Cell Biol Toxicol (2011) 27:69–81 Turner et al. 1996; Zager and Iwata 1997; Xu et al. 2005). Formation of epithelia is accompanied by kera- tinocyte differentiation, during which the pattern of keratin synthesis is changed. Keratins form inter- mediate filaments in a dynamic equilibrium be- tween a soluble and a fibrillar status. More than 20 keratins have been identified in human tissues (Moll et al. 1982). In stratified epithelia, K15 is expressed (Leube et al. 1988; Lloyd et al. 1995; Waseem et al. 1999; Werner and Munz 2000), as well as polypeptides K1/K10 and K4/K13 as pairs (Kirfel et al. 2003; Hatakeyama et al. 2004). K19 is generally expressed by simple epithelial types, though it may occasionally be expressed in stratified epithelia too. The transcriptional regulation of keratins involves calcium ions, cytokines and growth factors, and hormones. Specific promoter binding proteins are identified, including members of the enhancer binding protein family (c/EBPs; Maytin et al. 1999; Shim et al. 2005). Cultured HaCaT cells, derived from human skin, proliferate in vitro under synthesis of a keratin pattern that corresponds to the basal cell layer of epithelia, i.e., proliferating cells of low degree of differentiation. Notably, K1, K10, and K13 are expressed just like in normal epidermis (Boukamp et al. 1988). After having attained confluency, HaCaT cells spontaneously form stratified layers, thereby changing their keratin pattern to that of differentiated keratinocytes, particularly inducing K15 (Lloyd et al. 1995; Werner and Munz 2000). Certain pharmacological agents have the capacity to suppress HaCaT differentiation and alter keratin expression (Hatakeyama et al. 2004). HaCaT differ- entiation can be suppressed, and keratin expression can be altered by external effectors (Pi et al. 2008). At present, HaCaT cells represent the only clonal cell line that exhibits this type of differentiation. Here, we study fluoride effects on HaCaT cells. Such a model to study fluoride effects on epithelia has so far not been available. Our results indicated that fluoride at high dose retarded differentiation of HaCaT cells. An altered expression pattern of keratin species was detected, and cells concomitantly failed to assemble stratified layers. We concluded that fluoride at high dose could influence cell differenti- ation, which may help to explain fluoride-induced tissue disturbances. 71 Materials and methods Cell culture HaCaT cells were kindly provided by Dr. N.E. Fusenig, Division of Carcinogenesis and Differentia- tion, German Cancer Research Center, Heidelberg. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with glutaMax™, supple- mented with 10% fetal bovine serum (FBS), 100 IU/ ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin (GIBCO). Cells were seeded at 10−5 cell/ml, and medium was changed every other day up to confluence (90% epithelialized dishes). Sodium fluoride (NaF; Sigma) was added as a stock solution of 1 M in demineralized sterile water. Cells were treated at different concentrations from 0.5 to 5 mM of NaF for periods of 1, 2, and 3 days under serum-free condition, unless otherwise stated. These concentrations are just lower than the 50% toxicity of NaF for HaCaT (TC50 = 6 mM) as reported by (Dogan et al. 2002). To test the reversibility of fluoride effects, fluoride treatment for 24 and 72 h was terminated by changing the culture medium (DMEM supplemented with 10% FBS, no fluoride) and allowing further growth for 24 h. RT-PCR HaCaT cells were treated with low or high dose of NaF during 24, 48, and 72 h. Cells were then harvested, and total RNA was extracted from cell culture with Tri- Reagent (Molecular Research Center Inc., Cincinnati, OH, USA) according to the manufacturer's instructions. RNA concentration was adjusted to 1 μg/μl. Total RNA was reverse-transcribed using superscript™ II Reverse Transcriptase (Invitrogen, Groningen, Nether- lands) according to the manufacturer's instructions, and complementary deoxyribonucleic acid (cDNA) was amplified for each keratin, for c/EBP alpha, and for HSC70 by using specific couple of primers (Table 1). The polymerase chain reactions (PCR) contained 2 μl of cDNA in 25 μl reaction solution, 2.5 μl PCR 5× buffer, 250 μM dNTP, 2 mM MgCl2, 0,5 μM forward and reverse primers, and 0.05 U/μl Taq polymerase (Eurobio, Les Ulis, France). The thermocycler was programmed for 25 cycles of 30 s at the specific annealing temperature (Ta in Table 1). RNA without previous reverse transcription (RT)