Effects of nicotine on proliferation, cell cycle, and differentiation in immortalized and malignant oral keratinocytes.
BACKGROUND
Numerous epidemiological studies have reported that tobacco smoking is a major risk factor for oral cancer, but relatively little is known about the effect of nicotine, a major product of cigarette smoking, on immortalized oral keratinocytes and cancer cells.


METHODS
We investigated the effects of nicotine on the growth and differentiation of immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12), and human skin keratinocytes (HaCaT), in the monolayer and in the three-dimensional (3D) raft cultures using the MTT assay, Western blotting, and cell cycle analysis.


RESULTS
Nicotine inhibited the proliferation of immortalized and malignant keratinocytes in dose- and time-dependent manners as determined by MTT assay. The 3D organotypic culture showed that nicotine at high concentration (300 microM) inhibits epithelial maturation, surface keratinization, and decreased epithelial thickness. Flow cytometry showed that nicotine inhibited cell cycle progression by inducing G(0)/G(1) arrest of HaCaT, IHOK, HN4, and HN12 cells without causing apoptosis. Nicotine treatment increased p21 expression in immortalized cells (HaCaT, IHOK) and oral cancer cells (HN4, HN12), but decreased pRb and p53 expression in oral cancer cells. Moreover, after high-dose nicotine treatment, the involucrin expression increased markedly in immortalized cells, but not in oral cancer cells.


CONCLUSIONS
We demonstrated that nicotine inhibits growth through cell cycle arrest at G(0)/G(1) phase probably by increasing the expression of p21(WAF1/CIP1). Nicotine also affects epithelial differentiation in immortalized and malignant oral keratinocytes. Malignant oral keratinocytes appear to be more resistant to the effects of nicotine on epithelial growth and differentiation as compared to the immortalized cells.
Lee
Hwa-Jeong
HJ
Department of Oral and Maxillofacial Pathology, College of Dentistry, Wonkwang University, Iksan, Korea.
Guo
Han-Young
HY
Lee
Sun-Kyung
SK
Jeon
Byung-Hun
BH
Jun
Chang-Duk
CD
Lee
Suk-Keun
SK
Park
Myung-Hee
MH
Kim
Eun-Cheol
EC
D002294:Carcinoma, Squamous Cell / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects*; D002454:Cell Differentiation / Q000187:drug effects*; D045744:Cell Line, Tumor / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects*; D009062:Mouth Neoplasms / Q000473:pathology; D009538:Nicotine / Q000494:pharmacology*
D009538:Nicotine