title: KRJ-I and BON cell lines: defining an appropriate enterochromaffin cell neuroendocrine tumor model.
abstract: BACKGROUND
  Neuroendocrine tumors (NETs) of the gastrointestinal (GI) system are increasing in incidence with minimal improvement in prognosis. Although the cell of origin has been identified as the enterochromaffin (EC) cell, its secretory and proliferative regulation has not been defined at a mechanistic level. To date, the BON cell line has been the most widely used in vitro EC cell model despite its pancreatic origin. Using whole-genome mathematical analysis as well as secretory and proliferative studies, we compared the BON cell line to the small intestine (SI) EC cell-derived NET cell line, KRJ-I, to assess individual cell line validity and applicability for the investigation of GI-NET disease.
  
  
  RESULTS
  Principal component analysis and ANOVA of KRJ-I and BON transcriptomes (U133 Plus 2) identified substantially different (<10%) overlap in transcripts with minimal (R(2) = 0.24) correlation in gene expression profiles. RT-PCR detected large variability (>12%) in neuroendocrine (NE) marker transcripts in the BON cell line and the absence of Tph-2, DDC, TGFbetaR2, and M3 transcripts in KRJ-I. The KRJ-I cell line secreted serotonin (5-HT) in response to isoproterenol (EC(50) = 100 nM), noradrenaline (EC(50) = 1.7 nM), and pituitary adenylate cyclase (PACAP, EC(50) = 0.03 nM). Cholecystokinin (IC(50) = 430 nM), somatostatin (IC(50) = 400 nM), acetylcholine (IC(50) = 3.7 nM), and gamma-aminobutyric acid A (GABA(A), IC(50) = 2 nM) all inhibited 5-HT release, while gastrin and bombesin had no effect. 5-HT secretion in the BON cell line was stimulated by isoproterenol (EC(50) = 900 nM), noradrenaline (EC(50) = 20 nM), cholecystokinin (EC(50) = 130 nM), PACAP (EC(50) = 0.12 nM), bombesin (EC(50) = 15 nM), and acetylcholine (EC(50) = 0.2 nM). It was inhibited by somatostatin (IC(50) = 300 nM) but not GABA(A). KRJ-I responded with proliferation to connective tissue growth factor (CTGF, EC(50) = 0.002 ng/ml), transforming growth factor-alpha (TGFalpha, EC(50) = 0.63 ng/ml) and transforming growth factor-beta (TGFbeta, EC(50) = 0.63 ng/ml). Epidermal growth factor (EGF) and somatostatin had no significant effect. BON cell proliferation was stimulated only by EGF and TGFalpha (EC(50) = 15.8 and 10 ng/ml). TGFbeta (IC(50) = 0.16 ng/ml), MZ-4-147 (IC(50) = 0.5 nM), and BIM23A761 (IC(50) = 0.06 nM) all inhibited proliferation. CTGF and somatostatin had no effect.
  
  
  CONCLUSIONS
  KRJ-I and BON cell lines demonstrate substantial differences in gene level transcripts, inconsistent receptor profile expression, wide variability in NE marker transcript levels, and significantly differential proliferative and secretory responses. Given the EC cell origin of KRJ-I, these results provide evidence that the BON cell line does not represent an EC cell system and is not a valid study model of (carcinoid) EC cell-derived NET.
authors.affiliation: Gastrointestinal Pathobiology Research Group, Yale University School of Medicine New Haven, New Haven, CT 06520-8062, USA.
authors.forename: Zakiya-Luna
authors.initials: ZL
authors.lastname: Siddique
authors.forename: Ignat
authors.initials: I
authors.lastname: Drozdov
authors.forename: Jared
authors.initials: J
authors.lastname: Floch
authors.forename: Bjorn I
authors.initials: BI
authors.lastname: Gustafsson
authors.forename: Kamilla
authors.initials: K
authors.lastname: Stunes
authors.forename: Roswitha
authors.initials: R
authors.lastname: Pfragner
authors.forename: Mark
authors.initials: M
authors.lastname: Kidd
authors.forename: Irvin M
authors.initials: IM
authors.lastname: Modlin
mesh_terms: D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004759:Enterochromaffin Cells / Q000187:drug effects / Q000502:physiology*; D006131:Growth Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology / Q000627:therapeutic use; D018358:Neuroendocrine Tumors / Q000188:drug therapy / Q000503:physiopathology*
chemical_list: D006131:Growth Inhibitors; D036341:Intercellular Signaling Peptides and Proteins