Cellular and biochemical characterization of VX-710 as a chemosensitizer: reversal of P-glycoprotein-mediated multidrug resistance in vitro.

VX-710 or (S)-N[2-Oxo-2-(3,4,5-trimethoxyphenyl)acetyl]-piperidine-2-carboxylic acid 1,7-bis(3-pyridyl)-4-heptyl ester, a novel non-macrocyclic ligand of the FK506-binding protein FKBP12, was evaluated for its ability to reverse P-glycoprotein-mediated multidrug resistance in vitro. VX-710 at 0.5-5 microM restored sensitivity of a variety of multidrug resistant cells to the cytotoxic action of doxorubicin, vincristine, etoposide or paclitaxel, including drug-selected human myeloma and epithelial carcinoma cells, and human MDR1 cDNA-transfected mouse leukemia and fibroblast cells. Uptake experiments showed that VX-710 at 0.5-2.5 microM fully restored intracellular accumulation of [14C]doxorubicin in multidrug resistant cells, suggesting that VX-710 inhibits the drug efflux activity of P-glycoprotein. VX-710 effectively inhibited photoaffinity labeling of P-glycoprotein by [3H]azidopine or [125I]iodoaryl azidoprazosin with EC50 values of 0.75 and 0.55 microM. Moreover, P-glycoprotein was specifically labeled by a tritiated photoaffinity analog of VX-710 and unlabeled VX-710 inhibited analog binding with an EC50 of 0.75 microM. VX-710 also stimulated the vanadate-inhibitable P-glycoprotein ATPase activity 2- to 3-fold in a concentration-dependent manner with an apparent k(a) of 0.1 microM. These data indicate that a direct, high-affinity interaction of VX-710 with P-glycoprotein prevents efflux of cytotoxic drugs by the MDR1 gene product in multidrug resistant tumor cells.

Chemosensitization and drug accumulation effects of VX-710, verapamil, cyclosporin A, MS-209 and GF120918 in multidrug resistant HL60/ADR cells expressing the multidrug resistance-associated protein MRP.

Overexpression of the multidrug resistance MDR1 gene product P-glycoprotein and/or the multidrug resistance-associated protein MRP confers multidrug resistance to cancer cells. The pipecolinate derivative VX-710 has previously been demonstrated to reverse MDR1-mediated multidrug resistance at concentrations of 0.5-2.5 microM by direct interaction with P-glycoprotein and inhibition of its drug efflux activity. In this study we investigated whether VX-710 as well as four other known MDR1 modulators could also reverse multidrug resistance mediated by MRP. VX-710 at 0.5-5 microM restored senstivity of MRP-expressing HL60/ADR promyelocytic leukemia cells to the cytotoxic action of doxorubicin, etoposide and vincristine. VX-710 was approximately 2-fold more effective than verapamil, MS-209 and CsA in modulating MRP-mediated multidrug resistance, whereas GF120918 had no significant effect. VX-710 was also more effective than verapamil, MS-209 and CsA in restoring the daunorubicin accumulation deficit in HL60/ADR cells and in increasing calcein uptake. A photoaffinity analog of VX-710, [3H]VF-13,159, specifically photo labeled the MRP protein and unlabeled VX-710 inhibited this binding in a concentration-dependent manner. These data suggest that VX-710 is not only a potent modulator of P-glycoprotein-mediated multidrug resistance, but also affects multidrug resistance in MRP-expressing cells and may exert its action, at least in part, by binding directly to MRP.

Basement membrane assembly and differentiation of cultured corneal cells: importance of culture environment and endothelial cell interaction.

A three-dimensional corneal tissue construct was used to examine the effect of culture environment and endothelial cell interaction on epithelial differentiation and basement membrane assembly. Rabbit corneal epithelial cells were cultured over rabbit stromal fibroblasts in a collagen matrix with or without an underlying layer of immortalized mouse corneal endothelial cells (Muragaki, Shiota, Inoue, Ooshima, Olsen, and Ninomiya. (1992) Eur. J. Biochem. 207, 895-902). The cultures were grown submerged or at a dry or moist interface. Basement membrane, anchoring fibril, and hemidesmosome assembly was monitored using transmission electron microscopy as well as indirect immunofluorescence microscopy of laminin, type VII collagen, and alpha 6 integrin. Antibodies against keratin 3 (K3) and alpha-enolase marked differentiated and undifferentiated corneal epithelial cells, respectively. When all three cell types were cultured at a moist interface, hemidesmosomes, anchoring fibrils, and a continuous basement membrane were observed 2 wk after lifting the cultures to an air-liquid interface (air-lift). The distribution of alpha-enolase and K3 was identical to patterns seen in the limbal region of the cornea. Air-lifted tissue constructs lacking the endothelial cell layer showed only limited distribution of laminin and type VII collagen at the epithelial-matrix junction. alpha 6 Integrin was present along the entire plasma membrane of the basal cells; epithelial differentiation was not complete as alpha-enolase was seen in basal and two to three layers of suprabasal cells. Submerged cultures without endothelial cells did not express differentiation markers or basement membrane components. These data indicate that endothelial cell interaction dramatically enhances the amount and quality of epithelial basement membrane assembly and that epithelial differentiation is influenced by the type of interface between tissue, liquid, and air.

The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function.

We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal layer to a more in vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct also increased with time reaching a permeability to water of less than 2%-h after approximately 2 weeks at the air-liquid interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used for in vitro research and testing and is also being tested in clinical applications.

Epidermis generated in vitro: practical considerations and applications.

The technology for culture of epidermis is one of the most advanced to date for generation of a tissue in vitro. Cultured epidermis is already used for a number of applications ranging from use as a permanent skin replacement to use as an organotypic culture model for toxicity testing and basic research. While simple epidermal sheets have been grafted successfully, more advanced models for skin replacement consisting of both dermal and epidermal components are in development and being tested in a number of laboratories. One of the most advanced in vitro models is the living skin equivalent, an organotypic model consisting of a collagen lattice contracted and nourished by dermal fibroblasts overlaid with a fully formed epidermis.