Involvement of CDKI in Growth Inhibition by Overexpression of Beta-defensins

Objective: Human beta-defensins (hBDs) are epithelial antimicrobial peptides. We recently observed that overexpression of hBDs in a squamous cell carcinoma cell line (KB cells) caused inhibition of cell growth. It is still not clear how overexpression of hBDs in the cells inhibit their growth. Cyclin-dependent kinase inhibitors (CDKI) are involved in inhibition of cell growth. The present study investigated whether CDKI are involved in the growth inhibition of hBDs-transfected KB cells. Methods: KB cells, a squamous cell carcinoma cell line, were grown in DMEM supplemented with 10% fetal bovine serum. For generating KB cells stably transfected with hBDs, the Flp-InTM system (Invitrogen, CA) was used. KB cells were transfected with the pFRT/lacZeo vector using the Lipofection method (EffectenTM, Qiagen, Germany). Transfected clones were selected in 0.1mg/ml zeocin (invitrogen, CA). The pcDNA/FRT vector containing hBD-1, -2 and -3 were cotransfected with pOG44 into the zeocin-selected KB cells. KB cells transfected with hBDs were selected in 0.2mg/ml hygromycin B (invitrogen, CA). Expression of hBD-1, -2 and -3 in transfected KB cells (KB-hBD1, KB-hBD2 and KB-hBD3) was confirmed by RT-PCR. Cell growth was evaluated by direct cell counting and XTT assays. Expression of the CDKI, p21waf1 and p27kip1, were observed by RT-PCR, real-time PCR (Roche Molecular Biochemicals, Germany) and Western blots. The results were compared between CAT-transfected KB (control cells) and hBDs-transfected KB cells. The statistical significance of the difference was analyzed using the Student t-test and Scheffes test. Results: The cell growth rate of KB-hBDs (-1, -2 and -3) was significantly lower than that of control cells (p<0.05). Expression of p21waf1 in KB-hBD2 and KB-hBD3 was observed. Expression of p27kip1 was detected in KB-hBD3. KB-hBD1 and control cells showed neither p21waf1 nor p27kip1 expression. Conclusion: The results suggest that inhibition of cell growth by overexpression of hBD-2 and hBD-3 occur via CDKI.