title: In vitro cyclooxygenase-2 protein expression and enzymatic activity in neoplastic cells.
abstract: BACKGROUND
  Cyclooxygenase-2 (COX-2) and its principle enzymatic metabolite, prostaglandin E2 (PGE2), are implicated in cancer progression. Based upon immunohistochemical (IHC) evidence that several tumor types in animals overexpress COX-2 protein, COX-2 inhibitors are used as anticancer agents in dogs and cats.
  
  
  OBJECTIVE
  IHC is inaccurate for assessing tumor-associated COX-2 protein and enzymatic activity.
  
  
  METHODS
  Five mammalian cell lines were assessed for COX-2 protein expression by IHC and Western blot analysis (WB), and functional COX-2 activity was based upon PGE2 production.
  
  
  RESULTS
  Detection of COX-2 protein by IHC and WB were in agreement in 4 of 5 cell lines. In 1 cell line that lacked COX-2 gene transcription because of promoter hypermethylation (HCT-116), IHC produced false-positive staining for COX-2 protein expression. Functional COX-2 enzymatic activity was dissociated from relative IHC-based COX-2 protein expression in 2 cell lines (RPMI 2650 and SCCF1). The RPMI 2650 cell line demonstrated strong COX-2 protein expression but minimal PGE2 production.
  
  
  CONCLUSIONS
  Western blot is more accurate than IHC for the detection of COX-2 protein in the cell lines studied. Furthermore, the semiquantitative identification of COX-2 protein by IHC or WB does not necessarily correlate with enzymatic activity. Based upon the potential inaccuracy of IHC and dissociation of COX-2 protein expression from enzymatic activity, the practice of instituting treatment of tumors with COX-2 inhibitors based solely on IHC results should be reconsidered.
authors.affiliation: Departments of Veterinary Clinical Medicine, Comparative Oncology Research Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Heller
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Fan
authors.forename: Louis-Philippe
authors.initials: LP
authors.lastname: de Lorimier
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Charney
authors.forename: Anne M
authors.initials: AM
authors.lastname: Barger
authors.forename: Sarah H
authors.initials: SH
authors.lastname: Tannehill-Gregg
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Rosol
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Wallig
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D002371:Cat Diseases / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D002415:Cats; D045744:Cell Line, Tumor; D051546:Cyclooxygenase 2 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015232:Dinoprostone / Q000032:analysis; D004283:Dog Diseases / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D004285:Dogs; D045325:HCT116 Cells; D006801:Humans; D007150:Immunohistochemistry / Q000662:veterinary; D051379:Mice; D009369:Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology / Q000662:veterinary*; D010766:Phosphorylation; D012334:RNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D017422:Sequence Analysis, DNA
chemical_list: D012334:RNA, Neoplasm; D051546:Cyclooxygenase 2; D015232:Dinoprostone