UCLA

Bisphosphonates (BPs) are the most widely used anti-resorptive agents for osteoporotic and cancer patients. With frequent use of BPs in multiple clinical settings, a growing body of evidence supports a notion that some BPs cause side effects that are specific to soft tissues. Previously, we reported that BPs inhibit proliferation and induce senescence in normal human oral keratinocytes (NHOKs). Cell cycle analysis in BP-treated NHOKs revealed an increase in the S phase cell cycle arrest (Kim et al, 2011). In this study, we further examined the underlying mechanisms by which BPs inhibit the proliferation of NHOKs.

Primary NHOKs were established from normal human oral epithelium. These cells were treated with pamidronate (PAM) and zoledronate (ZOL) and subjected to microarray gene profiling. Microarray analysis showed that genes associated with cell cycle control of chromosomal replication were significantly dysregulated. Among the validated genes, expression of CCNA2, a cyclin that is associated with S-phase in the cell cycle, was drastically inhibited by both PAM and ZOL as determined by qRT-PCR and Western blotting. Expression of CCND1, Cdk6, and Cdk2 were not affected by PAM and ZOL. The luciferase reporter assay using CCNA2 promoter showed suppression of luciferase activity in ZOL-treated NHOK, indicating that the loss of cyclin A2 expression is transcriptionally regulated. Immunohistochemistry also showed the inhibition of cyclin A2 expression in NHOK in ZOL-treated oral mucosa tissue constructs. Our study, through the microarray gene expression profiling, demonstrates that inhibition of NHOK proliferation by BPs is, in part, mediated by suppressing CCNA2 expression at the transcriptional level, which may explain the underlying mechanisms of soft tissue toxicity by BPs.