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HPAF-II, a Cell Culture Model to Study Pancreatic Epithelial Cell Structure and Function

Rajasekaran, Sigrid A. PhD*; Gopal, Jegan BS*; Espineda, Cromwell BS*; Ryazantsev, Sergey PhD; Schneeberger, Eveline E. MD; Rajasekaran, Ayyappan K. PhD*

Original Article
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Objectives: Epithelial cells have distinct apical and basolateral plasma membrane domains separated by tight junctions. This phenotype is essential for the directional transport functions of epithelial cells. Here we characterized a well-differentiated pancreatic epithelial cell line to establish a useful model for understanding the mechanisms involved in the regulation of junctional complexes, polarity, and disease processes in the pancreas.

Methods: Immunofluorescence of cell junction marker proteins and electron microscopy were used to determine the presence of tight junctions, adherens junctions, and desmosomes. The functionality of tight junctions was tested by transepithelial resistance measurements and transepithelial permeability studies of nonionic molecules. Tight junction function in polarity was determined by laser scanning confocal microscopy.

Results: Immunofluorescence analysis in HPAF-II cells revealed tight junction localization of ZO-1, occludin, and claudin-4; adherens junction localization of E-cadherin and β-catenin; and desmosomal localization of desmocollin. Transmission electron microscopy showed the presence of tight junctions, adherens junctions, and des-mosomes, and freeze-fracture electron microscopy revealed the presence of distinct anastomosing tight junction strands. Transepithelial electrical resistance and permeability measurements revealed functional tight junctions. In addition, 3-dimensional images of the monolayer generated by laser scanning confocal microscopy revealed that HPAF-II cells show polarity. Immunoblotting and RT-PCR analyses revealed high expression levels of E-cadherin and Na,K-ATPase β-subunit but low levels of the transcription factor Snail in HPAF-II cells compared with MiaPaCa-2 cells.

Conclusion: The HPAF-II cell line is a well-differentiated human pancreatic carcinoma cell line that should be useful as a model for studies aimed at understanding epithelial polarity, regulation of junctional complexes, and disease processes in pancreas.

From the *Department of Pathology and Laboratory Medicine, UCLA David Geffen School of Medicine, Los Angeles, California; †Department of Biological Chemistry, UCLA David Geffen School of Medicine, Los Angeles, CA; and ‡Molecular Pathology Unit, Massachusetts General Hospital East, Charlestown, MA.

Received for publication May 13, 2004; accepted July 2, 2004.

This work was supported by DK56216 (A.K.R.), NCI1F31CA 93084-01 (C.E.E.), and NRSA T32CA09056 (S.A.R.).

Reprints: Ayyappan K. Rajasekaran, PhD, Department of Pathology and Laboratory Medicine, Room 13-344 CHS, UCLA, Los Angeles, CA 90095 (e-mail: arajasekaran@mednet.ucla.edu).

© 2004 Lippincott Williams & Wilkins, Inc.