Reversible disassembly of an intestinal epithelial monolayer by prolonged exposure to phorbol ester

Abstract

This article describes a model of reversible disassembly of a cultured human intestinal epithelial monolayer by prolonged exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Prolonged phorbol ester exposure reduces protein kinase C (PKC) levels in numerous cell types including T84, as shown here. Under PKC-downregulated conditions, T84 monolayers, which simulate the highly organized structure of native intestinal crypt cells, become disassembled into 2 or 3 layers of rounded cells. Proliferation does not account for these morphological changes as assessed by thymidine incorporation studies. The effects of structural disorganization on epithelial barrier function was also examined. The permeability of disassembled monolayers was significantly greater than that of controls. Flux studies localized the permeability defect to the tight junction. PKC-associated alterations in the perijunctional ring of actin and myosin, one of the putative regulators of flow across the tight junction, were found to correlate with the observed functional changes. Most interesting was the fact that monolayer reassembly to the original columnar epithelial phenotype and reestablishment of barrier function occurred upon normalization of PKC levels. This model of reversible monolayer disassembly will allow investigation into the relationship between epithelial structure and function and examination of factors that govern monolayer formation.

Protein kinase C activation downregulates the expression and function of the basolateral Na+/K+/2Cl? cotransporter

“…6B). Extended PMA exposure caused the cell monolayers to disassemble and form multilayers, an event associated with loss of normal actin cytoskeletal architecture, confirming previous findings by Hecht et al (1994).…”

Protein kinase Cα inhibitor enhances the sensitivity of human pancreatic cancer HPAC cells to Clostridium perfringens enterotoxin via claudin-4

“…In the present study, TPA-induced PKC activation has also been shown to affect the epithelial barrier function via the downregulation in the formation of Ecadherin and β-catenin of adherens junctions and actinbased cytoskeletal disorganization in HPAC cells (see Supplemental data 1), as previously reported (Hecht et al 1994). Recently, transforming growth factor-β1-induced drug resistance in pancreatic cancer cells has been reported to be associated with PKCα expression ).…”

Proteolysis of Enteric Cell Villin by Entamoeba histolytica Cysteine Proteinases

“…Cultures-Caco-2 clone 1 cells (obtained from Dr. E. Pringault, Pasteur Institute, Paris, France) were isolated from a human colorectal cancer cell line through selection for homogeneity and high degree of terminal differentiation (17,19 (20) and HCT-8/E11 (21) were grown in a 1:1 mixture of DMEM and Ham's F-12 medium (Invitrogen) and in RPMI 1640 medium, respectively, ϩ 10% fetal calf serum at 37°C under a 10 and 5% CO 2 atmosphere, respectively. Trophozoites of E. histolytica strain HM1:IMSS were grown axenically in TYI-S-33 (7), supplemented with 60 g/ml G418 for pSA8 (18) or with small amounts of viable Crithidia fasciculata for E. dispar SAW760 trophozoites (22).…”