SCREENING METHOD FOR TYROSINASE INHIBITORS 517 derived from moderately pigmented neonatal foreskins, were obtained from Cascade Bio- logics (Portland, OR) and cultured with Medium 254 supplemented with human mel- anocyte growth supplement (Cascade Biologics), 100 U ml-1 penicillin, 0.1 mg ml-1 streptomycin, and 0.25 μg ml-1 amphotericin B. DNA CONSTRUCTS The pING-TYR plasmid that encodes human TYR under the control of the cytomegalo- virus promoter (12,13) was kindly provided by Dr. Alan Houghton from Memorial Sloan- Kettering Cancer Center, and used as the template for the polymerase chain reaction (PCR)-amplifi cation of the entire coding sequence of human TYR, using primers 5′-GGG AAG CTT GCC ACC ATG CTC CTG GCT GTT TTG TAC TGC C-3′ and 5′-GGG TCT AGA TTA TAA ATG GCT CTG ATA CAA GCT-3′ (HindIII and XbaI restriction sites were incorporated into the sense and antisense primers, respectively, as underlined). The sense primer also contained a Kozak sequence shown in bold font. PCR was done in a reaction mixture (50 μl) that contained 10 pmol of each primer, 1.25 units of Taq DNA polymerase, and 2.5 mM of dNTPs. The conditions for PCR were set as 1 min at 98°C, 35 cycles of 10 s at 98°C, 10 s at 65°C, and 3 min at 72°C, with a fi nal extension step of 3 min at 72°C. The amplifi ed product was purifi ed by a PCR purifi ca- tion kit (SolGent, Daejeon, Korea). The amplifi ed product was digested with restriction enzymes HindIII (New England Biolabs, Ipswich, US) and XbaI (Elpis-Biotech, Daejeon, Korea), and ligated into the same restriction sites of pcDNA3.1+ (Invitrogen, Grand Is- land, CA) using T4 DNA ligase (Elpis-Biotech) to generate a pcDNA-TYR construct. The construct was propagated in the E. coli strain DH5α (Elpis-Biotech) and purifi ed us- ing Endo-free Maxi-Prep DNA purifi cation kits (Qiagen, Valencia, CA). The purifi ed plasmid DNA was linearized by digesting it with MfeI followed by purifi cation with a gel-purifi cation kit (SolGent, Daejeon, Korea). GENERATION OF A STABLE CELL LINE THAT EXPRESSES HUMAN TYR HEK293 cells were transfected with the linearized plasmid construct using Lipofectamine 2000 (Invitrogen) as previously described (14). Briefl y, cells at about 20% confl uency in a 100-mm culture dish were washed with PBS and treated with a mixture of 5 μg of linearized DNA and 10 μl of Lipofectamine in 5 ml of Opti-MEM (Invitrogen) for 18 h. The transfected cells were cultured in a normal growth medium for one day. Then the cells were subcultured in a medium supplemented with 1 mg ml-1of G418 (Geneticin®, Invit- rogen). The culture medium was changed every four days, and after a total of three weeks, the culture dishes were examined for surviving colonies. Healthy-looking colonies that seemed to have dark granules were harvested and distributed to 96-well plates at a density of one cell per well. Among the several colonies that grew rapidly, the most dark-colored colony was chosen and replated on 96-well plates for the second-round selection. The most rap- idly proliferating colony was expanded further into a cell line named HEK293-TYR. IN VITRO ASSAY FOR TYR ACTIVITY USING CELL-FREE EXTRACTS Cells were homogenized in an ice-cold lysis buffer (10 mM Tris-Cl, pH 7.4 120 mM NaCl 25 mM KCl 2.0 mM EGTA 1.0 mM EDTA and 0.5% Triton X-100 and

JOURNAL OF COSMETIC SCIENCE 518 protease inhibitor cocktail). The cell homogenates were centrifuged at 13,000 × g for 15 min at 4°C to obtain cell-free extracts. The in vitro TYR assay was done on a 96-well microplate with the reaction mixture (200 μl) containing 100 mM sodium phosphate buffer (pH 6.8), cell-free extracts (40 μg protein), 0.5 mM L-tyrosine, and 1 μM DOPA. When indicated, 1~1000 μM of test samples were included in the reaction mixture. The reaction mixture was incubated for 120 min at 37°C, and DOPA chrome formation was estimated at 490 nm by a BioRad Model 680 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA). WESTERN BLOTTING Cell-free extracts containing 40 μg of protein were diluted in Laemmli buffer containing 2% SDS and 1.2% dithiothreitol, and heat-denatured at 95°C for fi ve minutes. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the separated proteins were transferred to a polyvinylidene fl uoride membrane (Pall Cor- poration, Port Washington, NY). The membrane was incubated with a primary antibody overnight at 4°C, and then with a secondary antibody conjugated with horseradish per- oxidase for one hour at room temperature. The bands were detected using Enhanced Peroxidase Detection reagents (Elpis-Biotech) according to the manufacturer’s instruc- tions and subjected to densitometry analysis. Primary antibodies for TYR and glyc- eraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ASSAY OF THE TYR-DEPENDENT MELANIN SYNTHESIS IN CELLS Cells were cultured with or without addition of 1.0 mM L-tyrosine. The medium was replaced every other day and the cells were cultured for a total of six days. In some experiments, HEMs were pretreated with 100 μM of TYR inhibitors 60 min prior to the addition of 1.0 mM L-tyrosine. Accumulation of melanin-like materials inside of cells was highlighted by Fontana–Masson staining (15). Cells were fi xed in 4% p-formaldehyde for 10 min at room temperature and stained for melanin using a Fontana–Masson staining kit from American Master*Tech Scientifi c, Inc. (Lodi, CA). Cells were stained with ammoniacal silver solution for 30 min at 60°C, followed by incubation in 0.1% gold chloride solution and then in 5% sodium thiosulfate solu- tion. Cell morphology and pigmentation were examined under a phase-contrast mi- croscope (Eclipse TE2000U, Nikon Instruments Inc., Melville, NY). Intracellular melanin was extracted with 100 mM NaOH at 60°C for 60 min. The optical density at 490 nm for melanin was normalized for the protein content determined by Bio-Rad DC assay. Cell viability was assayed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe- nyltetrazolium bromide (16,17). RESULTS AND DISCUSSION In order to establish a cell line that grows rapidly and expresses human TYR constitu- tively, HEK293 cells were transfected with a human TYR construct and cloned based on