title: TNF-α induced secretion of HMGB1 from non-immune canine mammary epithelial cells (MTH53A).
abstract: BACKGROUND
  Mammary neoplasias are one of the most frequent and spontaneously occurring malignancies in dogs and humans. Due to the similar anatomy of the mammary gland in both species, the dog has become an important animal model for this cancer entity. In human breast carcinomas, the overexpression of a protein named high-mobility group box 1 (HMGB1) was reported. Cells of the immune system were described to release HMGB1 actively exerting cytokine function. Thereby it is involved in the immune system activation, tissue repair, and cell migration. Passive release of HMGB1 by necrotic cells at sites of tissue damage or in necrotic hypoxic regions of tumors induces cellular responses e.g. release of proinflammatory cytokines leading to elevated inflammatory response and neo-vascularization of necrotic tumor areas. Herein we investigated if a time-dependent stimulation with the separately applied proinflammatory cytokines TNF-α and IFN-γ can cause secretion of HMGB1 in a non-immune related HMGB1-non-secreting epithelial canine mammary cell line (MTH53A) derived from non-neoplastic tissue.
  
  
  METHODS
  The canine cell line was transfected with recombinant HMGB1 bicistronic expression vectors and stimulated after transfection with the respective cytokine independently for 6, 24 and 48 h. HMGB1 protein detection was performed by Western blot analysis and quantified a by enzyme-linked immunosorbent assay. Live cell laser scanning multiphoton microscopy of MTH53A cells expressing a HMGB1-GFP fusion protein was performed in order to examine, if secretion of HMGB1 under cytokine stimulating conditions is also visible by fluorescence imaging.
  
  
  RESULTS
  The observed HMGB1 release kinetics showed a clearly time-dependent manner with a peak release 24h after TNF-α stimulation, while stimulation with IFN-γ had only small effects on the HMGB1 release. Multiphoton HMGB1 live cell microscopy showed diffuse cell membrane structure changes 29 h after cytokine-stimulation but no clear secretion of HMGB1-GFP after TNF-α stimulation was visible.
  
  
  CONCLUSIONS
  Our results demonstrate that non-immune HMGB1-non-secreting cells of epithelial origin derived from mammary non-neoplastic tissue can be induced to release HMGB1 by single cytokine application. This indicates that tumor and surrounding tissue can be stimulated by tumor present inflammatory and necrotic cytokines to release HMGB1 acting as neo-vascularizing factor thus promoting tumor growth.
authors.affiliation: Small Animal Clinic and Research Cluster of Excellence "REBIRTH", University of Veterinary Medicine Hannover, Buenteweg 9, D-30559 Hannover, Germany. saskia.willenbrock@tiho-hannover.de
authors.forename: Saskia
authors.initials: S
authors.lastname: Willenbrock
authors.forename: Olga
authors.initials: O
authors.lastname: Braun
authors.forename: Judith
authors.initials: J
authors.lastname: Baumgart
authors.forename: Sandra
authors.initials: S
authors.lastname: Lange
authors.forename: Christian
authors.initials: C
authors.lastname: Junghanss
authors.forename: Alexander
authors.initials: A
authors.lastname: Heisterkamp
authors.forename: Ingo
authors.initials: I
authors.lastname: Nolte
authors.forename: Jörn
authors.initials: J
authors.lastname: Bullerdiek
authors.forename: Hugo
authors.initials: H
authors.lastname: Murua Escobar
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002457:Cell Extracts; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism*; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D024243:HMGB1 Protein / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007834:Lasers; D008321:Mammary Glands, Animal / Q000166:cytology* / Q000276:immunology*; D008856:Microscopy, Fluorescence; D009336:Necrosis; D015203:Reproducibility of Results; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D002457:Cell Extracts; D017077:Culture Media, Conditioned; D024243:HMGB1 Protein; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma