Diphenylhydantoin plays a role in gene expression related to cytoskeleton and protein adhesion in human normal palate fibroblasts.
OBJECTIVE
Morphogenetic processes during palate development are related to extracellular matrix composition. The cell-extracellular matrix relation plays a role in cell activity and in gene expression. We studied the effect of diphenylhydantoin, a teratogen known to induce cleft palate in human newborns, on extracellular matrix production. We investigated whether diphenylhydantoin treatment caused any differences in glycosaminoglycans, collagen synthesis and gene expression in human normal palate fibroblasts.


METHODS
Human palate fibroblasts were maintained for 24 hours in serum-free 199 medium containing 5 microg/mL (3)H-glucosamine or (3)H proline hydrochloride. Collagen and glycosaminoglycan classes were then measured using biochemical methods, gene expression with microarray analysis and cytoskeleton components with immunofluorescent antibodies and computer analysis.


RESULTS
In normal fibroblasts diphenylhydantoin reduced collagen and glycosaminoglycan synthesis with a marked effect on sulphated glycosaminoglycans. There were also substantial decreases in tubulin, vimentin and alpha-actin staining and an increase of vinculin compared to controls. Diphenylhydantoin acted on several genes related to the synthesis of cytoskeleton and adhesion membrane proteins. It inhibited caderin, caveolin, RTK and alpha-actin, and increased nectin, cytoplasmatic FRG vinculin, ITGA, ITGB extracellular matrix ligand and EDG2 gene expression. DNA binding gene expression, which plays a role in cell growth and senescence, was activated.


CONCLUSIONS
Since cell activity is dependent on the cell morphology and extracellular matrix composition, these findings indicate that in human normal palate fibroblasts diphenylhydantoin can modify cytoskeletal components and extracellular matrix-cell adhesion, with consequent effects on gene expression. These changes might be related to anomalous palate development.
Pezzetti
Furio
F
Histology, Embryology and Applied Biology Department, University of Bologna, Bologna, Italy.
Carinci
Francesco
F
Palmieri
Annalisa
A
Vizzotto
Laura
L
Moscheni
Claudia
C
Vertemati
Maurizio
M
Calastrini
Carla
C
Pellati
Agnese
A
Stabellini
Giordano
G
D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000187:drug effects*; D002478:Cells, Cultured; D002675:Child, Preschool; D003094:Collagen / Q000096:biosynthesis / Q000187:drug effects; D003599:Cytoskeleton / Q000187:drug effects*; D005260:Female; D005347:Fibroblasts / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects*; D006025:Glycosaminoglycans / Q000096:biosynthesis; D006801:Humans; D008297:Male; D046529:Microscopy, Electron, Transmission; D020411:Oligonucleotide Array Sequence Analysis; D010159:Palate / Q000187:drug effects* / Q000378:metabolism; D010672:Phenytoin / Q000633:toxicity*; D013723:Teratogens / Q000633:toxicity*
D015815:Cell Adhesion Molecules; D006025:Glycosaminoglycans; D013723:Teratogens; D010672:Phenytoin; D003094:Collagen