Nitrogen-bisphosphonates block retinoblastoma phosphorylation and cell growth by inhibiting the cholesterol biosynthetic pathway in a keratinocyte model for esophageal irritation

The surprising discovery that nitrogen-containing bisphosphonates (N-BPs) a ct via inhibition of the mevalonate-to-cholesterol pathway raised the possi bility that esophageal irritation by N-BPs is mechanism-based. We used norm al human epidermal keratinocytes (NHEKs) to model N-BP effects on stratifie d squamous epithelium of the esophagus. The N-BPs alendronate and risedrona te inhibited NHEK growth in a dose-dependent manner without inducing apopto sis. N-BPs (30 muM) caused accumulation of cells in S phase and increased b inucleation (inhibited cytokinesis). Consistent with N-BP inhibition of iso prenylation, geranylgeraniol or farnesol prevented accumulation in S phase. Binucleation was also induced by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor lovastatin and by the squalene synthase inhibitor zara gozic acid A and was prevented by adding low-density lipoprotein. At 300 mu M, N-BPs reduced expression of cyclin-dependent kinase (cdk) 2 and cdk4 and enhanced expression of p21(waf1) and p27(kip1) and their binding to cdks w ith corollary hypophosphorylation of retinoblastoma. Lovastatin and zaragoz ic acid A produced similar effects, except that p21(waf1) expression and bi nding to cdks was not induced. Growth inhibition, but not binucleation, was also caused by the geranylgeranyl transferase I inhibitor, GGTI-298, which also enhanced cdk2 and cdk4 association with p27(kip1). These findings are consistent with suppression of epithelial cell growth by N-BPs via inhibit ion of the mevalonate pathway and the consequent reduction in cholesterol s ynthesis, which blocks cytokinesis, and in geranylgeranylation, which inter feres with progression through the cell cycle.