Studies of interactions should therefore be more informative than mere detection of expressed proteins. Preferably, such studies should be performed in material that is as biologically and clinically relevant as possible, i.e. in primary cells and tissues. In addition, to be able to take into account the heterogeneity of such samples, the analyses should be performed in situ to retain information on the sub-cellular localization where the interactions occur, enabling determination of the activity status of individual cells and allowing discrimination between e.g. tumor cells and surrounding stroma. This requires assays with an utmost level of sensitivity and selectivity.

    Taking these issues into consideration, the in situ proximity-ligation assay (in situ PLA) was developed, providing localized detection of proteins, protein-protein interactions and post-translational modifications in fixed cells and tissues. The high sensitivity and selectivity afforded by the assay's requirement for dual target recognition in combination with powerful signal amplification enables visualization of single protein molecules in intact single cells and tissue sections.

    To further increase the usefulness and application of in situ PLA, the assay was adapted to high content analysis techniques such as flow cytometry and high content screening. The use of in situ PLA in flow cytometry offers the possibility for high-throughput analysis of cells in solution with the unique characteristics offered by the assay. For high content screening, it was demonstrated that in situ PLA can enable cell-based drug screening of compounds affecting post-translational modifications and protein-protein interactions in primary cells, offering superior abilities over current assays.

    The methods presented in this thesis provide powerful new tools to study proteins in genetically unmodified cells and tissues, and should offer exciting new possibilities for molecular biology, diagnostics and drug discovery.

    

    List of papers
    1. Direct observation of individual endogenous protein complexes in situ by proximity ligation
    Open this publication in new window or tab >>Direct observation of individual endogenous protein complexes in situ by proximity ligation
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    2006 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 3, no 12, p. 995-1000Article in journal (Refereed) Published
    Abstract [en]

    Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes-oligonucleotides attached to antibodies against the two target proteins-guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-gamma (IFN-gamma) signaling and low-molecular-weight inhibitors.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-16092 (URN)10.1038/nmeth947 (DOI)000242213900018 ()17072308 (PubMedID)
    Available from: 2008-06-25 Created: 2008-06-25 Last updated: 2017-12-08Bibliographically approved
    2. In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method
    Open this publication in new window or tab >>In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method
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    2007 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, no 9, p. 1500-1509Article in journal (Refereed) Published
    Abstract [en]

    Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor β (PDGFRβ) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRβ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRβ in porcine aortic endothelial cells transfected with the β-receptor, but not in cells transfected with the α-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRβ. We furthermore visualized tyrosine phosphorylated PDGFRβ in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-12659 (URN)10.1074/mcp.M700166-MCP200 (DOI)000249237200004 ()17565975 (PubMedID)
    Available from: 2008-05-28 Created: 2008-05-28 Last updated: 2017-12-11Bibliographically approved
    3. Flow cytometric in situ proximity ligation analyses of protein interactions and post-translational modification of the epidermal growth factor receptor family
    Open this publication in new window or tab >>Flow cytometric in situ proximity ligation analyses of protein interactions and post-translational modification of the epidermal growth factor receptor family
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    2009 (English)In: Cytometry: Part A, ISSN 1552-4922, Vol. 75A, no 10, p. 833-839Article in journal (Refereed) Published
    Abstract [en]

    Interactions between members of the epidermal growth factor receptor (EGFR) family mediates cellular responses to ligand stimulation. Measurement of these interactions could provide important information and may prove useful as prognostic markers in malignancy. Therefore, to develop methods to study these interactions in genetically unmodified cells, such as clinical samples, in a sensitive and selective way, with good statistical accuracy, is important. The in situ proximity ligation assay (in situ PLA) was used to quantify homo- and heteromeric interactions between EGFR and HER2 in cultured cells, using flow cytometry as the readout method. Cells were monitored for changes in dimerization patterns and phosphorylation status upon stimulation. The different cell lines displayed varying amounts of interactions between EGFR and HER2, but the amount of dimerization was not found to be affected significantly upon stimulation by EGF. Activation of EGFR could be visualized by in situ PLA, but not by immunofluorescence staining. In situ PLA was successfully used to study receptor dimerization and activation of the EGF-receptor family with high selectivity and sensitivity. The combination of in situ PLA and flow cytometry provided a statistically powerful way of analyzing protein-protein interactions and post-translational modifications on a single-cell basis.

    Keywords
    proximity ligation, flow cytometry, protein interactions, epidermal growth factor receptor, HER2
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-112106 (URN)10.1002/cyto.a.20771 (DOI)000270419700004 ()19650109 (PubMedID)
    Available from: 2010-01-08 Created: 2010-01-08 Last updated: 2018-12-04
    4. High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation
    Open this publication in new window or tab >>High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation
    Show others...
    2010 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 1, p. 178-183Article in journal (Refereed) Published
    Abstract [en]

    The cost of developing new drugs is a major obstacle for pharmaceutical companies and academia with many drugs identified in the drug discovery process failing approval for clinical use due to lack of intended effect or because of severe side effects. Since the early 1990 s, high throughput screening of drug compounds has increased enormously in capacity but has not resulted in a higher success rate of the identified drugs. Thus, there is a need for methods that can identify biologically relevant compounds and more accurately predict in vivo effects early in the drug discovery process. To address this, we developed a proximity ligation-based assay for high content screening of drug effects on signaling pathways. As a proof of concept, we used the assay to screen through a library of previously identified kinase inhibitors, including six clinically used tyrosine kinase inhibitors, to identify compounds that inhibited the platelet-derived growth factor (PDGF) receptor beta signaling pathway in stimulated primary human fibroblasts. Thirteen of the 80 compounds were identified as hits, and the dose responses of these compounds were measured. The assay exhibited a very high Z' factor (0.71) and signal to noise ratio (11.7), demonstrating excellent ability to identify compounds interfering with the specific signaling event. A comparison with regular immunofluorescence detection of phosphorylated PDGF receptor demonstrated a far superior ability by the in situ proximity ligation assay to reveal inhibition of receptor phosphorylation. In addition, inhibitor-induced perturbation of protein-protein interactions of the PDGF signaling pathway could be quantified, further demonstrating the usefulness of the assay in drug discovery.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-112102 (URN)10.1074/mcp.M900331-MCP200 (DOI)000275505900014 ()19864249 (PubMedID)
    Available from: 2010-01-08 Created: 2010-01-08 Last updated: 2017-12-12Bibliographically approved
  • 10.
    Pinidiyaarachchi, Amalka
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis.
    Zieba, Agata
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Allalou, Amin
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis.
    Pardali, Katerina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis.
    A detailed analysis of 3D subcellular signal localization2009In: Cytometry Part A, ISSN 1552-4922, Vol. 75A, no 4, p. 319-328Article in journal (Refereed)
    Abstract [en]

    Detection and localization of fluorescent signals in relation to other subcellular structures is an important task in various biological studies. Many methods for analysis of fluorescence microscopy image data are limited to 2D. As cells are in fact 3D structures, there is a growing need for robust methods for analysis of 3D data. This article presents an approach for detecting point-like fluorescent signals and analyzing their subnuclear position. Cell nuclei are delineated using marker-controlled (seeded) 3D watershed segmentation. User-defined object and background seeds are given as input, and gradient information defines merging and splitting criteria. Point-like signals are detected using a modified stable wave detector and localized in relation to the nuclear membrane using distance shells. The method was applied to a set of biological data studying the localization of Smad2-Smad4 protein complexes in relation to the nuclear membrane. Smad complexes appear as early as 1 min after stimulation while the highest signal concentration is observed 45 min after stimulation, followed by a concentration decrease. The robust 3D signal detection and concentration measures obtained using the proposed method agree with previous observations while also revealing new information regarding the complex formation.

  • 11. Szado, Tania
    et al.
    Vanderheyden, Veerle
    Parys, Jan B
    De Smedt, Humbert
    Rietdorf, Katja
    Kotelevets, Larissa
    Chastre, Eric
    Khan, Farid
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Bootman, Martin D
    Roderick, H Llewelyn
    Phosphorylation of inositol 1,4,5-trisphosphate receptors by protein kinase B/Akt inhibits Ca2+ release and apoptosis.2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 7, p. 2427-2432Article in journal (Refereed)
    Abstract [en]

    Imbalance of signals that control cell survival and death results in pathologies, including cancer and neurodegeneration. Two pathways that are integral to setting the balance between cell survival and cell death are controlled by lipid-activated protein kinase B (PKB)/Akt and Ca2+. PKB elicits its effects through the phosphorylation and inactivation of proapoptotic factors. Ca2+ stimulates many prodeath pathways, among which is mitochondrial permeability transition. We identified Ca2+ release through inositol 1,4,5-trisphosphate receptor (InsP(3)R) intracellular channels as a prosurvival target of PKB. We demonstrated that in response to survival signals, PKB interacts with and phosphorylates InsP(3)Rs, significantly reducing their Ca2+ release activity. Moreover, phosphorylation of InsP(3)Rs by PKB reduced cellular sensitivity to apoptotic stimuli through a mechanism that involved diminished Ca2+ flux from the endoplasmic reticulum to the mitochondria. In glioblastoma cells that exhibit hyperactive PKB, the same prosurvival effect of PKIB on InsP(3)R was found to be responsible for the insensitivity of these cells to apoptotic stimuli. We propose that PKIB-mediated abolition of InsP(3)-induced Ca2+ release may afford tumor cells a survival advantage.

  • 12.
    Thunberg, Ulf
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Tobin, Gerard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Johnson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Padyukov, Leonid
    Hultdin, Magnus
    Klareskog, Lars
    Enblad, Gunilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Sundström, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular and Morphological Pathology.
    Roos, Göran
    Rosenquist, Richard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Polymorphism in the P2X7 receptor gene and survival in chronic lymphocytic leukaemia2002In: The Lancet, ISSN 0140-6736, E-ISSN 1474-547X, Vol. 360, no 9349, p. 1935-1939Article in journal (Refereed)
    Abstract [en]

    BACKGROUND:

    The P2X7 receptor is a ligand-gated cation channel that mediates ATP-induced apoptotic death in haemopoietic and chronic lymphocytic leukaemia (CLL) cells. We aimed to investigate the clinical effect of a single nucleotide polymorphism in the P2X7 receptor gene (1513A-->C) and correlate findings with the immunoglobulin heavy chain variable (V(H)) gene mutation status, a prognostic marker in CLL.

    METHODS:

    We investigated tumour DNA in 170 patients with CLL using PCR-RFLP analysis with HhaI restriction enzyme cleavage to screen for the polymorphism in the P2X7 receptor gene. The VH gene mutation status was assessed in 165 patients by PCR amplification and nucleotide sequencing. We correlated the findings of the P2X7 receptor genotype with the V(H) gene mutation data and overall survival and screened for the P2X7 receptor polymorphism in 200 healthy controls.

    FINDINGS:

    Of the 170 patients, 35 (21%) were heterozygous and one (1%) homozygous for the 1513C allele; whereas 134 (79%) had the 1513A/A genotype of the P2X7 receptor gene. Overall survival was significantly longer for patients with CLL heterozygous for the 1513C allele than those with the 1513A/A genotype. Median survival for heterozygous patients was 104 months (range 33-467) and 72 months (1-190) for homozygous patients (p=0.009). Of the 165 patients with CLL in whom we assessed the V(H) gene mutation status, the V(H) genes were mutated in 71 (43%) patients and unmutated in 94; 18 (25%) of the 71 patients with mutated genes had 1513C allele compared with 17 (18%) of 94 who had unmutated genes. In patients with mutated VH genes, those with CLL who were 1513C positive had 53 months' longer median survival than did those with the 1513A/A genotype (151 vs 98 months, p=0.011).

    INTERPRETATION:

    The P2X7 polymorphism could affect clinical outcome in CLL, especially in patients with mutated V(H) genes. Studies are necessary to elucidate the biological role of the P2X7 polymorphism in CLL in vivo.

  • 13.
    Tobin, Gerard
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Thunberg, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Johnson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Botling, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular and Morphological Pathology.
    Enblad, Gunilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Sällström, Jan
    Sundström, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular and Morphological Pathology.
    Roos, Göran
    Rosenquist, Richard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Response to "Do B-cell chronic lymphocytic leukemia patients with Ig VH3-21 genes constitute a new subset of chronic lymphocytic leukemia?": IgVH3-21 gene usage in chronic lymphocytic leukemia2002In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 100, no 3, p. 1098-1099Article in journal (Other academic)
  • 14. Wedrén, Sara
    et al.
    Lovmar, Lovisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
    Humphreys, Keith
    Magnusson, Cecilia
    Melhus, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Syvänen, Ann-Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
    Kindmark, Andreas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Fermér, Maria Lagerström
    Stiger, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Persson, Ingemar
    Baron, John A.
    Weiderpass, Elisabete
    Estrogen receptor alpha gene polymorphism and endometrial cancer risk: a case-control study2008In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 8, p. 322-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND:

    Estrogen is an established endometrial carcinogen. One of the most important mediators of estrogenic action is the estrogen receptor alpha. We have investigated whether polymorphic variation in the estrogen receptor alpha gene (ESR1) is associated with endometrial cancer risk.

    METHODS:

    In 702 cases with invasive endometrial cancer and 1563 controls, we genotyped five markers in ESR1 and used logistic regression models to estimate odds ratios (OR) and 95 percent confidence intervals (CI).

    RESULTS:

    We found an association between rs2234670, rs2234693, as well as rs9340799, markers in strong linkage disequilibrium (LD), and endometrial cancer risk. The association with rs9340799 was the strongest, OR 0.75 (CI 0.60-0.93) for heterozygous and OR 0.53 (CI 0.37-0.77) for homozygous rare compared to those homozygous for the most common allele. Haplotype models did not fit better to the data than single marker models.

    CONCLUSION:

    We found that intronic variation in ESR1 was associated with endometrial cancer risk.

1 - 14 of 14
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