Nitrogen-Bisphosphonates Block Retinoblastoma Phosphorylation and Cell Growth by Inhibiting the Cholesterol Biosynthetic Pathway in a Keratinocyte Model for Esophageal Irritation
- Department of Bone Biology and Osteoporosis Research, Merck Research Laboratories, West Point, Pennsylvania
Abstract
The surprising discovery that nitrogen-containing bisphosphonates (N-BPs) act via inhibition of the mevalonate-to-cholesterol pathway raised the possibility that esophageal irritation by N-BPs is mechanism-based. We used normal human epidermal keratinocytes (NHEKs) to model N-BP effects on stratified squamous epithelium of the esophagus. The N-BPs alendronate and risedronate inhibited NHEK growth in a dose-dependent manner without inducing apoptosis. N-BPs (30 μM) caused accumulation of cells in S phase and increased binucleation (inhibited cytokinesis). Consistent with N-BP inhibition of isoprenylation, 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 zaragozic acid A and was prevented by adding low-density lipoprotein. At 300 μM, N-BPs reduced expression of cyclin-dependent kinase (cdk) 2 and cdk4 and enhanced expression of p21waf1 and p27kip1 and their binding to cdks with corollary hypophosphorylation of retinoblastoma. Lovastatin and zaragozic acid A produced similar effects, except that p21waf1 expression and binding 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 p27kip1. These findings are consistent with suppression of epithelial cell growth by N-BPs via inhibition of the mevalonate pathway and the consequent reduction in cholesterol synthesis, which blocks cytokinesis, and in geranylgeranylation, which interferes with progression through the cell cycle.
Footnotes
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Send reprint requests to: Dr. Alfred A. Reszka, Department of Bone Biology, WP26A-1000, Merck Research Laboratories, West Point, PA, 19486. E-mail: alfred_reszka{at}merck.com
- Abbreviations:
- HMG
- 3-hydroxy-3-methylglutaryl
- CoA
- coenzyme A
- BP
- bisphosphonate
- N-BP
- nitrogen-containing bisphosphonate
- ALN
- alendronate
- RIS
- risedronate
- FPP
- farnesyl diphosphate
- NHEK
- normal human epidermal keratinocyte
- pRb
- retinoblastoma
- LOV
- lovastatin
- ANOVA
- analysis of variance
- FACS
- fluorescence-activated cell sorting
- HBS
- HEPES-buffered saline
- cdk
- cyclin-dependent kinase
- Zara-A
- zaragozic acid A
- GGTI
- geranylgeranyl transferase
- FTI
- farnesyl transferase
- FTI-1
- 5(S)-n-butyl-4-[1-(4-cyanobenzyl)imidazol-5-ylmethyl]-1-(3-trifluoromethoxyphenyl)piperazin-2-one
- LDL
- low-density lipoprotein
- GGOH
- geranylgeraniol
- FOH
- farnesol
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- Received August 24, 2000.
- Accepted October 6, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
