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  • 1.
    Ansell, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Farnebo, Lovisa
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Grénman, Reidar
    Department of Otorhinolaryngology, Head & Neck Surgery, and Medical Biochemistry, University of Turku, Finland.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Thunell, Lena
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Polymorphism of FGFR4 in cancer development and sensitivity to cisplatin and radiation in head and neck cancer2009In: Oral Oncology, ISSN 1368-8375, E-ISSN 1879-0593, Vol. 45, no 1, p. 23-29Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to investigate the predisposition of the FGFR4 Gly/Arg polymorphism for development of head and neck squamous cell carcinoma (HNSCC) and, furthermore, to examine if the FGFR4 Arg(388) allele can be associated with resistance to chemo-and radiotherapy.

    When analysing 110 tumour biopsies a significant 1.7-fold increased risk to develop HNSCC in individuals carrying the Gly(388) allele (p = 0.026) was found. Moreover a 2-fold increased risk for mates harbouring the Gly(388) allele (p = 0.031) to develop HNSCC was detected. In 39 HNSCC cell lines the role of the Arg(388) allele for radiation and cisplatin sensitivity was investigated. Our results show no rote of the Arg(388) allele for the radiosensitivity (p = 0.996) but indicate a tendency to increased cisplatin sensitivity (p = 0.141). When screening the transmembrane and kinase domains in the FGFR4 gene a novel mutation, probably generating a truncated protein lacking exons 14-18, was found in six of eight selected cell lines.

    Taken together, we have here identified a marker that predicts the risk to develop HNSCC and possibly the sensitivity to cisplatin as well as a novel. mutation in the FGFR4 gene.

  • 2.
    Ansell, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
    Jedlinski, Adam
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    Johansson, Ann-Charlotte
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
    Roberg, Karin
    Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping. Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science.
    Epidermal growth factor is a biomarker for poor cetuximab response in tongue cancer cells2016In: Journal of Oral Pathology & Medicine, ISSN 0904-2512, E-ISSN 1600-0714, Vol. 45, no 1, p. 9-16Article in journal (Refereed)
    Abstract [en]

    Background: Epidermal growth factor receptor (EGFR) is a target for treatment in tongue cancer. Here, EGFR ligands were evaluated for their potential uses as predictive biomarkers of cetuximab treatment response.

    Methods: In three tongue cancer cell lines the influences of epidermal growth factor (EGF), amphiregulin (AR), and epiregulin (EPR) on tumour cell proliferation and cetuximab response were evaluated by the addition of recombinant human (rh) proteins or the siRNA-mediated downregulation of endogenous ligand production.

    Results: EGF or AR downregulation suppressed the proliferation of all investigated cell lines. Furthermore, all cell lines displayed increased cetuximab resistance upon the addition of rhEGF, whereas EGF silencing resulted in an improved cetuximab response in one cell line.

    Conclusions: Our data suggest that EGF and AR are critical components of the EGFR signalling network required for full proliferative potential. Moreover, EGF is a potential predictive biomarker of poor cetuximab response and a possible treatment target.

  • 3.
    Ansell, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Jerhammar, Fredrik
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Ceder, R
    Karolinska Institute, Institute Environm Med, Div Biochem Toxicol and Expt Canc Research, S-10401 Stockholm, Sweden .
    Grenman, R
    Turku University, Department Otorhinolaryngol Head and Neck Surg, Cent Hospital, Turku, Finland .
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Matrix metalloproteinase-7 and-13 predict response to cisplatin in head and neck cancer in ORAL ONCOLOGY, vol , issue , pp 94-942009In: ORAL ONCOLOGY, 2009, p. 94-94Conference paper (Refereed)
    Abstract [en]

    n/a

  • 4.
    Ansell, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Jerhammar, Fredrik
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Ceder, Rebecca
    Karolinska Institute, Stockholm, Sweden.
    Grafström, Roland
    VTT Technical Research Centre of Finland.
    Grénman, Reidar
    VTT Technical Research Centre of Finland.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Matrix metalloproteinase-7 and -13 expression associate to cisplatin resistance in head and neck cancer cell lines.2009In: Oral Oncology, ISSN 1368-8375, E-ISSN 1879-0593, Vol. 45, no 10, p. 866-871Article in journal (Refereed)
    Abstract [en]

    Concomitant chemoradiotherapy is a common treatment for advanced head and neck squamous cell carcinomas (HNSCC). Cisplatin is the backbone of chemotherapy regimens used to treat HNSCC. Therefore, the aim of this study was to identify predictive markers for cisplatin treatment outcome in HNSCC. The intrinsic cisplatin sensitivity (ICS) was determined in a panel of tumour cell lines. From this panel, one sensitive and two resistant cell lines were selected for comparative transcript profiling using microarray analysis. The enrichment of Gene Ontology (GO) categories in sensitive versus resistant cell lines were assessed using the Gene Ontology Tree Machine bioinformatics tool. In total, 781 transcripts were found to be differentially expressed and 11 GO categories were enriched. Transcripts contributing to this enrichment were further analyzed using Ingenuity Pathway Analysis (IPA) for identification of key regulator genes. IPA recognized 20 key regulator genes of which five were differentially expressed in sensitive versus resistant cell lines. The mRNA level of these five genes was further assessed in a panel of 25 HNSCC cell lines using quantitative real-time PCR. Among these key regulators, MMP-7 and MMP-13 are implicated as potential biomarkers of ICS. Taken together, genome-wide transcriptional analysis identified single genes, GO categories as well as molecular networks that are differentially expressed in HNSCC cell lines with different ICS. Furthermore, two novel predictive biomarkers for cisplatin resistance, MMP-7 and MMP-13, were identified.

  • 5.
    Ansell, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences.
    Jerhammar, Fredrik
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences.
    Ceder, Rebecca
    Institute of Environmental Medicine, Division of Biochemical Toxicology and Experimental Cancer Research, Karolinska Institution, Stockholm, Sweden.
    Grénman, Reidar
    Department of Otorhinolaryngology, Head & Neck Surgery, Turku University Central Hospital, Finland.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Matrix metalloproteinase-7 and -13 predict response to cisplatin in head and neck cancerManuscript (preprint) (Other academic)
    Abstract [en]

    Purpose: To identify gene ontology categories and key regulators with impact on the intrinsic cisplatin sensitivity (ICS) in head and neck squamous cell carcinoma (HNSCC).

    Experimental design: The ICS was determined in 35 HNSCC cell lines. Three of these cell lines, one sensitive and two resistant, were selected for microarray analysis. Gene Ontology (GO) categories were assessed using the gene ontology tree machine (GOTM) tool, and transcripts included in these categories were further analyzed using Ingenuity Pathway Analysis (IPA) for detection of key regulator genes. A group of key regulators were verified at protein level by Western blot analysis and on mRNA level using quantitative real-time PCR (qPCR).

    Results: 781 transcripts were detected as significantly differently expressed for the resistant cell lines compared to the sensitive cell line. A total of ten different categories were enriched in GOTM by these transcripts and a transcriptional profile was made from the 20 key regulators identified in the IPA analysis. Five key regulator genes, apolipoprotein E (APOE), catenin beta1 (CTNNB1), matrix metalloproteinase-7 (MMP-7), matrix metalloproteinase-13 (MMP-13), and thrombospondin 1 (THBS1), were verified in 25 HNSCC cell lines on mRNA level using qPCR. The results confirmed MMP-7 (p=0.0013) and implied MMP-13 (p=0.058) as potential biomarkers of ICS.

    Conclusions: We conclude that genome-wide transcriptional analysis and appropriate bioinformatics enable the identification of genes with impact on treatment response. Furthermore, we propose MMP-7 and MMP-13 as predictive markers of cisplatin resistance in HNSCC.

  • 6.
    Ansell, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuroscience. Linköping University, Faculty of Health Sciences.
    Kankainen, M.
    Institute of Biomedicine, Medical Biochemistry and Developmental Biology, Genome-Scale Biology, Research Program, University of Helsinki, Helsinki, Finland.
    Jönsson, Jan-Ingvar
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Monni, O.
    Institute of Biomedicine, Medical Biochemistry and Developmental Biology, Genome-Scale Biology, Research Program, University of Helsinki, Helsinki, Finland.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuroscience. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    Johansson, Ann-Charlotte
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuroscience. Linköping University, Faculty of Health Sciences.
    Molecular cross-talk between head and neck squamous cell carcinoma cells and cancer-associated fibroblasts2013Manuscript (preprint) (Other academic)
    Abstract [en]

    Cancer-associated fibroblasts (CAFs) are one of the main components of the tumor stroma and are known to increase tumor growth and stimulate  invasion and metastasis. Increasing evidence suggests that CAFs may also be an important determinant of the response to various treatments. In this study we aimed to characterize the molecular cross-talk between CAFs and head and neck squamous cell carcinoma (HNSCC) cells.

    HNSCC cell lines were co-cultured with their patient-matched CAFs for seven days, after which the gene expression of tumor cells was investigated by Affymetrix microarray. 58 protein coding genes were found to be differentially expressed (Q≤0.05) in tumor cells cocultured with CAFs when compared to tumor cells cultured alone. The top functions of these genes were cancer, cellular movement, and embryonic development as analyzed by Ingenuity Pathway Analysis. Nine genes were upregulated by ≥1.5-fold while the expression of 35 genes was found to be reduced by ≤ 0.67-fold. Several of the differentially expressed genes have been associated with epithelial-to-mesenchymal transition (EMT). The change in the expression of POSTN, GREM1, COL1A2, VIM, and MMP7 was verified by qPCR analysis. Moreover, the influence of CAFs on the proliferation, migration and cetuximab sensitivity of tumor cells was investigated, and was found to vary among the tumor cell-CAF pairs.

    In conclusion, we demonstrate that CAF-derived signals cause changes in the expression of multiple genes, several of which are associated with an EMT phenotype of tumor cells. Furthermore, CAFs modulate the proliferation, migration and cetuximab treatment response of tumor cells.

  • 7.
    Ceder, Rebecca
    et al.
    Karolinska Institute.
    Haig, Ylva
    Karolinska Institute.
    Merne, Marina
    Karolinska Institute.
    Hansson, Annette
    Karolinska Institute.
    Zheng, Xi
    Karolinska Institute.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL.
    Nees, Matthias
    VTT Technical Research Centre Finland.
    Iljin, Kristiina
    VTT Technical Research Centre Finland.
    Bloor, Balvinder K
    Kings College London.
    Morgan, Peter R
    Kings College London.
    Fadeel, Bengt
    Karolinska Institute.
    Grafstrom, Roland C
    Karolinska Institute.
    Differentiation-Promoting Culture of Competent and Noncompetent Keratinocytes Identifies Biomarkers for Head and Neck Cancer2012In: American Journal of Pathology, ISSN 0002-9440, E-ISSN 1525-2191, Vol. 180, no 2, p. 457-472Article in journal (Refereed)
    Abstract [en]

    Aberrant contact-inhibited proliferation and differentiation induction couple with tumor severity, albeit with an imprecise association with prognosis. Assessment of contact inhibition and differentiation-promoting culture in this study of normal and immortalized oral keratinocytes (NOK and SVpgC2a, respectively) demonstrated elevated cloning ability and saturation density in the immortalized versus normal state, including consistent absence of differentiated morphological features. Transcriptomic analysis implicated 48 gene ontology categories, 8 molecular networks, and 10 key regulator genes in confluency-induced differentiation of NOK, all of which remained nonregulated in SVpgC2a. The SVpgC2a versus NOK transcriptome enriched 52 gene ontology categories altogether, 18 molecular networks, and 39 key regulator genes, several of which were associated with epithelial-mesenchymal transition. Assessment of the previously described gene sets relative to training data sets of head and neck squamous cell carcinoma samples, one including data on tumor differentiation and patient outcome and one present in the Human Gene Expression Map, identified four genes with association to poor survival (COX7A1, MFAP5, MPDU1, and POLD1). This gene set predicted poor outcome in an independent data set of 71 head and neck squamous cell carcinomas. The present study defines, for the first time to our knowledge, the broad gene spectrum that couples to induction, and loss, of oral keratinocyte differentiation. Bioinformatics assessments of the results relative to clinical data generated novel differentiation-related tumor biomarkers relevant to patient outcome.

  • 8. D`Herde, Katharina
    et al.
    Mussche, Sylvie
    Roberg, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology.
    Morphological changes in dying cells2003In: Cell proliferation & apoptosis / [ed] D Hughes, FIMLS, H Mehmet;, Linköping: Linköpings universitet , 2003, p. 201-231Chapter in book (Other academic)
    Abstract [en]

    Cell Proliferation and Apoptosis provides a detailed and novel practical guide to cell proliferation and apoptosis detection methods.

  • 9.
    Eriksson, Ida
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Joosten, M.
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    Öllinger, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.
    The histone deacetylase inhibitor trichostatin A reduces lysosomal pH and enhances cisplatin-induced apoptosis2013In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 319, no 1, p. 12-20Article in journal (Refereed)
    Abstract [en]

    High activity of histone deacetylases (HDACs) has been documented in several types of cancer and may be associated with survival advantage. In a head and neck squamous cell carcinoma cell line, cisplatin-induced apoptosis was augmented by pretreatment with the HDAC inhibitor trichostatin Apoptosis was accompanied by lysosomal membrane permeabilization (LMP), as shown by immunoblotting of the lysosomal marker protease cathepsin B in extracted cytosol and by immunofluorescence. Moreover, LAMP-2 (lysosomal associated membrane protein-2) was translocated from lysosomal membranes and found in a digitonin extractable fraction together with cytosolic proteins and pretreatment with trichostatin A potentiated the release. Overall, protein level of LAMP-2 was decreased during cell death and, interestingly, inhibition of cysteine cathepsins, by the pan-cysteine cathepsin inhibitor zFA-FMK, prevented loss of LAMP-2. The importance of LAMP-2 for lysosomal membrane stability, was confirmed by showing that LAMP-2 knockout MEFs (mouse embryonic fibroblasts) were more sensitive to cisplatin as compared to the corresponding wildtype cells. Trichostatin A reduced lysosomal pH from 4.46 to 4.25 and cell death was prevented when lysosomal pH was increased by NH4Cl, or when inhibiting the activity of lysosomal proteases. We conclude that trichostatin A enhances cisplatin induced cell death by decreasing lysosomal pH, which augments cathepsin activity resulting in reduced LAMP-2 level, and might promote LMP.

  • 10.
    Farnebo, Lovisa
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Jedlinski, Adam
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Ansell, Anna
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences.
    Vainikka, Linda
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Thunell, Lena
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Grenman, Reidar
    University of Turku.
    Johansson, Ann-Charlotte
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Proteins and single nucleotide polymorphisms involved in apoptosis, growth control, and DNA repair predict cisplatin sensitivity in head and neck cancer cell lines2009In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 24, no 4, p. 549-556Article in journal (Refereed)
    Abstract [en]

    The present study was undertaken to evaluate the possibility of using a panel of proteins and single nucleotide polymorphisms (SNPs) involved in apoptosis, growth control, and DNA repair as predictive markers for cisplatin sensitivity. For this purpose the intrinsic cisplatin sensitivity (ICS) was determined in 39 cell lines derived from squamous cell carcinomas of the head and neck using a colony-forming assay. In these cell lines and in normal oral keratinocytes (NOK), the expression of epidermal growth factor receptor (EGFR), Hsp70, Bax, Bcl-2, Bcl-XL, survivin, and COX-2 was determined. Moreover, the p53, MDM2, FGFR4, XPC, XPD, XRCC1, and XRCC3 genes were analyzed for the presence of specific single nucleotide polymorphisms (SNPs). Pearsons correlation test showed that EGFR was the only protein that was significantly correlated to the ICS (r=0.388, p=0.015). The combination of EGFR, Hsp70, Bax, and Bcl-2 gave the strongest correlation (r=0.566, p andlt;= 0.001), whereas Bax alone had the second highest influence on the ICS. Furthermore, all four SNPs within genes involved in DNA repair, i.e. XPC, XPD, XRCC1, and XRCC3, tended to influence the ICS. In order to find the combination of factors, on both protein and gene levels, with the highest correlation to ICS, a multivariate statistical calculation was performed. Our results indicate that SNPs in DNA repair genes (XRCC3(241) and XPD751) influence the ICS and together with the expression of EGFR, Hsp70, Bax, and Bcl-2, they could predict the cisplatin sensitivity of head and neck cancer cell lines (r=0.614, p andlt;= 0.001).

  • 11.
    Farnebo, Lovisa
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Jerhammar, Fredrik
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Ceder, Rebecca
    Institute of Environmental Medicine, Division of Biochemical Toxicology and Experimental Cancer Research, Karolinska Institute, Stockholm, Sweden.
    Grafström, Roland C
    Institute of Environmental Medicine, Division of Biochemical Toxicology and Experimental Cancer Research, Karolinska Institute, Stockholm, Sweden.
    Vainikka, Linda
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Thunell, Lena
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Grénman, Reidar
    Medical Biochemistry, University of Turku, Finland.
    Johansson, Ann-Charlotte
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Combining factors on protein and gene level to predict radioresponse in head and neck cancer cell lines2011In: Journal of Oral Pathology & Medicine, ISSN 0904-2512, E-ISSN 1600-0714, Vol. 40, no 10, p. 739-746Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Radiotherapy is the main therapy for head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems, highlighting the need for predictive markers. In this study, we evaluated selected proteins, mutations, and single nucleotide polymorphisms (SNPs) involved in apoptosis, cell proliferation, and DNA repair alone or combined as predictive markers for radioresponse in 42 HNSCC cell lines.

    METHODS: The expression of epidermal growth factor receptor, survivin, Bax, Bcl-2, Bcl-XL, cyclooxygenase-2, and heat shock protein 70 was analyzed by ELISA. Furthermore, mutations and SNPs in the p53 gene as well as SNPs in the MDM2, XRCC1, and XRCC3 genes were analyzed for their relation to radioresponse. To enable the evaluation of the predictive value of several factors combined, each cell line was allocated points based on the number of negative points (NNP) system, and the NNP sum was correlated with radioresponse.

    RESULTS: Survivin was the only factor that alone was significantly correlated with the intrinsic radiosensitivity (r=0.36, p=0.02). The combination of survivin, Bax, Bcl-2, Bcl-XL, cyclooxygenase-2, and the p53 Arg72Pro polymorphism was found to most strongly correlate with radioresponse (r=0.553, p<0.001).

    CONCLUSION: These data indicate that the intrinsic radiosensitivity of 42 HNSCC cell lines can be predicted by a panel of factors on both the protein and gene levels. Moreover, among the investigated factors, survivin was the most promising biomarker of radioresponse.

  • 12.
    Farnebo, Lovisa
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Jerhammar, Fredrik
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences.
    Vainikka, Linda
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Grénman, Reidar
    Department of Otorhinolaryngology, Head and Neck Surgery, Central Hospital and University of Turku and Medical Biochemistry, University of Turku, Finland.
    Norberg-Spaak, Lena
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences.
    Number of negative points: a novel method for predicting radiosensitivity in head and neck tumor cell lines.2008In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 20, no 2, p. 453-461Article in journal (Refereed)
    Abstract [en]

    The present study was aimed at establishing a method that combines multiple factors of protein and genetic changes that enables prediction of radiosensitivity in the head and neck squamous cell carcinoma (HNSCC) cell lines. In nine HNSCC cell lines, the quantity of protein expression and the type of genetic alterations were translated into a point system, called the Number of Negative Points. The expression of 14 proteins involved in growth control and/or apoptosis was quantified using a densitometric assessment of Western blots. The blots were adjusted to actin and standardised to normal oral keratinocytes classifying them into four groups depending on the amount of protein expressed (0-3 points). Mutations of the p53 gene were classified into three groups and each mutation was given one point. Since the cell lines each had a known intrinsic radiosensitivity, a multivariate statistical calculation could then be performed to select for the combination of factors having the strongest correlation to radiosensitivity. The strongest correlation of the investigated factors was the combination of epidermal growth factor receptor, survivin and splice site/missense p53 mutations (R=0.990 and P<0.0001). No single factor had a significant correlation to the intrinsic radiosensitivity. Since a significant correlation to the intrinsic radiosensitivity was achieved only when two or more factors were combined, we conclude that a method such as the Number of Negative Points is necessary for prediction of treatment response. We present a novel method to combine factors which enables the prediction of radiosensitivity of HNSCC cell lines.

  • 13.
    Farnebo, Lovisa
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    Tiefenböck, Katharina
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Ansell, Anna
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuroscience. Linköping University, Faculty of Health Sciences.
    Thunell, Lena
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Garvin, Stina
    Linköping University, Department of Clinical and Experimental Medicine, Oncology. Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuroscience. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    Strong expression of survivin is associated with positive response to radiotherapy and improved overall survival in head and neck squamous cell carcinoma patients2013In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 133, no 8, p. 1994-2003Article in journal (Refereed)
    Abstract [en]

    Head and neck squamous cell carcinoma (HNSCC) is a malignancy that is associated with severe mortality despite advances in therapy. Todays standard treatment most commonly includes radiotherapy, often combined with chemotherapy or surgery. There are so far no established biomarkers to predict response to radiation, and thus the aim of this study was to investigate a series of markers that could potentially identify HNSCC patients who would benefit from radiotherapy. The selected markers, both proteins (epidermal growth factor receptor, survivin and p53), and single nucleotide polymorphisms (SNPs) in the genes of XRCC3, XRCC1, XPC, XPD, MDM2, p53 and FGFR4 were correlated to the response to radiotherapy and overall survival. Investigations were performed on pretreatment tumor biopsies from patients classified as responders or nonresponders to radiotherapy. Protein expression was examined using immunohistochemistry and the genotyping of specific SNPs was analyzed using PCR-RFLP or pyrosequencing. We found that survivin expression was significantly stronger in the responder group (p = 0.003) and that patients with a strong survivin expression had a significantly better overall survival (p andlt; 0.001). Moreover, downregulation of survivin by siRNA in two HNSCC cell lines significantly decreased their sensitivity to radiation. Among the SNPs analyzed, patients with the XPD Lys751Gln SNP had a significantly shorter overall survival (p = 0.048), and patients with the FGFR4 Gly388Arg SNP had a significantly longer overall survival (p = 0.010). In conclusion, our results suggest that survivin plays an important role in the response to radiotherapy and may be a useful marker for predicting radiotherapy response in patients with HNSCC. less thanbrgreater than less thanbrgreater thanWhats new? Resistance to radiation therapy is a significant problem in the treatment of head and neck squamous cell carcinoma (HNSCC) and has created a need for the discovery of markers predictive of radiotherapy response. One promising marker is survivin, an inhibitor of apoptosis. Here, in pre-treatment biopsies from 40 patients with HNSCC, strong survivin expression was significantly associated with response to radiotherapy and increased overall survival. The data also indicate that single nucleotide polymorphisms in the genes XPD and FGFR4 are other possible predictors of overall survival after radiotherapy.

  • 14. Garner, B.
    et al.
    Roberg, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Qian, M.
    Brunk, Ulf
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Eaton, J W
    Truscott, RJW
    Redox availability of lens iron and copper: Implication for HO generation in cataract.1999In: Redox report, ISSN 1351-0002, E-ISSN 1743-2928, Vol. 4, p. 313-315Article in journal (Refereed)
  • 15. Garner, B
    et al.
    Roberg, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Qian, M
    Eaton, JW
    Truscott, RJW
    Distribution of ferritin and redox-active transition metals in normal and cataractous human lenses2000In: Experimental Eye Research, ISSN 0014-4835, E-ISSN 1096-0007, Vol. 71, no 6, p. 599-607Article in journal (Refereed)
    Abstract [en]

    Previous studies have shown that lenticular levels of Fe and Cu are elevated in age-related cataract. However, it is not known if these metals are present in a state that is permissive for redox reactions that may lead to the formation of free radicals. In addition, there is little data available concerning the concentration and lenticular distribution of ferritin, the major intracellular Fe-sequestering protein, in the lens. The aim of the present work was therefore to determine the distribution of ferritin and the redox-availability of Fe and Cu in healthy and cataractous lenses. Lens ferritin distribution was assessed by ELISA and immunohistochemistry. A modified ELISA detected ferritin in an 'insoluble' lens protein fraction. Ferritin levels were not significantly different in the cortex vs nucleus of healthy lenses. In contrast, ferritin levels in the cataractous lens nuclei appeared to be 70 % lower compared to the cortex. This was at least partially due to the presence of ferritin within an insoluble protein fraction of the homogenized lenses. In normal lenses, ferritin staining was most intense in the epithelium, with diffuse staining observed throughout the cortex and nucleus. The redox-availability of lenticular metals was determined using: (1) autometallography, (2) Ferene-S as a chromogenic Fe chelator, and (3) NO release from nitrosocysteine to probe for redox-active Cu. The antometallography studies showed that the cataractous lenses stained more heavily for redox-active metals in both the nucleus and cortex when compared to age-matched control lenses. Chelatable Fe was detected in homogenized control lenses after incubation with Ferene-S, with almost three-fold higher levels detected in the cataractous lenses on average. The Cu-catalysed liberation of NO from added nitrosocysteine was not demonstrated in any lens sample. When exogenous Cu (50 nM) was added to the lenses, it was rapidly chelated. The cataractous samples were approximately twice as effective at redox-inactivation of added Cu. These studies provide evidence that a chelatable pool of potentially redox-active Fe is present at increased concentrations in human cataractous lenses. In contrast, it seems that lenticular Cu may not be readily available for participation in redox reactions. (C) 2000 Academic Press.

  • 16.
    Garvin, Stina
    et al.
    Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics. Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    Tiefenböck, Katharina
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    Farnebo, Lovisa
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    Thunell, Lena
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Farnebo, Marianne
    Karolinska Institute, Sweden.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    Nuclear expression of WRAP53 beta is associated with a positive response to radiotherapy and improved overall survival in patients with head and neck squamous cell carcinoma2015In: Oral Oncology, ISSN 1368-8375, E-ISSN 1879-0593, Vol. 51, no 1, p. 24-30Article in journal (Refereed)
    Abstract [en]

    Objectives: Today there are no reliable predictive markers for radiotherapy response in head and neck squamous cell carcinoma (HNSCC), leading to both under-and over-treatment of patients, personal suffering, and negative socioeconomic effects. Inherited mutation in WRAP53 beta (WD40 encoding RNA Antisense to p53), a protein involved in intracellular trafficking, dramatically increases the risk of developing HNSCC. The purpose of this study was to investigate whether WRAP53 beta can predict response to radiotherapy in patients with HNSCC. Materials and methods: Tumor biopsies from patients with HNSCC classified as responders or non-responders to radiotherapy were examined for the expression of the WRAP53 beta protein and single nucleotide polymorphisms in the corresponding gene employing immunohistochemistry and allelic discrimination, respectively. In addition, the effect of RNAi-mediated downregulation of WRAP53 beta on the intrinsic radiosensitivity of two HNSCC cell lines was assed using crystal violet and clonogenic assays. Results: Nuclear expression of WRAP53 beta was significantly associated with better response to radiotherapy and improved patient survival. Downregulation of WRAP53 beta with siRNA in vitro enhanced cellular resistance to radiation. Conclusions: Our findings suggest that nuclear expression of WRAP53 beta promotes tumor cell death in response to radiotherapy and is a promising predictor of radiotherapy response in patients with HNSCC.

  • 17.
    Grafstrom, Roland C.
    et al.
    Karolinska Institute, Sweden .
    Ceder, Rebecca
    Karolinska Institute, Sweden .
    Fadeel, Bengt
    Karolinska Institute, Sweden .
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL.
    Willighagen, Egon
    Karolinska Institute, Sweden .
    Bioinformatics-based cancer research have wide toxicological applicability in TOXICOLOGY LETTERS, vol 211, issue , pp S160-S1602012In: TOXICOLOGY LETTERS, Elsevier , 2012, Vol. 211, p. S160-S160Conference paper (Refereed)
    Abstract [en]

    n/a

  • 18.
    Hultcrantz, Elisabeth
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Harder, Lena
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Zetterlund, Eva-Lena
    Östergötlands Läns Landsting, Anaesthesiology and Surgical Centre, Department of Anaesthesiology and Intensive Care VHN.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    To treat snoring with nasal steroids - effects on more than one level?2010In: ACTA OTO-LARYNGOLOGICA, ISSN 0001-6489, Vol. 130, no 1, p. 124-131Article in journal (Refereed)
    Abstract [en]

    Conclusion. An inflammatory swelling in the uvula and nose due to vibration might be a contributing factor in snoring. The presence of corticosteroid receptors in the uvula indicates the possibility for treatment with local steroids. Use of mometasone furoate (MF) for 3 months reduced snoring and related symptoms in some patients. Objective. To investigate the effect of a nasal steroid, MF, on snoring and related discomfort. Subjects and methods. In the first part of the study, uvular and nasal biopsies from six patients with social snoring were examined using immunohistochemistry to evaluate whether corticosteroid receptors were present. Then 100 snoring patients were invited to participate in the second part of the study. In all, 72 men and 22 women with a mean age of 47 years and BMI 27 answered a questionnaire about symptoms, had ENT status assessed and reported sleep and related variables for a 7 day period. After randomization to placebo or MF, they used a nasal spray for 3 months at a dosage of 200 mu g. Thereafter the procedure was repeated. Results. Corticosteroid receptors were present in the mucous membranes and around the blood vessels in all uvulas examined. A total of 84 patients were evaluated. No decrease in mean snoring score was seen. Daytime sleepiness showed a slight improvement in the MF group and partners were less disturbed. Minor side effects were equal for both groups.

  • 19.
    Jain, Mayur Vilas
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Paczulla, Anna M.
    University of Tubingen, Germany .
    Klonisch, Thomas
    University of Manitoba, Canada .
    Dimgba, Florence N.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Rao, Sahana
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    Schweizer, Frank
    University of Manitoba, Canada .
    Lengerke, Claudia
    University of Tubingen, Germany .
    Davoodpour, Padideh
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Health Sciences.
    Palicharla, Vivek R.
    Centre DNA Fingerprinting and Diagnost CDFD, India .
    Maddika, Subbareddy
    Centre DNA Fingerprinting and Diagnost CDFD, India .
    Los, Marek Jan
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Interconnections between apoptotic, autophagic and necrotic pathways: implications for cancer therapy development2013In: Journal of Cellular and Molecular Medicine (Print), ISSN 1582-1838, E-ISSN 1582-4934, Vol. 17, no 1, p. 12-29Article, review/survey (Refereed)
    Abstract [en]

    The rapid accumulation of knowledge on apoptosis regulation in the 1990s was followed by the development of several experimental anticancer- and anti-ischaemia (stroke or myocardial infarction) drugs. Activation of apoptotic pathways or the removal of cellular apoptotic inhibitors has been suggested to aid cancer therapy and the inhibition of apoptosis was thought to limit ischaemia-induced damage. However, initial clinical studies on apoptosis-modulating drugs led to unexpected results in different clinical conditions and this may have been due to co-effects on non-apoptotic interconnected cell death mechanisms and the yin-yang role of autophagy in survival versus cell death. In this review, we extend the analysis of cell death beyond apoptosis. Upon introduction of molecular pathways governing autophagy and necrosis (also called necroptosis or programmed necrosis), we focus on the interconnected character of cell death signals and on the shared cell death processes involving mitochondria (e.g. mitophagy and mitoptosis) and molecular signals playing prominent roles in multiple pathways (e.g. Bcl2-family members and p53). We also briefly highlight stress-induced cell senescence that plays a role not only in organismal ageing but also offers the development of novel anticancer strategies. Finally, we briefly illustrate the interconnected character of cell death forms in clinical settings while discussing irradiation-induced mitotic catastrophe. The signalling pathways are discussed in their relation to cancer biology and treatment approaches.

  • 20.
    Jangamreddy, Jaganmohan Reddy
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Jain, Mayur V.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Hallbeck, Anna-Lotta
    Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology. Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    Lotfi, Kourosh
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Pharmacology.
    Los, Marek Jan
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences. Pomeranian Medical University, Szczecin, Poland.
    Glucose starvation-mediated inhibition of salinomycin induced autophagy amplifies cancer cell specific cell death2015In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 6, no 12, p. 10134-10145Article in journal (Refereed)
    Abstract [en]

    Salinomycin has been used as treatment for malignant tumors in a small number of humans, causing far less side effects than standard chemotherapy. Several studies show that Salinomycin targets cancer-initiating cells (cancer stem cells, or CSC) resistant to conventional therapies. Numerous studies show that Salinomycin not only reduces tumor volume, but also decreases tumor recurrence when used as an adjuvant to standard treatments. In this study we show that starvation triggered different stress responses in cancer cells and primary normal cells, which further improved the preferential targeting of cancer cells by Salinomycin. Our in vitro studies further demonstrate that the combined use of 2-Fluoro 2-deoxy D-glucose, or 2-deoxy D-glucose with Salinomycin is lethal in cancer cells while the use of Oxamate does not improve cell death-inducing properties of Salinomycin. Furthermore, we show that treatment of cancer cells with Salinomycin under starvation conditions not only increases the apoptotic caspase activity, but also diminishes the protective autophagy normally triggered by the treatment with Salinomycin alone. Thus, this study underlines the potential use of Salinomycin as a cancer treatment, possibly in combination with short-term starvation or starvation-mimicking pharmacologic intervention.

  • 21.
    Jedlinski, Adam
    et al.
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL.
    Ansell, Anna
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Johansson, Ann-Charlotte
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL.
    EGFR status and EGFR ligand expression influence the treatment response of head and neck cancer cell lines2013In: Journal of Oral Pathology & Medicine, ISSN 0904-2512, E-ISSN 1600-0714, Vol. 42, no 1, p. 26-36Article in journal (Refereed)
    Abstract [en]

    Background: Combination treatment (chemoradiotherapy) is the standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems. A high level of epidermal growth factor receptor (EGFR) has been associated with a more aggressive phenotype as well as decreased responsiveness to radio- or chemotherapy. We examined the role of EGFR status and EGFR ligand expression for the treatment response. Methods: Intrinsic sensitivity to radiotherapy, cisplatin, and cetuximab treatments was investigated in 25 HNSCC cell lines. EGFR gene copy number, mRNA and protein expression, EGFR and Akt phosphorylation status, and mRNA expression of the EGFR ligands were analyzed using quantitative PCR and ELISA and assessed for their impact on treatment sensitivity. Results: Different treatment modalities yielded great diversity in outcome; of note, cetuximab treatment stimulated growth in one cell line. When treatments were combined primarily additive effects were observed. While radioresistance tended to be associated with a high level of phosphorylated EGFR (pEGFR; P = 0.09), cetuximab-resistant cells had low levels of pEGFR (P = 0.13). The three most cetuximab-sensitive cell lines had high EGFR gene copy numbers. Furthermore, cetuximab treatment response was significantly correlated with epiregulin mRNA expression (r = -0.408, P = 0.043). Cisplatin-resistant tumor cells expressed significantly lower levels of EGFR protein (P = 0.04) compared to cisplatin-sensitive cells and tended to have lower levels of phosphorylated Akt (pAkt; P = 0.13) and lower expression levels of amphiregulin (P = 0.18). Conclusions: Epidermal growth factor receptor status and ligand expression influence the treatment sensitivity of HNSCC cells and may be useful as predictive markers.

  • 22.
    Jedlinski, Adam
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Divison of Neurobiology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    Garvin, Stina
    Linköping University, Department of Clinical and Experimental Medicine, Divison of Neurobiology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Clinical pathology.
    Johansson, Ann-Charlotte
    Linköping University, Department of Clinical and Experimental Medicine, Center for Social and Affective Neuroscience. Linköping University, Faculty of Medicine and Health Sciences.
    Edqvist, Per-Henrik
    Uppsala University, Uppsala, Sweden.
    Pontén, Fredrik
    Uppsala University, Uppsala, Sweden.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    Cetuximab sensitivity of head and neck squamous cell carcinoma xenografts is associated with treatment-induced reduction of EGFR, pEGFR, and pSrc2017In: Journal of Oral Pathology & Medicine, ISSN 0904-2512, E-ISSN 1600-0714, no 9, p. 717-724Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The aim of this study was to validate in vitro drug sensitivity testing of head and neck squamous cell carcinoma (HNSCC)cell lines in an in vivo xenograft model, and to identify treatment-induced changes in the EGFR signaling pathway that could be used as markersfor cetuximab treatment response.

    METHODS: The in vitro cetuximab sensitivity of two HNSCC cell lines, UT-SCC-14 and UTSCC-45, was assessed using a crystal violet assay. In order to determine the corresponding in vivo sensitivity, UT-SCC-14 and UT-SCC-45 xenografts were generated in female BALB/c (nu/nu) nude mice. Mice were given three injections of intraperitoneal cetuximab or PBS and the tumor volume was recorded continuously. The expression of epidermal growth factor receptor (EGFR), phosphorylated EGFR (pEGFR), phosphorylated Src (pSrc), and Ki67 was investigated by immunohistochemistry.

    RESULTS: The treatment sensitive UT-SCC-14 cells were found to have an intrinsic cetuximab sensitivity (ICmabS) of 0.15 whereas the ICmabS of the insensitive cell line UT-SCC-45 was 0.78. The corresponding size ratio between untreated and cetuximab treated xenografts was 0.22 and 0.83 for UT-SCC-14 and UT-SCC-45, respectively. UT-SCC-14 cells had a higher baseline expression of pEGFR as compared to UT-SCC-45. Furthermore, in UT-SCC-14 xenografts there was a decrease in EGFR, pEGFR and pSrc upon cetuximab treatment. In contrast, a slight cetuximab-induced increase in EGFR, pEGFR and pSrc was observed in treatment-resistant UT-SCC-45 xenografts.

    CONCLUSIONS: The in vitro treatment sensitivity was reproduced in the in vivo model and cetuximab sensitivity was found to associate with a treatment-induced reduction in pEGFR and pSrc.

  • 23.
    Jerhammar, Fredrik
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Ceder, Rebecca
    Karolinska Institute, Stockholm.
    Garvin, Stina
    Linköping University, Department of Clinical and Experimental Medicine, Oncology. Linköping University, Faculty of Health Sciences.
    Grenman, Reidar
    Turku University Hospital, Finland.
    C Grafstrom, Roland
    Karolinska Institute, Stockholm.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Fibronectin 1 is a potential biomarker for radioresistance in head and neck squamous cell carcinoma2010In: CANCER BIOLOGY and THERAPY, ISSN 1538-4047, Vol. 10, no 12, p. 1244-1251Article in journal (Refereed)
    Abstract [en]

    Radiotherapy remains the backbone of head and neck cancer therapy but response is sometimes impeded by tumor radioresistance. Identifying predictive biomarkers of radiotherapy response is a crucial step towards personalized therapy. The aim of this study was to explore gene expression data in search of biomarkers predictive of the response to radiotherapy in head and neck squamous cell carcinoma (HNSCC). Microarray analysis was performed on five cell lines with various intrinsic radiosensitivity, selected from a panel of 29 HNSCC cell lines. The bioinformatics approach included Gene Ontology (GO) enrichment profiling and Ingenuity Pathway Analysis (IPA). The GO-analysis detected 16 deregulated categories from which development, receptor activity and extracellular region represented the largest groups. Fourteen hub genes (CEBPA, CEBPB, CTNNB1, FN1, MYC, MYCN, PLAU, SDC4, SERPINE1, SP1, TAF4B, THBS1, TP53 and VLDLR) were identified from the IPA network analysis. The hub genes in the highest ranked network, (FN1, SERPINE1, THBS1 and VLDLR) were further subjected to qPCR analysis in the complete panel of 29 cell lines. Of these genes, high FN1 expression associated to high intrinsic radiosensitivity (p = 0.047). In conclusion, gene ontologies and hub genes of importance for intrinsic radiosensitivity were defined. The overall results suggest that FN1 should be explored as a potential novel biomarker for radioresistance.

  • 24.
    Jerhammar, Fredrik
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences.
    Ceder, Rebecca
    Institute of Environmental Medicine, Division of Biochemical Toxicology and Experimental Cancer Research, Karolinska Institute, Stockholm, Sweden.
    Grénman, Reidar
    Department of Otorhinolaryngology-Head and Neck Surgery, Turku University Central Hospital, University of Turku, Turku, Finland/Department of Medical Biochemistry, University of Turku, Turku, Finland.
    Grafström, Roland C.
    Institute of Environmental Medicine, Division of Biochemical Toxicology and Experimental Cancer Research, Karolinska Institute, Stockholm, Sweden.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Identification of Key Regulator Genes Linked to Radioresistance in Head and Neck Squamous Cell Carcinoma by Bioinformatic Processing of Transcript DataManuscript (preprint) (Other academic)
    Abstract [en]

    Purpose: We analyzed basal expression patterns of cell lines with different intrinsic radiosensitivity to discover predictive markers of radiotherapy response.

    Experimental Design: Five head and neck squamous cell carcinoma (HNSCC) cell lines were selected for microarray analysis. Two cell lines showed high resistance to radiation, two cell lines showed an intermediate resistance and one cell line was sensitive and therefore used as reference to other cell lines. Three gene lists were generated from this analysis; one list with commonly deregulated genes in all cell lines compared to the reference and two lists with deregulated genes for the intermediate and highly resistant cell lines compared to the reference, respectively. Gene Ontology enrichment profiling and Ingenuity Pathway Analysis was applied on all gene lists. Key transcript findings were verified at the protein level by Western blot.

    Results: Expression analysis of the high and intermediate resistant cell lines compared to the reference resulted in approximately 1300 significantly altered transcripts, respectively; 552 transcripts were found commonly differently expressed. The deregulated transcripts enriched several GO-categories under biological process, cellular component and molecular function as well as multiple molecular networks in Ingenuity Pathway Analysis. A transcriptional profile of 28 key-regulator genes from the molecular networks was generated from the four resistant lines compared to the reference. Finally, immunoblot analysis supported deregulation at the protein level of markers implicated from the transcriptional-profile.

    Conclusions: Novel markers for prediction of radiation sensitivity could be proposed from bioinformatic processing of gene-expression profiles in HNSCC carcinoma cells.

  • 25.
    Jerhammar, Fredrik
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuroscience. Linköping University, Faculty of Health Sciences.
    Johansson, Ann-Charlotte
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuroscience. Linköping University, Faculty of Health Sciences. Not Found:Linkoping Univ, Fac Hlth Sci, Dept Clin and Expt Med, Div Otorhinolaryngol and Head and Neck Surg, Linkoping, Sweden .
    Ceder, Rebecca
    Karolinska Institute, Sweden .
    Welander, Jenny
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Jansson, Agneta
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences.
    Grafstrom, Roland C.
    Karolinska Institute, Sweden VTT Technical Research Centre Finland, Finland .
    Söderkvist, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuroscience. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Otorhinolaryngology in Linköping.
    YAP1 is a potential biomarker for cetuximab resistance in head and neck cancer2014In: Oral Oncology, ISSN 1368-8375, E-ISSN 1879-0593, Vol. 50, no 9, p. 832-839Article in journal (Refereed)
    Abstract [en]

    Objectives: Targeted therapy against the epidermal growth factor receptor (EGFR) only variably represents a therapeutic advance in head and neck squamous cell carcinoma (HNSCC). This study addresses the need of biomarkers of treatment response to the EGFR-targeting antibody cetuximab (Erbitux (R)). Materials and Methods: The intrinsic cetuximab sensitivity of HNSCC cell lines was assessed by a crystal violet assay. Gene copy number analysis of five resistant and five sensitive cell lines was performed using the Affymetrix SNP 6.0 platform. Quantitative real-time PCR was used for verification of selected copy number alterations and assessment of mRNA expression. The functional importance of the findings on the gene and mRNA level was investigated employing siRNA technology. The data was statistically evaluated using Mann-Whitney U-test and Spearmans correlation test. Results: Analysis of the intrinsic cetuximab sensitivity of 32 HNSCC cell lines characterized five and nine lines as cetuximab sensitive or resistant, respectively. Gene copy number analysis of five resistant versus five sensitive cell lines identified 39 amplified protein-coding genes, including YAP1, in the genomic regions 11q22.1 or 5p13-15. Assessment using qPCR verified that YAP1 amplification associated with cetuximab resistance. Amplification of YAP1 correlated to higher mRNA levels, and RNA knockdown resulted in increased cetuximab sensitivity. Assessment of several independent clinical data sets in the public domain confirmed YAP1 amplifications in multiple tumor types including HNSCC, along with highly differential expression in a subset of HNSCC patients. Conclusion: Taken together, we provide evidence that YAP1 could represent a novel biomarker gene of cetuximab resistance in HNSCC cell lines.

  • 26.
    Jerhammar, Fredrik
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Johansson, Ann-Charlotte
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Jansson, Agneta
    Linköping University, Department of Clinical and Experimental Medicine, Oncology. Linköping University, Faculty of Health Sciences.
    Welander, Jenny
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Söderkvist, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL.
    YAP1 Gene Amplification is a Marker for Cetuximab Resistance in Head and Neck CancerManuscript (preprint) (Other academic)
    Abstract [en]

    The epidermal growth factor receptor (EGFR) is commonly overexpressed in head and neck squamous cell carcinomas (HNSCC). The monoclonal antibody cetuximab (Erbitux®) inhibits its signaling and has been approved for treatment of HNSCC. However, since many patients do not benefit from cetuximab treatment, predictive biomarkers of cetuximab response are required. The present study aims at finding novel markers of cetuximab resistance.

    The intrinsic cetuximab sensitivity of 35 HNSCC cell lines was determined, and revealed a great variation in the response between cell lines. Five cell lines (14%) were cetuximab sensitive, and 12 (34%) were resistant. Interestingly, two cell lines proliferated after cetuximab treatment.

    10 cell lines (five cetuximab sensitive and five cetuximab resistant) were selected for gene copy number array analysis on the Affymetrix SNP 6.0 platform. 39 protein coding genes were amplified in cetuximab resistant cells and normal in sensitive cells, all present on genomic regions 11q22.1 or 5p13-15. Five genes were selected for quantitative PCR  verification, namely, YAP1 and TRPC6 (11q22.1) and PDCD6, TPPP, and PTGER4 (5p13-15). An extended panel of totally 10 cetuximab resistant and 10 sensitive cell lines verified that YAP1 amplified cells are cetuximab resistant.

    YAP1 gene amplification was highly correlated to the YAP1 mRNA expression, which was significantly higher in cetuximab resistant cells than in sensitive. YAP1 downregulation resulted in increased cetuximab sensitivity in one of two cetuximab resistant cell lines investigated and growth inhibition in another. We conclude that YAP1 is a marker for cetuximab resistance in head and neck cancer.

  • 27.
    Jerhammar, Fredrik
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL. Östergötlands Läns Landsting.
    Variation of intrinsic cetuximab sensitivity in head and neck squamous cell carcinomas in EJC SUPPLEMENTS, vol 8, issue 5, pp 50-502010In: EJC SUPPLEMENTS, Elsevier Science B.V., Amsterdam. , 2010, Vol. 8, no 5, p. 50-50Conference paper (Refereed)
    Abstract [en]

    n/a

  • 28.
    Jerhammar, Fredrik
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Welander, Jenny
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Johansson, A C
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Söderkvist, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL.
    Gene Copy Number as Predictive Marker for Cetuximab Resistance in Head and Neck Squamous Cell Carcinomas in EUROPEAN JOURNAL OF CANCER, vol 47, issue , pp S571-S5712011In: EUROPEAN JOURNAL OF CANCER, Elsevier , 2011, Vol. 47, p. S571-S571Conference paper (Refereed)
    Abstract [en]

    n/a

  • 29.
    Johansson, A
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Ansell, Anna
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Ostman, A
    Karolinska Institute.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL. Östergötlands Läns Landsting.
    Cancer-associated fibroblasts desensitizes head and neck squamous cell carcinoma cells to epidermal growth factor receptor-targeted therapy in EJC SUPPLEMENTS, vol 8, issue 5, pp 134-1342010In: EJC SUPPLEMENTS, Elsevier Science B.V., Amsterdam. , 2010, Vol. 8, no 5, p. 134-134Conference paper (Refereed)
    Abstract [en]

    n/a

  • 30.
    Johansson, Ann-Charlotte
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Ansell, Anna
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Jerhammar, Fredrik
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Bradic Lindh, Maja
    Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL.
    Grenman, Reidar
    Turku University Hospital, Finland University of Turku, Finland .
    Munck-Wikland, Eva
    Karolinska University Hospital, Sweden .
    Ostman, Arne
    Karolinska Institute, Sweden .
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL.
    Cancer-Associated Fibroblasts Induce Matrix Metalloproteinase-Mediated Cetuximab Resistance in Head and Neck Squamous Cell Carcinoma Cells2012In: Molecular Cancer Research, ISSN 1541-7786, E-ISSN 1557-3125, Vol. 10, no 9, p. 1158-1168Article in journal (Refereed)
    Abstract [en]

    A growing body of evidence suggests that components of the tumor microenvironment, including cancer-associated fibroblasts (CAF), may modulate the treatment sensitivity of tumor cells. Here, we investigated the possible influence of CAFs on the sensitivity of head and neck squamous cell carcinoma (HNSCC) cell lines to cetuximab, an antagonistic epidermal growth factor receptor (EGFR) antibody. Cetuximab treatment caused a reduction in the proliferation rate of HNSCC cell lines, whereas the growth of HNSCC-derived CAF cultures was unaffected. When tumor cells were cocultured with CAFs in a transwell system, the cetuximab-induced growth inhibition was reduced, and a complete protection from growth inhibition was observed in one of the tumor cell lines investigated. Media that had been conditioned by CAFs offered protection from cetuximab treatment in a concentration-dependent manner, suggesting that the resistance to treatment was mediated by CAF-derived soluble factors. The coculture of HNSCC cell lines with CAFs resulted in an elevated expression of matrix metalloproteinase-1 (MMP-1) in both the tumor cells and CAFs. Moreover, the CAF-induced resistance was partly abolished by the presence of an MMP inhibitor. However, CAFs treated with siRNA targeting MMP-1 still protected tumor cells from cetuximab treatment, suggesting that several MMPs may cooperate to facilitate resistance or that the protective effect is mediated by another member of the MMP family. These results identify a novel CAF-dependent modulation of cetuximab sensitivity and suggest that inhibiting MMPs may improve the effects of EGFR-targeted therapy.

  • 31.
    Johansson, Ann-Charlotte
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Appelqvist, Hanna
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Nilsson, Cathrine
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Kågedal, Katarina
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Öllinger, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Regulation of apoptosis-associated lysosomal membrane permeabilization2010In: APOPTOSIS, ISSN 1360-8185, Vol. 15, no 5, p. 527-540Article in journal (Refereed)
    Abstract [en]

    Lysosomal membrane permeabilization (LMP) occurs in response to a large variety of cell death stimuli causing release of cathepsins from the lysosomal lumen into the cytosol where they participate in apoptosis signaling. In some settings, apoptosis induction is dependent on an early release of cathepsins, while under other circumstances LMP occurs late in the cell death process and contributes to amplification of the death signal. The mechanism underlying LMP is still incompletely understood; however, a growing body of evidence suggests that LMP may be governed by several distinct mechanisms that are likely engaged in a death stimulus- and cell-type-dependent fashion. In this review, factors contributing to permeabilization of the lysosomal membrane including reactive oxygen species, lysosomal membrane lipid composition, proteases, p53, and Bcl-2 family proteins, are described. Potential mechanisms to safeguard lysosomal integrity and confer resistance to lysosome-dependent cell death are also discussed.

  • 32.
    Johansson, Ann-Charlotte
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Linköping University, Faculty of Health Sciences.
    Norberg-Spaak, Lena
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences.
    Role of lysosomal cathepsins in naphthazarin- and Fas-induced apoptosis in oral squamous cell carcinoma cells2006In: Acta Oto-Laryngologica, ISSN 0001-6489, Vol. 126, no 1, p. 70-81Article in journal (Refereed)
    Abstract [en]

    Conclusion. Intracellular cysteine cathepsins are pro-apoptotic factors involved in activation of caspases in two oral squamous cell carcinoma (SCC) cell lines.

    Objective. To study the possible involvement of lysosomal cathepsins in oral SCC cell apoptosis.

    Material and methods. Apoptosis was induced in the two human oral SCC cell lines UT-SCC-20A and UT-SCC-24A using naphthazarin or anti-Fas antibodies, and was studied by analysis of caspase activity and nuclear morphology. Involvement of lysosomal cathepsins was investigated using the cysteine cathepsin inhibitor z-FA-FMK and the cathepsin D inhibitor pepstatin A. The amounts of cellular and soluble Fas death receptor were determined by ELISA.

    Results. Release of cathepsins from the lysosomes to the cytosol was observed early in apoptosis. Cysteine cathepsins were found to be involved in activation of caspases in response to treatment with naphthazarin or anti-Fas antibodies, but inhibition of cysteine cathepsin activity was not sufficient to prevent cell death. Moreover, inhibition of cysteine cathepsin activity resulted in increased expression of the Fas death receptor, suggesting involvement of extracellular cysteine cathepsins in death receptor shedding.

  • 33.
    Johansson, Ann-Charlotte
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Linköping University, Faculty of Health Sciences.
    Steen, Håkan
    Öllinger, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine2003In: Cell Death and Differentiation, ISSN 1350-9047, Vol. 10, no 11, p. 1253-1259Article in journal (Refereed)
    Abstract [en]

    There is increasing evidence that proteases other than caspases, for example, the lysosomal cathepsins B, D and L, are involved in apoptotic cell death. In the present study, we present data that suggest a role for cathepsin D in staurosporine-induced apoptosis in human foreskin fibroblasts. Cathepsin D and cytochrome c were detected partially released to the cytosol after exposure to 0.1 µM staurosporine for 1 h. After 4 h, activation of caspase-9 and -3 was initiated and later caspase-8 activation and a decrease in full-length Bid were detected. Pretreatment of cells with the cathepsin D inhibitor, pepstatin A, prevented cytochrome c release and caspase activation, and delayed cell death. These results imply that cytosolic cathepsin D is a key mediator in staurosporine-induced apoptosis. Analysis of the relative sequence of apoptotic events indicates that, in this cell type, cathepsin D acts upstream of cytochrome c release and caspase activation.

  • 34.
    Kågedal, Katarina
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Johansson, Ann-Charlotte
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Johansson, Uno
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Heimlich, Gerd
    Institute for Medical Microbiology, Immunology and Hygiene, University of Köln, Köln, Germany.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Wang, Nancy S.
    Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
    Jürgensmeier, Juliane M.
    Institute for Medical Microbiology, Immunology and Hygiene, University of Köln, Köln, Germany.
    Öllinger, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Lysosomal membrane permeabilization during apoptosis: Involvement of Bax?2005In: International journal of experimental pathology (Print), ISSN 0959-9673, E-ISSN 1365-2613, Vol. 86, no 5, p. 309-321Article in journal (Refereed)
    Abstract [en]

    Bcl-2 family members have long been known to control permeabilization of the mitochondrial membrane during apoptosis, but involvement of these proteins in lysosomal membrane permeabilization (LMP) was not considered until recently. The aim of this study was to investigate the mechanism underlying the release of lysosomal proteases to the cytosol seen during apoptosis, with special emphasis on the role of Bax. In human fibroblasts, exposed to the apoptosis-inducing drug staurosporine (STS), the release of the lysosomal protease cathepsin D to the cytosol was observed by immunocytochemistry. In response to STS treatment, there was a shift in Bax immunostaining from a diffuse to a punctate pattern. Confocal microscopy showed co-localization of Bax with both lysosomes and mitochondria in dying cells. Presence of Bax at the lysosomal membrane was confirmed by immuno-electron microscopy. Furthermore, when recombinant Bax was incubated with pure lysosomal fractions, Bax inserted into the lysosomal membrane and induced the release of lysosomal enzymes. Thus, we suggest that Bax is a mediator of LMP, possibly promoting the release of lysosomal enzymes to the cytosol during apoptosis.

  • 35.
    La Fleur, Linnea
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Johansson, Ann-Charlotte
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL.
    A CD44(high)/EGFR(low) Subpopulation within Head and Neck Cancer Cell Lines Shows an Epithelial-Mesenchymal Transition Phenotype and Resistance to Treatment2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 9Article in journal (Refereed)
    Abstract [en]

    Mortality in head and neck squamous cell carcinoma (HNSCC) is high due to emergence of therapy resistance which results in local and regional recurrences that may have their origin in resistant cancer stem cells (CSCs) or cells with an epithelial-mesenchymal transition (EMT) phenotype. In the present study, we investigate the possibility of using the cell surface expression of CD44 and epidermal growth factor receptor (EGFR), both of which have been used as stem cell markers, to identify subpopulations within HNSCC cell lines that differ with respect to phenotype and treatment sensitivity. Three subpopulations, consisting of CD44(high)/EGFR(low), CD44(high)/EGFR(high) and CD44(low) cells, respectively, were collected by fluorescence-activated cell sorting. The CD44(high)/EGFR(low) population showed a spindle-shaped EMT-like morphology, while the CD44(low) population was dominated by cobblestone-shaped cells. The CD44(high)/EGFR(low) population was enriched with cells in G0/G1 and showed a relatively low proliferation rate and a high plating efficiency. Using a real time PCR array, 27 genes, of which 14 were related to an EMT phenotype and two with stemness, were found to be differentially expressed in CD44(high)/EGFR(low) cells in comparison to CD44(low) cells. Moreover, CD44(high)/EGFR(low) cells showed a low sensitivity to radiation, cisplatin, cetuximab and gefitinib, and a high sensitivity to dasatinib relative to its CD44(high)/EGFR(high) and CD44(low) counterparts. In conclusion, our results show that the combination of CD44 (high) and EGFR (low) cell surface expression can be used to identify a treatment resistant subpopulation with an EMT phenotype in HNSCC cell lines.

  • 36.
    Mahmoudi, S
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Farnebo, M
    Karolinska Institute.
    WRAP53 promotes cancer cell survival and is a potential target for cancer therapy2011In: CELL DEATH and DISEASE, ISSN 2041-4889, Vol. 2, no e114Article in journal (Refereed)
    Abstract [en]

    We previously identified WRAP53 as an antisense transcript that regulates the p53 tumor suppressor. The WRAP53 gene also encodes a protein essential for Cajal body formation and involved in cellular trafficking of the survival of motor neuron complex, the telomerase enzyme and small Cajal body-specific RNAs to Cajal bodies. Here, we show that the WRAP53 protein is overexpressed in a variety of cancer cell lines of different origin and that WRAP53 overexpression promotes cellular transformation. Knockdown of the WRAP53 protein triggers massive apoptosis through the mitochondrial pathway, as demonstrated by Bax/Bak activation, loss of mitochondrial membrane potential and cytochrome c release. The apoptosis induced by WRAP53 knockdown could moreover be blocked by Bcl-2 overexpression. Interestingly, human tumor cells are more sensitive to WRAP53 depletion as compared with normal human cells indicating that cancer cells in particular depends on WRAP53 expression for their survival. In agreement with this, we found that high levels of WRAP53 correlate with poor prognosis of head and neck cancer. Together these observations propose a role of WRAP53 in carcinogenesis and identify WRAP53 as a novel molecular target for a large fraction of malignancies.

  • 37.
    Nilsson, C
    et al.
    Östergötlands Läns Landsting.
    La Fleur, L
    Östergötlands Läns Landsting.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of ENT - Head and Neck Surgery UHL. Östergötlands Läns Landsting.
    Differences in radiation, cisplatin, and cetuximab sensitivity between subpopulations of head and neck cancer cells in EJC SUPPLEMENTS, vol 8, issue 5, pp 107-1072010In: EJC SUPPLEMENTS, Elsevier Science B.V., Amsterdam. , 2010, Vol. 8, no 5, p. 107-107Conference paper (Refereed)
    Abstract [en]

    n/a

  • 38.
    Nilsson, Cathrine
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Grafström, Roland C.
    Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden.
    Öllinger, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Intrinsic differences in cisplatin sensitivity of head and neck cancer celllines correlates to lysosomal pH2010In: Head and Neck, ISSN 1043-3074, E-ISSN 1097-0347, Vol. 32, no 9, p. 1185-1194Article in journal (Refereed)
    Abstract [en]

    Cisplatin is part of the treatment regime of head and neck squamous cell carcinomas (HNSCC). In order to predict the clinical outcome of the treatment, markers for evaluation of the intrinsic cisplatin sensitivity are inquired. In this study we characterize the lysosomal compartment and compare cisplatin sensitivity in five HNSCC lines and normal oral keratinocytes (NOKs). Cisplatin sensitivity differed 3-fold between the least and most sensitive cell lines, and the cisplatin LD50 correlated significantly to lysosomal pH, which varied from 4.3 in NOKs to 4.9 in the most resistant HNSCC line. Lysosomes are acidified by the V0V1-ATPase complex located in the lysosomal membrane. Interestingly, in cell lines exhibiting high lysosomal pH, we found decreased expression of the V0V1-ATPase B2 subunit, possibly explaining the defective acidification. In all cell lines, exposure to cisplatin caused activation of caspase-3. Cisplatin exposure was accompanied by lysosomal membrane permeabilization and inhibition of the llysosomal cathepsins B, D and L partly prevented cell death. No correlation between cisplatin sensitivity and expression of cathepsins B, D and L or secretion of their respective proforms into the culture medium was found in the cell lines studied. We conclude that lysosomal pH and expression of V0V1-ATPase subunits are possible future markers of intrinsic cisplatin sensitivity.

  • 39.
    Perskvist, Nasrin
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Roberg, Karin
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Kulyte, Agné
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Stendahl, Olle
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Rab5a GTPase regulates fusion between pathogen-containing phagosomes and cytoplasmic organelles in human neutrophils2002In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 115, no 6, p. 1321-1330Article in journal (Refereed)
    Abstract [en]

    Biogenesis of phagolysosomes proceeds through a sequential series of interactions with endocytic organelles, a process known to be regulated by Rab and SNARE proteins. The molecular mechanisms underlying phagosome maturation in neutrophils are, however, not clearly understood. We investigated fusion between phagosomes containing the intracellular pathogen Mycobacterium tuberculosis versus the extracellular pathogen Staphylococcus aureus (designated MCP for mycobacteria-containing phagosome and SCP for S. aureus-containing phagosome) and cytoplasmic compartments in human neutrophils. Western blot analysis of phagosomes isolated after internalisation revealed that lactoferrin (a constituent of secondary granules) and LAMP-1 were incorporated into both SCP and MCP, whereas hck (marker of azurophil granules) interacted solely with SCP. The subcellular distribution of the proteins Rab5a and syntaxin-4 suggested a role in docking of granules and/or endosomes to the target membrane in the neutrophil. We observed that during phagocytosis, Rab5a in GTP-bound form interacted with syntaxin-4 on the membrane of MCP and were retained for up to 90 minutes, whereas the complex was recruited to the SCP within 5 minutes but was selectively depleted from these vacuoles after 30 minutes of phagocytosis. Downregulation of Rab5a by antisense oligonucleotides efficiently reduced the synthesis of Rab5a, the binding of syntaxin-4 to MCP and SCP and the capacity for fusion exhibited by the pathogen-containing phagosomes, but it had no effect on bacteria internalisation. These data indicate that the difference in granule fusion is correlated with a difference in the association of Rab5a and syntaxin-4 with the phagosomes. Intracellular pathogen-containing phagosomes retain Rab5a and syntaxin-4, whereas extracellular pathogen-containing phagosomes bind briefly to this complex. These results also identified Rab5a as a key regulator of phagolysosome maturation in human neutrophils.

  • 40.
    Roberg, Karin
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Immunocytochemical visualization of cathepsin D during oxidative stress1998Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cathepsin D is the major aspartic protease in lysosomes, and it is found in almost all animal cells. In the present studies, a pre-embedding immunocytochemical method was used to visualize cathepsin D in cultured cells. The protein can be detected at both light and electron microscopic levels by neutral-pH silver enhancement of ultrasmall (0.8 nm) gold particles conjugated to the antibodies. The best morphological preservation and the highest labeling density were achieved by initial fixation in 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde (GA) in 0.15 M sodium cacodylate buffer, followed by membrane permeabilization in sodium borohydride for 20 min at 4° c. Three cell types were used: human foreskin fibroblasts, mouse histiocytic lymphoma (1774) cells, and primary rat heart myocytes. Cathepsin D was found in lysosome-like structures in all three cell types.

    Rat heart myocytes were treated with the redox cycling, xenobiotic compound naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) to induce oxidative stress. Immunofluorescence staining and light rnicroscopy (LM) 30 min after exposure showed that cathepsin D fluorescence had changed from a mainly lysosomal pattern in control cells to a partly cytosolic location in cells exposed to oxidative stress. Electron microscopic morphometry 15, 30, and 45 min after exposure to naphthazarin confirmed that a statistically significant amount of cathepsin D was transferred from lysosome-like structures to the cytosol. Exposure to naphthazarin also caused a decrease in intracellular pH and ATP, whereas lipid peroxidation was not detected within the same time frame.

    Following exposure to naphthazarin, cells were washed and normal culture conditions were reestablished for another 18 h. Thereafter many cells displayed apoptotic morphology (i.e. cellular shrinkage and chromatin condensation; analyzed by Giemsa staining). Also, 41% of the cells stained positive with the TUNEL technique, and DNA fragmentation was detected. Pretreatment of the cultures with α-tocopherol succinate largely prevented relocation of cathepsin D (shown by immunofluorescence) and also significantly decreased apoptosis.

  • 41.
    Roberg, Karin
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Relocalization of Cathepsin D and Cytochrome c Early in Apoptosis Revealed by Immunoelectron Microscopy2001In: Laboratory Investigation, ISSN 0023-6837, E-ISSN 1530-0307, Vol. 81, no 2, p. 149-158Article in journal (Refereed)
    Abstract [en]

    Cathepsin D was translocated from lysosomal structures to the cytosol in primary cultures of neonatal rat cardiomyocytes exposed to oxidative stress, and these cells underwent apoptotic death during subsequent incubation. Temporal aspects of cathepsin D relocalization, cytochrome c release, and decrease in mitochondrial transmembrane potential (Δψm) were studied in myocytes exposed to the redox-cycling xenobiotic naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Immunofluorescence labeling revealed that cathepsin D was translocated to the cytosol after 30 minutes of naphthazarin treatment, and cytochrome c was released from mitochondria to the cytosol after 2 hours. Western blotting and immunoelectron microscopy indicated a minor release of cytochrome c after only 30 minutes and 1 hour, respectively. Thereafter, a decrease in Δψm was detected using the Δψm-sensitive dye JC-1 and confocal microscopy, and ultrastructural analysis indicated apoptotic morphology. Pretreatment of the cultures with the cathepsin D inhibitor pepstatin A prevented release of cytochrome c from mitochondria and maintained the Δψm. Moreover, ultrastructural examination showed no apoptotic morphology. These findings suggest that lysosomal destabilization (detected as the release of cathepsin D) and release of cytochrome c from mitochondria take place early in apoptosis. Also, the former event probably occurs before the latter during apoptosis induced by oxidative stress because pretreatment with pepstatin A prevented release of cytochrome c and loss of Δψm in cardiomyocytes exposed to naphthazarin.

  • 42.
    Roberg, Karin
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    The role of cathepsin D in apoptosis induced by oxidative stress2001Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The lysosomal protease cathepsin D is translocated from lysosomes to the cytosol during apoptosis induced by oxidative stress. In the present studies, the redox-cycling, xenobiotic compound naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) was used to create oxidative stress in rat cardiomyocytes and human foreskin fibroblasts. In naphthazarin exposed cells, lysosomal release of cathepsin D preceded liberation of cytochrome c from mitochondria and a decrease in the mitochondrial transmembrane potential (ΔΨm).

    A pre-embedding immunocytochemical method was used for ultrastructural examination of cathepsin D and cytochrome c in cultured cells. Electron microscopic morphometry confirmed that a statistically significant amount of cathepsin D was transferred from lysosome-like structures to the cytosol before any biochemical or morphological signs of apoptosis were detected. Pretreatment of the cells with atocopherol succinate largely prevented translocation of cathepsin D and also significantly decreased apoptosis. Electron microscopy also revealed that, during exposure to naphthazarin, a minor release of cytochrome c has occured after one hour and a more extensive release after two hours, and these results were verified by Western blotting. After the translocation of cathepsin D and cytochrome c, a decrease in ΔΨm was detected using the ΔΨm-sensitive probe JC-1 and confocal microscopy or measured by flow cytornetry. Pretreatment with the cathepsin D inhibitor pepstatin A prevented release of cytochrome c from mitochondria, maintained the ΔΨm and inhibited apoptosis.

    In conclusion, these findings show that translocation of cathepsin D precedes important incidents in mitochondria, such as release of cytochrome c and loss of ΔΨm during apoptosis induced by oxidative stress. Moreover, inhibition of cathepsin D prevented the apoptosis and the mitochondrial changes, which indicates that cathepsin D is an inducer of apoptosis upstream of cytochrome c release.

    List of papers
    1. A Pre-embedding Technique for Immunocytochemical Visualization of Cathepsin D in Cultured Cells Subjected to Oxidative Stress
    Open this publication in new window or tab >>A Pre-embedding Technique for Immunocytochemical Visualization of Cathepsin D in Cultured Cells Subjected to Oxidative Stress
    1998 (English)In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 46, no 3, p. 411-418Article in journal (Refereed) Published
    Abstract [en]

    We describe a pre-embedding immunocytochemical method for visualization of the lysosomal enzyme cathepsin D in cultured cells. The protein was demonstrated at both light and electron microscopic levels by neutral-pH silver enhancement of ultrasmall (0.8-nm) gold particles conjugated to the antibodies. The best morphological preservation and the highest labeling density were achieved by initial fixation for 20 min at 4C in 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde (GA) in 0.15 M sodium cacodylate buffer, followed by permeabilization in sodium borohydride. Three cell types were used: human foreskin fibroblasts, histocytic lymphoma (J-774) cells, and primary rat heart myocytes. In all three, cathepsin D was demonstrated in lysosome-like structures. The rat heart myocytes were also exposed to the redox cycling substance naphthazarin (5,8-dihydroxy-1,4-naph-thoquinone) to induce oxidative stress. This was done for such a short period of time that the cells initially did not show any signs of morphological damage and retained normal plasma membrane stability, although an early and clear redistribution of cathepsin D from membrane-bound structures to the cytosol was apparent. This redistribution was followed by cell degeneration and, eventually, by cell death.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-80993 (URN)10.1177/002215549804600316 (DOI)
    Available from: 2012-09-05 Created: 2012-09-05 Last updated: 2017-12-07Bibliographically approved
    2. Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes
    Open this publication in new window or tab >>Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes
    1998 (English)In: American Journal of Pathology, ISSN 0002-9440, E-ISSN 1525-2191, Vol. 152, no 5, p. 1151-1156Article in journal (Refereed) Published
    Abstract [en]

    Exposing neonatal rat heart myocytes to the redox cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) for 15 to 45 minutes led to a time-dependent release of cathepsin D from many secondary lysosomes to the cytosol, as analyzed by morphometry. Cathepsin D was detected electron microscopically using a pre-embedding immunostaining technique that utilizes antibodies conjugated to ultra-small (0.8-nm) gold particles and subsequent silver enhancement. The exposure to naphthazarin also caused a decrease in both the pH and the ATP level of the cells within the same time frame. Lipid peroxidation was, however, not detected. Pretreatment of the cultures with alpha-tocopherol succinate prevented cathepsin D relocation, as shown by immunofluorescence. After exposure to naphthazarin, cells were washed, and normal culture conditions were re-established for 18 hours. Many cells then showed apoptotic morphology (ie, cellular shrinkage and chromatin condensation) as analyzed by Giemsa staining. Also, 41% of the cells stained positive with the TUNEL technique, and DNA fragmentation was detected by separation of intact and fragmented DNA. Apoptosis was significantly decreased in cultures pretreated with alpha-tocopherol succinate.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-80994 (URN)9588882 (PubMedID)
    Available from: 2012-09-05 Created: 2012-09-05 Last updated: 2017-12-07Bibliographically approved
    3. Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress
    Open this publication in new window or tab >>Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress
    1999 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 27, no 11-12, p. 1228-1237Article in journal (Refereed) Published
    Abstract [en]

    Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential–sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (ΔΨm) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of ΔΨm, and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-80995 (URN)10.1016/S0891-5849(99)00146-X (DOI)
    Available from: 2012-09-05 Created: 2012-09-05 Last updated: 2017-12-07Bibliographically approved
    4. Relocalization of Cathepsin D and Cytochrome c Early in Apoptosis Revealed by Immunoelectron Microscopy
    Open this publication in new window or tab >>Relocalization of Cathepsin D and Cytochrome c Early in Apoptosis Revealed by Immunoelectron Microscopy
    2001 (English)In: Laboratory Investigation, ISSN 0023-6837, E-ISSN 1530-0307, Vol. 81, no 2, p. 149-158Article in journal (Refereed) Published
    Abstract [en]

    Cathepsin D was translocated from lysosomal structures to the cytosol in primary cultures of neonatal rat cardiomyocytes exposed to oxidative stress, and these cells underwent apoptotic death during subsequent incubation. Temporal aspects of cathepsin D relocalization, cytochrome c release, and decrease in mitochondrial transmembrane potential (Δψm) were studied in myocytes exposed to the redox-cycling xenobiotic naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Immunofluorescence labeling revealed that cathepsin D was translocated to the cytosol after 30 minutes of naphthazarin treatment, and cytochrome c was released from mitochondria to the cytosol after 2 hours. Western blotting and immunoelectron microscopy indicated a minor release of cytochrome c after only 30 minutes and 1 hour, respectively. Thereafter, a decrease in Δψm was detected using the Δψm-sensitive dye JC-1 and confocal microscopy, and ultrastructural analysis indicated apoptotic morphology. Pretreatment of the cultures with the cathepsin D inhibitor pepstatin A prevented release of cytochrome c from mitochondria and maintained the Δψm. Moreover, ultrastructural examination showed no apoptotic morphology. These findings suggest that lysosomal destabilization (detected as the release of cathepsin D) and release of cytochrome c from mitochondria take place early in apoptosis. Also, the former event probably occurs before the latter during apoptosis induced by oxidative stress because pretreatment with pepstatin A prevented release of cytochrome c and loss of Δψm in cardiomyocytes exposed to naphthazarin.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-27927 (URN)12688 (Local ID)12688 (Archive number)12688 (OAI)
    Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
  • 43.
    Roberg, Karin
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Ceder, Rebecca
    Division of Biochemical Toxicology and Experimental Cancer Research, Institute of Environmental Medicine, Karolinska Institute.
    Farnebo, Lovisa
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Norberg-Spaak, Lena
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Grafström, Roland
    Division of Biochemical Toxicology and Experimental Cancer Research, Institute of Environmental Medicine, Karolinska Institute.
    Multiple genotypic aberrances associate to terminal differentiation-deficiency of an oral squamous cell carcinoma in serum-free culture2008In: Differentiation, ISSN 0301-4681, E-ISSN 1432-0436, Vol. 76, no 8, p. 868-880Article in journal (Refereed)
    Abstract [en]

    Oral squamous cell carcinoma (OSCC) lines proliferative in the serum-free conditions devised for normal oral keratinocytes (NOK) are virtually absent, complicating studies of carcinogenesis. A tongue squamous cell carcinoma generated under conditions for normal cell culture an apparently immortal line (termed LK0412) that has undergone ≥200 population doublings from over a year in culture. LK0412 exhibited epithelial morphology, intermediate filaments, desmosomes, and cytokeratin. Soft agar growth and tumorigenicity in athymic nude mice indicated the malignant phenotype. Compared with NOK, LK0412 exhibited increased indices for proliferation and apoptosis, and a decreased terminal differentiation index. Fetal bovine serum inhibited growth and increased apoptosis but failed to induce terminal differentiation of LK0412; the latter outcome differed clearly from that in NOK. Gene ontology assessment of transcript profiles implicated multiple alterations in biological processes, molecular functions, and cellular components in LK0412. Genetic changes, some that were confirmed to the protein level, included previously proposed OSCC markers, i.e., BAX, CDC2, and TP53, as well as multiple cancer-associated genes not considered for OSCC, e.g., BST2, CRIP1, ISG15, KLRC1, NEDD9, NNMT, and TWIST1. Elevation of p53 protein agreed with a missense mutation detectable in both the LK0412 line and the original tumor specimen. Moderate differentiation characterized the original tumor as well as tumors generated from inoculation of LK0412 in mice. Overall, the results suggest that the LK0412 cell line represent a subgroup of OSCC with unique genomic and phenotypic profiles. LK0412 should be useful to exploration of OSCC development, particularly the deregulated growth and differentiation responsiveness to serum factors.

  • 44.
    Roberg, Karin
    et al.
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Johansson, Uno
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Öllinger, Karin
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress1999In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 27, no 11-12, p. 1228-1237Article in journal (Refereed)
    Abstract [en]

    Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential–sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (ΔΨm) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of ΔΨm, and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.

  • 45.
    Roberg, Karin
    et al.
    Linköping University, Department of Neuroscience and Locomotion, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Jonsson, Ann-Charlotte
    Linköping University, Department of Medicine and Care, Radiation Physics. Linköping University, Faculty of Health Sciences.
    Grénman, Reidar
    Department of Otorhinolaryngology, Head and Neck Surgery, Central Hospital and University of Turku and Medical Biochemistry, University of Turku, Finland.
    Norberg-Spaak, Lena
    Linköping University, Department of Neuroscience and Locomotion, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Radiotherapy response in oral squamous carcinoma cell lines: Evaluation of apoptotic proteins as prognostic factors2007In: Head and Neck, ISSN 1043-3074, E-ISSN 1097-0347, Vol. 29, no 4, p. 325-334Article in journal (Refereed)
    Abstract [en]

    Background. In this study, we investigated the importance of apoptosis for cell death after radiotherapy, and whether the expression of pro- and anti-apoptotic proteins has any correlation to the radiosensitivity.

    Methods. Three oral squamous cell carcinoma cell lines, UT-SCC-2, UT-SCC-9 and UT-SCC-24A, were subjected to radiotherapy. After irradiation, viable and dead cells were counted to determine radiation sensitivity and apoptosis was analyzed by measurement of caspase-3 activity. The expressions of pro- and anti-apoptotic proteins were assessed using western blot analyses.

    Results and Conclusion. After irradiation, apoptotic morphology and caspase-3 activity were only detected in cell lines exhibiting high or moderate radiosensitivity. Western blot analysis indicates that survivin, epidermal growth factor receptor, cyclooxygenase-2, and Bcl-xL are critical components in irradiation resistance of the investigated cell lines. Moreover, our results suggest that apoptotic cell death and the balance between pro- and anti-apoptotic proteins are of importance for the outcome of radiotherapy.

  • 46.
    Roberg, Karin
    et al.
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Kågedal, Katarina
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Öllinger, Karin
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Microinjection of cathepsin D induces caspase-dependent apoptosis in fibroblasts2002In: American Journal of Pathology, ISSN 0002-9440, E-ISSN 1525-2191, Vol. 161, no 1, p. 89-96Article in journal (Refereed)
    Abstract [en]

    Recent reports have indicated that enzymes such as cathepsins D and B are translocated from lysosomal compartments to the cytosol early during apoptosis. We have previously noted that a translocation of cathepsins D and B occur before cytochrome c release and caspase activation in cardiomyocytes and human fibroblasts during oxidative stress-induced apoptosis. In the present report, we use a microinjection technique to investigate if cytosolic location of the cathepsins D and B are important for induction of apoptosis. We found that microinjection of cathepsin D into the cytosol of human fibroblasts caused apoptosis, which was detected as changes in distribution of cytochrome c, cell shrinkage, activation of caspases, chromatin condensation, and formation of pycnotic nuclei. No apoptosis was, however, induced by microinjection of cathepsin B. Moreover, apoptosis was prevented in fibroblasts pretreated with a caspase-3-like inhibitor, and also when microinjected with cathepsin D mixed with the cathepsin D inhibitor, pepstatin A. These results show that cytosolic cathepsin D can act as a proapoptotic mediator upstream of cytochrome c release and caspase activation in human fibroblasts.

  • 47.
    Roberg, Karin
    et al.
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Öllinger, Karin
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    A Pre-embedding Technique for Immunocytochemical Visualization of Cathepsin D in Cultured Cells Subjected to Oxidative Stress1998In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 46, no 3, p. 411-418Article in journal (Refereed)
    Abstract [en]

    We describe a pre-embedding immunocytochemical method for visualization of the lysosomal enzyme cathepsin D in cultured cells. The protein was demonstrated at both light and electron microscopic levels by neutral-pH silver enhancement of ultrasmall (0.8-nm) gold particles conjugated to the antibodies. The best morphological preservation and the highest labeling density were achieved by initial fixation for 20 min at 4C in 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde (GA) in 0.15 M sodium cacodylate buffer, followed by permeabilization in sodium borohydride. Three cell types were used: human foreskin fibroblasts, histocytic lymphoma (J-774) cells, and primary rat heart myocytes. In all three, cathepsin D was demonstrated in lysosome-like structures. The rat heart myocytes were also exposed to the redox cycling substance naphthazarin (5,8-dihydroxy-1,4-naph-thoquinone) to induce oxidative stress. This was done for such a short period of time that the cells initially did not show any signs of morphological damage and retained normal plasma membrane stability, although an early and clear redistribution of cathepsin D from membrane-bound structures to the cytosol was apparent. This redistribution was followed by cell degeneration and, eventually, by cell death.

  • 48.
    Roberg, Karin
    et al.
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Öllinger, Karin
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes1998In: American Journal of Pathology, ISSN 0002-9440, E-ISSN 1525-2191, Vol. 152, no 5, p. 1151-1156Article in journal (Refereed)
    Abstract [en]

    Exposing neonatal rat heart myocytes to the redox cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) for 15 to 45 minutes led to a time-dependent release of cathepsin D from many secondary lysosomes to the cytosol, as analyzed by morphometry. Cathepsin D was detected electron microscopically using a pre-embedding immunostaining technique that utilizes antibodies conjugated to ultra-small (0.8-nm) gold particles and subsequent silver enhancement. The exposure to naphthazarin also caused a decrease in both the pH and the ATP level of the cells within the same time frame. Lipid peroxidation was, however, not detected. Pretreatment of the cultures with alpha-tocopherol succinate prevented cathepsin D relocation, as shown by immunofluorescence. After exposure to naphthazarin, cells were washed, and normal culture conditions were re-established for 18 hours. Many cells then showed apoptotic morphology (ie, cellular shrinkage and chromatin condensation) as analyzed by Giemsa staining. Also, 41% of the cells stained positive with the TUNEL technique, and DNA fragmentation was detected by separation of intact and fragmented DNA. Apoptosis was significantly decreased in cultures pretreated with alpha-tocopherol succinate.

  • 49.
    Staab, C A
    et al.
    Karolinska Institute.
    Ceder, R
    Karolinska Institute.
    Roberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Grafstrom, R C
    Karolinska Institute.
    Hoog, J-O
    Karolinska Institute.
    Serum-responsive expression of carbonyl-metabolizing enzymes in normal and transformed human buccal keratinocytes2008In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 65, no 22, p. 3653-3663Article in journal (Refereed)
    Abstract [en]

    Gene expression of carbonyl-metabolizing enzymes (CMEs) was investigated in normal buccal keratinocytes (NBK) and the transformed buccal keratinocyte lines SVpgC2a and SqCC/Y1. Studies were performed at a serum concentration known to induce terminal squamous differentiation (TSD) in normal cells. Overall, 39 of 58 evaluated CMEs were found to be expressed at the transcript level. Together the transformed cell lines showed altered transcription of eight CME genes compared to NBK, substantiating earlier results. Serum increased transcript levels of ALDH1A3, DHRS3, HPGD and AKR1A1, and decreased those of ALDH4A1 in NBK; of these, the transformed, TSD-deficient cell lines partly retained regulation of ALDH1A3 and DHRS3. Activity measurements in crude cell lysates, including relevant enzymatic inhibitors, indicated significant capacity for CME-mediated xenobiotic metabolism among the cell lines, notably with an increase in serum-differentiated NBK. The results constitute the first evidence for differential CME gene expression and activity in non-differentiated and differentiated states of epithelial cells.

  • 50.
    Staab, Claudia A
    et al.
    Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
    Ceder, Rebecca
    Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
    Jägerbrink, Theres
    Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
    Nilsson, Jan-Anders
    Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
    Roberg, Karin
    Linköping University, Department of Neuroscience and Locomotion, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of ENT - Head and Neck Surgery UHL.
    Jörnvall, Hans
    Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
    Höög, Jan-Olov
    Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
    Grafström, Roland C
    Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
    Bioinformatics processing of protein and transcript profiles of normal and transformed cell lines indicates functional impairment of transcriptional regulators in buccal carcinoma2007In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 9, p. 3705-3717Article in journal (Refereed)
    Abstract [en]

    Normal and two transformed buccal keratinocyte lines were cultured under a standardized condition to explore mechanisms of carcinogenesis and tumor marker expression at transcript and protein levels. An approach combining three bioinformatic programs allowed coupling of abundant proteins and large-scale transcript data to low-abundance transcriptional regulators. The analysis identified previously proposed and suggested novel protein biomarkers, gene ontology categories, molecular networks, and functionally impaired key regulator genes for buccal/oral carcinoma. © 2007 American Chemical Society.

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