of 262, no. of 25, 4024-4033 d in a-melanocyte-stimulating ... · the journal of biological...

THE JOURNAL OF BIOLOGICAL CHEMISTRY D 1987 by The American Society of Biological Chemieta, Inc.

Vol. 262, No. 9, Issue of March 25, pp. 4024-4033 1987 Printed in r i ~ . ~ .

a-Melanocyte-stimulating Hormone Regulation of Tyrosinase in Cloudman S-91 Mouse Melanoma Cell Cultures*

(Received for publication, December 18, 7986)

Bryan 8. Fuller, Julie B. Lunsford, and Deborah S . Iman From the Department of Biochemistv and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 731 90

a - M S H (melanocyte-stimulating hormone) causes an increase in tyrosinase activity (O-diphenol-Oz oxidoreductase; EC 1.14.18.1) in Cloudman S-91 mouse melanoma cell cultures following a lag period of approximately 9 h. Treatment of cells with 2 X 10” M a-MSH for 6 days results in a 90-fold increase in the specific activity of the enzyme. The hormone-mediated increase in tyrosinase activity is dependent upon continued transcription since the enzyme induction is sup- pressed by either cordycepin (1 pg/ml) or a-amanitin (10 ~g/ml) . Immunoprecipitation analysis of pulse-labeled tyrosinase from control and MSH-treated cultures (48-h exposure) has demonstrated that MSH stimulates tyrosinase synthesis by approximately 4- fold, a level of induction which does not correspond to the observed 24-fold increase in enzyme activity. When immunotitration curves were developed from cell extracts of control and MSH-treated cultures using immunoprecipitation and competitive enzyme-linked immunosorbent assay protocols, evidence for the presence of immunologically active but catalytically less active enzyme in untreated melanoma cell cultures was demonstrated. Degradation rates of tyrosinase were found to be similar in control cultures or in cells treated with MSH for up to 48 h. Taken together, these results suggest that in addition to stimulating tyrosinase synthesis, MSH may also promote an increase in the catalytic efficiency of the enzyme.

CIoudman S-91 mouse melanoma cells demonstrate increased melanogenesis in response to treatment with melanocyte-stimulating hormone, MSH.’ This increase in melanin production has been shown to be the result of a hormonally induced rise in tyrosinase (0-diphenol-0, oxidoreductase, EC 1.14.18.1) activity, the rate-limiting enzyme in the melanin synthesis pathway (1). Various aspects of the melanoma cell response to MSH have been examined. MSH apparently exerts its melanogenic effect through the second messenger, cyclic AMP, since either dibutyryl CAMP (Z), or theophylline, an inhibitor of phosphodiesterase, will mimic MSH action (3). Furthermore, MSH stimulates adenylate cyclase activity (4, 5 ) and increases cellular levels of cyclic AMP (6). A lag

* This work was supported by United States Public Health Service Grants CA41425 and CA30393 from the Nationallnstitutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: MSH, or-melanocyte-stimulating hormone; PBS, phosphate-buffered saline; L-DOPA, L-dihydroxyphen- ylalanine; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay.

_I__

period of several hours exists following exposure of cells to MSH before any rise in tyrosinase activity is detectable. Studies with inhibitors of RNA and protein synthesis have provided evidence that MSH action requires continued transcription and translation (7). Although this observation suggests that MSH may increase the synthesis of tyrosinase, other reports have provided evidence that MSH may increase tyrosinase activity in melanoma cell cultures by activating pre-existing but catalytically inactive enzyme molecules (8). Results from one study have suggested, however, that MSH stimulates tyrosinase synthesis and that, in general, the level of tyrosinase activity in melanoma cells correlates with the abundance of the enzyme (9). The present studies were un- dertaken to more clearly characterize the molecular events involved in the MSH stimulation of tyrosinase in melanoma. In this report, we discuss various aspects of the kinetics of MSH action and provide evidence that MSH may act to both increase tyrosinase synthesis as well as promote the activation of pre-existing enzyme molecules.

EXPERIMENTAL PROCEDURES

Materials The Cloudman S-91 (CCL 53.1) melanoma cells were obtained

from the American Type Culture Collection Cell repository (Rock- ville, MD). All radiochemicals used were purchased from New Eng- land Nuclear and included the following: ~-[4,5-’HH]leucine, specific activity 50 Ci/rnmol; [ring-3,5-3H]tyrosine, specific activity 48 Ci/ mmol; I‘4C]tyrosine, specific activity 400 mCi/mmol; and [T-~~PIATP, specific activity 10-40 Ci/mmol. Horse serum was purchased from Sterile Systems (Logan, Utah), and Ham’s F-10 nutrient medium was obtained from Gibco (Grand Island, NY). a-MSH, dibutyryl cyclic AMP, goat anti-rabbit I@, cordycepin, and CAMP-dependent protein kinase were purchased from Sigma. @-Amanitin was purchased from Boehringer Mannheim. Peroxidase-labeled goat anti-rabbit I& was obtained from Cooper Biomedical (Malvern, PA). Trypsin (1:250) was purchased from Gibco (Grand Island, NY).

Cell Cultures Melanoma cells were grown in Ham’s F-10 nutrient medium for-

tified with 10% horse serum. Penicillin (100 units/ml) and strepto- mycin (100 Fg/ml) were also present in the medium. Stock cultures were maintained in l-liter Corning flasks and subcultured weekly. Cells were removed from flasks with Tyrode’s salt solution containing 5 T ~ M EDTA. Cell counts were made with a hemocytometer.

Cyclic AMP Measurements Intracellular levels of cyclic AMP were measured by the use of a

radioimmunoassay kit purchased from New England Nuclear. Cells were seeded (lo6 cells/flaak) into 25-cm2 culture flasks and allowed to attach overnight. MSH M) was then added and at various times thereafter medium was removed, the flasks washed twice in ice- cold Hank‘s balanced salt solution and 3 ml of cold 7% perchloric acid added immediately. The flasks were then left at 4 “C for 24 h after which time the perchloric acid fraction containing cyclic AMP was removed and neutralized with 1 N KOH. Following centrifugation

4024

MSH Regulation of Tyrosinase 4025

for 10 min, the supernatant was saved and assayed directly. The radioimmunoassay used in these studies is able to detect cyclic AMP levels as low as 0.005 pmole/reaction tube.

CAMP-dependent Protein Kinase Assay Cell extracts were assayed for CAMP-dependent protein kinase

activity as described by Haddox et al. (10). Cells were homogenized in 5.0 mM sodium phosphate buffer, pH 7.4, containing 10 mM EDTA, 0.5 mM l-methy1-3-isobutylxanthine, and 0.5% glycerol. Homoge- nates were centrifuged at 30,000 X g for 30 min at 4 "C. The supernatant (50 pl) was assayed for kinase activity in 25 pl of reaction mixture consisting of 20 mM sodium phosphate, pH 7.0,lO mM MgC12, 0.1 mM ATP, 0.5 mM l-methyl-3-isobutylxanthine, 50 pg of histone, 5 mM sodium fluoride, 0.5 pCi of [32P]ATP, and 10 pM CAMP. Reactions were allowed to proceed at 30 "C for the indicated times and the amount of incorporation of 32P into histone determined as described elsewhere (10).

Purification of Tyrosinase from Mouse Melanoma Tumors Preparation of Tumor Homogenntes-Tyrosinase was purified from

Cloudman S-91 mouse melanoma tumors raised in DBA/2 mice (Jackson Laboratories, Bar Harbor, Maine). Mice were injected subcutaneously with 2 X lo6 cells in 0.2 ml of Hank's balanced salt solution. Tumors were visible within 2 weeks and usually removed 3- 4 weeks after injection. Tumor tissue (10 g) was homogenized in 0.25 M sucrose (10% w/v) in a Virtis homogenizer at setting #6 for 4 1- min bursts followed each time by 1-min cooling periods. The resulting homogenate was centrifuged at 1,000 X g and the pellet discarded. The supernatant was made 0.5% Triton X-100 and stirred for 1 h at 4 "C. Trypsin was then added (0.2 mg/mg of protein) and stirring continued for 3 h at 37 "C. Following this tryptic digestion, the mixture was centrifuged at 50,000 X g for 20 min at 4 "C. The resulting supernatant was made 0.05 M Tris-HC1, pH 8.0, and 0.05 M KC1 and applied to a DEAE-Sephadex ion exchange column as described below.

DEAE-Sephadex Ion Exchange Chromatography-The 50,000 X g supernatant from the trypsin-treated melanoma cell homogenate (200 mg of protein in 50 ml) was adjusted to 0.05 M Tris-HC1, pH 8.0,0.05 M KCI, and applied to a 2.5 X 30-cm DEAE-Sephadex anion exchange column equilibrated with the same buffer. Following the addition of sample, the column was washed with a volume of starting buffer (0.05 M Tris-HC1, pH 8.0, with 0.05 M KCI) equal to the sample volume. Tyrosinase was then eluted with a linear gradient from 0.05 to 0.4 M KC1 (400 ml total gradient volume, 10 ml/tube). The fractions containing tyrosinase were visualized by incubating 200 pl from each tube with 0.5 ml of L-DOPA in 0.1 M sodium phosphate buffer, pH 6.8, a t 37 "C for 5 min. Tyrosinase fractions were pooled and concentrated.

Purification of Tyrosinase by Discontinuous Polyacrylamide Gel Electrophoresis

Discontinuous polyacrylamide gel electrophoresis was carried out by the method of Davis (11). For preparative applications, 3-mm thick slab gels were used (14 X 11 cm high). The trough former used for the stacking gel allowed up to 5 ml of sample to be applied. Samples were equilibrated with upper buffer prior to electrophoresis. Tyrosinase, partially purified by DEAE ion exchange chromatography, was loaded onto an 10% T, 5% C discontinuous polyacrylamide slab gel and electrophoresed at constant current (60 mA/3-mm thick slab) until the bromphenol blue tracking dye had migrated to within 5 mm of the bottom of the gel. Tyrosinase was visualized by first neutralizing the gel in 0.5 M potassium phosphate buffer, pH 6.5, for 5 min, and then incubating the gel at 37 "C in 0.1 M sodium phosphate buffer, pH 6.8, containing 0.2% L-DOPA. The section of gel containing the melanin band (detectable within 5 min) was cut out and immediately layered onto a 10% T, 2.7% C nondenaturing SDS- polyacrylamide gel, sealed in with 1% agarose (prepared in stacking gel buffer), and electrophoresed with the same run conditions as above. Following this electrophoresis, the gel was again stained with L-DOPA and the tyrosinase band cut out. Tyrosinase was recovered by placing the gel slice in Spectrapor dialysis tubing (molecular weight cut off, 8000) containing electroelution buffer (25 mM Tris, 150 mM glycine) and electroeluting in a Transfor Cell (Hoeffer Scientific, San Francisco, CA), containing the same buffer, at 40 volts (0.4 A) for 3 h. At the end of 3 h the electrodes were reversed for 10 min to release any enzyme from the dialysis membrane wall. The tyrosinase released from the gel was then concentrated by ultrafiltration on a PM-10

filtration membrane (Amicon Corp.). All preparative electrophoresis procedures were carried out in a Hoeffer SE600 slab gel system with an E-C 500 power supply (E-C Apparatus Corp., St. Petersburg, FL).

SDS-Polyacrylamide Gel Electrophoresis

SDS-gel electrophoresis was performed using the method of Laemmli (12). Samples for denaturing SDS gels (10% T, 2.7% C) were heated in boiling water for 90 s in 2% SDS, 10% glycerol, 5% p- mercaptoethanol prepared in 0.06 M Tris-HC1, pH 6.8. For nondenaturing SDS gels, tyrosinase preparations were incubated in the above buffer without 8-mercaptoethanol for 30 min at 37 "C. For both preparative discontinuous gel electrophoresis and SDS-gel electrophoresis, bromphenol blue was used as the tracking dye.

Preparation of Anti-tyrosinuse Antiserum

Purified tyrosinase (50 pg) was dissolved in 1 ml of PBS and emulsified in 1 ml of complete Freund's adjuvant. This emulsion was then injected into New Zealand White rabbits at 10 intradermal sites (0.1 ml/site) and into 5 intramuscular sites of 0.2 ml each. Rabbits were injected weekly for 3 weeks (incomplete Freund's adjuvant was used for all but the first immunization) and then boosted on the fifth week. Animals were bled starting at week 6. As an alternative immunization protocol, some animals were injected with homogenized polyacrylamide gel slices containing purified tyrosinase (approximately 20 pg/slice). Following staining of the gel with L-DOPA to visualize tyrosinase, the gel slice containing the enzyme was cut out and homogenized in 2 ml of PBS in a Dounce homogenizer. The homogenate was injected in 0.1-ml increments into 20 sites both intradermally and subcutaneously. A second injection of acrylamide homogenate was given 5 weeks later. Serum recovered from immu- nized rabbits was tested for reactivity and specificity against tyrosinase following the procedures discussed below.

Immunological Procedures

Antiserum against tyrosinase was detected by: 1, Ouchterlony gel diffusion; 2, immunoelectrophoresis; and by 3, immunoprecipitation. Ouchterlony gel diffusion was carried out on microscope slides layered with 1% agarose in Tris-buffered saline, pH 7.2. Immunoprecipitation bands were usually visible at 24 h and complete at 48 h. Slides were then rinsed in PBS for 2 days and either dried and stained with Coomassie R-250 or incubated with L-DOPA to visualize tyrosinase and then dried. Immunoelectrophoresis was carried out on 1 X 3-inch slides with 1% agarose in 0.06 M Barbital buffer, pH 8.6. Antigens were electrophoresed in a Bio-Rad 1405 horizontal electrophoresis cell at 3 mA/slide for 90 min. Antiserum was then added to the center trough and the slides incubated for 48 h at 4 "C. To immunoprecipitate tyrosinase, purified enzyme was incubated with rabbit anti- tyrosinase serum in immunoprecipitation buffer (IP buffer; 40 mM PBS, 0.1% BSA, 0.1% SDS, 0.5% Triton X-100) for 45 min at 37 "C and then at 4 "C overnight. Samples were then centrifuged at 15,000 X g in a microfuge for 5 min and the supernatants assayed for tyrosinase activity as described below.

Tyrosinme Assays

To determine in situ tyrosinase activity in cells, growth medium was supplemented with 1 pCi/ml of 13H]tyrosine (specific activity 48 Ci/mmol) and added to cells for various time periods (usually 24 h). The [3H]tyrosine was first dried under a stream of nitrogen gas to remove 3H20 prior to adding to the medium. The medium removed from the flasks was assayed for the presence of 3H20 formation using a modification of the charcoal absorption method of Pomerantz (13). Following absorption of [3H]tyrosine by charcoal (Norit SG activated charcoal, MCB Manufacturing Chemists, Cincinnati, OH), 1 ml of the liquid phase was passed over a Dowex 50W column (Bio-Rad) equilibrated with 0.1 M HCl. This step removed residual [3H]tyrosine. The eluent from the Dowex column was collected directly into scintillation vials, scintillation fluid added, and vials counted in a Beck- man LS-7500 scintillation spectrometer. To determine tyrosinase activity in cell extracts, two methods were used. To measure tyrosine hydroxylase activity of the enzyme, aliquots were incubated in 1 ml of a reaction mixture consisting of 0.1 mM tyrosine, 2 pCi/ml of [3H] tyrosine, and 0.1 mM L-DOPA in 0.1 M sodium phosphate buffer, pH 6.8. Incubations were carried out at 37 "C for the times indicated. To terminate the reaction, 1 ml of charcoal (10% w/v, in 0.1 N HCI) was added to each assay tube and the samples centrifuged at 2000 X g for

4026 MSH Regulation of Tyrosinase 10 min. The supernatants were passed over Dowex 50W columns and counted as described above.

To measure the conversion of ["Cltyrosine to melanin, the procedure of Hearing and Ekel (14) was used. Aliquots of cell extracts (20 pl) were incubated in 30 pl of a 0.1 M potassium phosphate buffer, pH 7.4, reaction mixture consisting of 10 p1 of ['4C]tyrosine (25 pCi/ ml, specific activity, 100 mCi/mmol), 10 pl of a solution containing chloramphenicol (1 mg/ml), cycloheximide (1 mg/ml), penicillin (1000 units/ml), bovine serum albumin (1 mg/ml), and 10 p1 of 0.25 mM L-DOPA. Incubations were carried out for the times indicated at 37 "C in microtiter plate wells. Reactions were terminated by spotting 20 el of each well onto Whatman 3MM filter discs and placing them in 10% trichloroacetic acid containing 50 mM tyrosine. The filters were washed as described by Hearing and Ekel (14).

Induction of Tyrosinase in Cell Cultures To stimulate tyrosinase activity in melanoma cultures, cells were

removed from stock culture flasks and seeded at the indicated den- sities into either 25-cm2 flasks for most studies or into 150-cm2 flasks for the ['Hlleucine labeling experiments. After allowingcells to attach to the growing surface overnight, cultures were then exposed to a final concentration of 2 X M MSH by adding an appropriate volume of a sterile aqueous solution of the hormone M) directly into the culture medium. Control cultures received an equivalent volume of sterile water. For some of the experiments the level of tyrosinase stimulation was determined in situ by supplementing the Ham's F-10 medium with 1 pCi/ml of sterile [3H]tyrosine and monitoring the amount of 3H20 formed at various times as described above. For other experiments, cells were removed from the flasks at the times indicated and centrifuged at 90 X g for 5 min. The cell pellets were resuspended in PBS containing 0.5% Triton X-100 and sonicated until no intact cells remained (30-60 s). The resulting cell extracts were assayed for tyrosinase activity as described above. In the ['Hlleucine-labeling studies, cell extracts were assayed for tyrosinase activity using the ['4C]tyrosine assay.

Incorporation of L-PHILeucine into Total Protein For pulse-labeling studies, melanoma cells were seeded into 20 150-

cm2 flasks at a density of 2 X lo6 cells/flask and 10 flasks were exposed to MSH (2 X 10" M) for the indicated times. The culture medium was then replaced with leucine-free Ham's nutrient medium. The medium was supplemented with 1% horse serum, 5 p~ leucine, and 10 pCi/ml of ['Hlleucine (specific activity, 50 Ci/mmol). This amount of leucine was sufficient to support maximal rates of protein synthesis for the duration of the 3-h labeling period (data not shown). Following the pulse with ['Hlleucine, cells were rinsed 3 times with Ham's F-LO medium, removed from the flasks and sonicated in 2 ml of PBS, 0.5% Triton X-100. Total protein synthesis was measured by the filter assay method of Mans and Novelli (15). To determine the amount of ['Hlleucine uptake into cells, homogenates were precipitated with 10% trichloroacetic acid and the trichloroacetic acid- soluble fraction counted.

Specific Rate of Synthesis of Tyrosinase in Melanoma Cells Following pulse labeling of control and MSH-treated cells with

['Hlleucine, aliquots of the sonicated cell extracts were assayed for tyrosinase activity by measuring the conversion of ["C]tyrosine to melanin. A second aliquot was used for the determination of trichloroacetic acid-precipitable protein as described above and the remaining extract was trypsinized for 3 h at 37 "C with 0.2 mg of trypsin/ mg of cell protein to solubilize the enzyme. Phenylmethylsulfonyl fluoride (final concentration, 1.0 mM) was then added and the extracts centrifuged at 50,000 X g for 20 min at 4 "C. Tyrosinase activity in the resultingsupernatant was again determined with the ["Cltyrosine assay. Normal rabbit serum was added to 0.3 ml of the trypsinized extract and after a 30-min incubation at 37 "C, goat anti-rabbit IgG was added to complete the precipitation. After centrifugation, rabbit anti-tyrosinase IgG was added in excess to the resulting "cleared" supernatant and the enzyme immunoprecipitated for 30 min at 37 "C followed by a 2-h incubation at 4 "C. Normal rabbit serum was added to duplicate samples to control any additional nonspecific immunoprecipitation. To complete the precipitation an amount of goat anti- rabbit IgG sufficient to precipitate the rabbit IgG was added and the tubes incubated for 1 h at 4 "C. The immunoprecipitates were then collected by centrifugation for 3 min in a Eppendorf microcentrifuge (Brinkmann Instruments). The supernatants were assayed for tyrosinase activity and the pellets were washed twice in PBS containing

0.5% Triton X-100, 0.1% SDS, and 0.1% BSA. The immunoprecipitates were then dissolved in 10% glycerol, 2% SDS, 5% P-mercaptoethanol at 100 "C and electrophoresed on 10% SDS-polyacrylamide gels (12). Following electrophoresis, the gels were stained with Coo- massie R-250 and the area corresponding to the purified tyrosinase marker (M, - 65,000) sliced out and the radioactivity determined. Alternatively, following electrophoresis, the gels were electroblotted to nitrocellulose in a Hoefer Transfor cell (Hoefer Scientific Instru- ments) a t 60 volts for 3 h in 20 mM Tris, 150 mM glycine, and 20% methanol. The nitrocellulose blot was stained with India ink (0.1% PBS, 0.3% Tween 20), cut into 2-mm sections, dissolved in 2-meth- oxyethanol, and counted. Nonspecific counts obtained from normal rabbit serum controls were subtracted from the anti-tyrosinase immunoprecipitate counts. As an additional step to ensure that all tyrosinase had been precipitated by the immune serum, the supernatants from the first immunoprecipitation were re-precipitated with tyrosinase antiserum. No additional radioactive counts were recovered at the M , = 65,500 position on SDS gels. The synthesis rate of tyrosinase was determined as described by Kumar et ai. (16). The counts corresponding to tyrosinase were divided by the number of units of tyrosinase precipitated from the cell extract giving counts/ min of tyrosinase/microunits of enzyme. This value was then multi- plied by the specific activity of tyrosinase in the culture (microunits/ mg of protein) yielding the specific rate of tyrosinase synthesis (counts/min of tyrosinase/mg of cell protein). .The relative rate was determined by dividing the specific rate of tyrosinase (counts/min of tyrosinase/mg of protein) by the rate of incorporation of ['Hlleucine into trichloroacetic acid-precipitable protein (counts/min of trichloroacetic acid-precipitable protein/mg of protein).

Determination of Tyrosinase Degradation Rates Cells were seeded into 75-cm2 flasks at 3 X los cells/flask and

allowed to attach overnight. Cells were rinsed three times in Ham's F-10 medium without leucine and then pulsed for 8 h in the same medium containing 50 pCi/ml of ['Hlleucine, 5 ~ L M leucine, and 1% horse serum. After the pulse cells were rinsed three times (over 20 min) in Ham's medium containing 2 mM leucine to terminate the incorporation of radioactive leucine. The cells were then incubated in complete Ham's medium containing 10% horse serum with or without MSH (2 X 10" M). A t the indicated times, cells were removed,

described above. homogenized, and tyrosinase immunoprecipitated and quantitated as

Immunochemical Titration of Tyrosinase To compare the levels of immunoreactive tyrosinase in control and

MSH-treated cells, equivalence point titration analysis was carried out as described by Feigelson and Greengard (17) with the following modifications. Cells were sonicated in PBS containing 0.5% Triton X-100 and the resulting extracts trypsinized for 3 h at 37 "C with 0.2 mg of trypsin/mg of protein. Phenylmethylsulfonyl fluoride was then added to a final concentration of 1.0 mM, the extracts centrifuged at 50,000 X g for 20 min at 4 "C, and the supernatants assayed for tyrosinase activity. Increasing amounts of tyrosinase activity were then added to the indicated amount of antiserum and the immune complex precipitated by the addition of Pansorbin, previously washed twice in PBS + 0.5% Triton X-100 (Behring Diagnostics). The supernatants were assayed for tyrosinase activity as described above.

Enzyme-linked Inmunosorbent Assay (ELISA) of Tyrosinase As a second method to determine the levels of immunoreactive

tyrosinase in melanoma cell cultures, a trypsinized cell extract was prepared as described above and used directly in competitive ELISA procedures. All of the assays were carried out in 96-well Dynatech polyvinyl microtiter plates. To coat the plates, 0.1 ml of purified tyrosinase (1 pg/ml in 0.04 M PBS, pH 7.2) was pipetted into each well and the plates incubated overnight at 4 "C. The antigen was removed and the wells "blocked" by adding 0.2 ml of 2% BSA in PBS to each well and incubating the plates for 30 min at room temperature. The wells were then washed three times with washing buffer (PBS + 0.05% Tween 20). Cell extracts (0.1 ml) to be tested were serially diluted (1:2) through 10 wells and 0.1 ml of diluted antiserum (1:2000 dilution in 0.04 M PBS containing 1% BSA and 0.05% Tween 20 (first antibody)) was added immediately. The plates were incubated for 3 h at room temperature and then washed three times. Peroxidase- conjugated goat anti-rabbit IgG (0.2 ml, 1:2000 dilution (second antibody)) was then added to each well and the plates incubated for 3 h at room temperature. The plates were again washed three times


and 0.2 ml of substrate solution (0.1 M citrate, pH 4.5, containing 0.1% 0-phenylenediamine and 0.05% H202) added to each well. The enzymatic reaction was stopped after 20 min by pipetting the contents of each well into 2 ml of 0.1 N NaOH. The absorbance was read in a Spectronic 2000 (Bausch and Lomb) double-beam spectrophotometer at 415 nm. The spectrophotometer was blanked with 0.2 ml of substrate solution diluted into 2 ml of 0.1 N NaOH. Controls for the ELISA assay included 1, wells coated with antigen, incubated with non-melanoma cell extracts (3T3 fibroblasts) as the competing antigen and treated with anti-tyrosinase serum and second antibody; 2, wells coated with antigen, treated with second antibody, but without anti-tyrosinase serum; 3, wells coated with a false antigen (mouse liver, kidney, spleen extract, 1 pg/ml) and exposed to rabbit anti- tyrosinase and second antibody; 4, wells coated with antigen but exposed to non-immune rabbit serum as the first antibody and with second antibody; and 5, wells which were not coated with antigen but which did receive first and second antibody. All assays were performed in triplicate.

Protein Determinations The amount of cellular protein was routinely determined using a

protein assay kit obtained from Bio-Rad. This kit is based on a protein assay described by Bradford (18). Bovine serum albumin (fraction V) was used as the reference standard.

RESULTS

Previous studies have shown that MSH stimulates tyrosinase activity after a lag period of approximately 9 h (6). To determine the magnitude of the induction of tyrosinase in melanoma cells treated with MSH, the experiments shown in Fig. 1 were carried out. We found that tyrosinase activity increased approximately linearly with time and by 6 days the level of enzyme activity was almost 90 times that of control levels (Fig. 1). To assure that the enzyme levels being measured by the tyrosine hydroxylase assay (3H20 production) accurately reflect the action of MSH on tyrosinase activity, enzyme levels were also determined by the [Ylmelanin assay method (14). As shown in Table I both assays record similar relative changes in tyrosinase activity in cells treated with MSH, although the activities recorded by the [14C]tyrosine assay are somewhat lower than the tyrosine hydroxylase assay. This difference in assays has been discussed by Hearing and Eke1 (14). As first demonstrated by Pawelek and co- workers (6 ) , MSH exerts its effects through cAMP since either theophylline or dibutyryl cAMP will mimic MSH action and MSH stimulates cyclic AMP production. The correlation between MSH dosage, cyclic AMP levels, and cyclic AMP- dependent protein kinase activity is shown in Fig. 2. The good correlation observed further suggests the involvement of cyclic AMP in mediating MSH action. We examined several aspects of the kinetics of the MSH stimulation of tyrosinase. We and others have previously shown that MSH requires a 6-9-h lag period before any rise in tyrosinase activity is observed (6, 7) . We have now examined the length of time cells must be continuously exposed to the hormone before any increase in tyrosinase can occur over a 24-h period. Continu- ous exposure to MSH for less than 2 h failed to elicit a rise in tyrosinase activity (Fig. 3). An 8-10-h exposure period seemed to produce a near-maximal response as compared to cells continuously exposed to MSH for the entire 24-h incubation period. To determine how rapidly the MSH stimulation of cAMP production was lost following removal of MSH from cell cultures, the experiments shown in Fig. 4 were carried out. Cyclic AMP levels were found to fall toward basal levels rapidly after removal of the hormone. Interestingly, the early large increase in cAMP decreases in these cells by 30 min whether or not MSH is removed (Fig. 4), an observation which has been made previously (3 ,7) and which may suggest that the MSH receptor-adenylate cyclase complex is being

1 I I

I- O a 06

I m 0.4

i /

/

I 2 3 5 6 DAYS

FIG. 1. Kinetics of tyrosinase stimulation by MSH. Cells were seeded at lo5 cells/flask into 75-cm2 flasks and allowed to attach overnight. MSH (2 X M) was added to the appropriate flasks and every 24 h starting at T = 0, one group of duplicate flasks (MSH and controls) were exposed to 1 pCi/ml of [3H]tyrosine. At the end of a 24-h exposure period the amount of 3Hz0 formed was determined as described under "Experimental Procedures." MSH was added fresh daily. To determine the amount of nonenzymatic 3Hz0 production, flasks without cells but containing [3H]tyrosine-labeled medium were assayed for 3H20 production every 24 h. Values are the average of six determinations t- S.E. A, control, 0 MSH.

TABLE I MSH effect on tyrosinase activity in mouse melanoma cells as

detected by tyrosine hydroxylase and radiolabeled melanin assays Treatment Tyrosine hydroxylase "C-Melanin

assay svnthesis assay microunitslmg

Experiment 1 Control" 3.11 f 0.07 0.96 t 0.02 MSH (24 h) 19.9 & 0.6 (6.4)* 7.39 f 0.1 (7.7)

Control 1.18 f 0.04 0.38 t 0.02 MSH (48 h) 13.9 f 0.7 (11.8) 5.15 t 0.2 (13.5)

Control 1.23 k 0.05 MSH (72 h) 27.2 k 0.6 (22)

0.41 f 0.01 9.34 f 0.2 (23)

Experiment 2

Experiment 3

For tyrosinase measurements, melanoma cells were seeded into 1-liter culture flasks at a cell density of lo6 cells/flask and allowed to attach overnight. MSH (2 X 10" M) was added to the indicated flasks for 24, 48, or 72 h. Cells were then homogenized in PBS containing 0.5% Triton X-100, proteins determined, and tyrosinase activity assayed with either [3H]tyrosine or ["Cltyrosine as the substrate as described under "Experimental Procedures." Values are the averages of four determinations & S.E.

The fold stimulation of tyrosinase for each experiment is shown in parentheses next to the MSH tyrosinase value.

internalized and degraded. We next examined the effect of removal of MSH at various times on the decay of tyrosinase activity in melanoma cell cultures. Cells exposed to MSH for 24 h continued to demonstrate an increase in tyrosinase

4028 MSH Regulation of Tyrosinase

FIG. 2. Dose-response for MSH effects on tyrosinase activity, cAMP production, and CAMP-dependent protein kinase. To determine tyrosinase activity, melanoma cells were seeded at 2 X lo5 cells/flask into 25-cm2 flasks and allowed to attach overnight. MSH was then added at the indicated concentrations for 24 h. The culture medium was fortified with 1 pCi/ml of [3H]tyrosine. The amount of 3H20 formed over the 24-h period was determined as described in the text. Cyclic AMP levels were determined in cell cultures exposed to the indicated concentrations of MSH for 30 min. Details for the preparation of cell extracts for cAMP measurements are described under “Experimental Procedures.” For protein kinase determinations, 3 X lo7 cells were seeded into 1-liter culture flasks and exposed to MSH at the indicated concentrations for 30 min. Following this incubation, cells were washed with PBS and scraped off with 5.0 mM sodium phosphate buffer, pH 7.4, containing 1 mM EDTA, 1 mM mercaptoethanol, 5% glycerol, and 0.5 mM methyliso- butylxanthine. Cells were homogenized, centrifuged for 30 min at 30,000 X g, and the supernatant assayed for CAMP-dependent protein kinase activity as described under “Experimental Procedures.” Each point is the average of four determinations rt S.E.

activity for an additional 24-h period following the removal of hormone after which time the enzyme levels dropped rapidly to basal values (Fig. 5). Continuous exposure of cells to MSH for 48 h did not prolong the induction past an additional 24 h.

We have previously shown that the MSH induction of tyrosinase activity in melanoma cell cultures requires continued transcription and translation (7). Our evidence for a transcriptional requirement for MSH action was based on studies with actinomycin D while a role for translation was based on studies with cycloheximide. We have now extended these studies on the transcriptional involvement for MSH action by examining the effects of a-amanitin, an RNA PO- lymerase I1 inhibitor, and cordycepin, an inhibitor of poly- adenylation, on the MSH induction of tyrosinase. Both inhibitors suppress the MSH-mediated increase in tyrosinase activity. a-Amanitin at 10 pg/ml inhibited MSH action com- pletely while cordycepin, at 1 pg/ml or greater, was effective in inhibiting tyrosinase activity by 80% (Fig. 6). These results implicate a role for RNA synthesis in the cellular response to MSH. To determine if MSH is inducing the synthesis of

HOURS OF EXPOSURE TO MSH

FIG. 3. Exposure time required for MSH stimulation of tyrosinase activity. Cells were seeded into 25-cm2 flasks at 5 X lo5 cells/flask. At T = 0 MSH (2 X 10’ M) was added for the times indicated. Following this exposure period, MSH-treated flasks, as well as their respective control flasks, were rinsed twice with medium and fresh medium containing 1 pCi/ml of [3H]tyrosine added for the remainder of the 24-h period. Medium was then assayed for the presence of 3H20. Values are the averages of four determinations j, S.E.

~.... .-“..-.“.~...I

4 I I I

I IO 20 x) 40 50 60 70 80 90

TIME (minutes) FIG. 4. Decay of cAMP following MSH removal. Cells were

seeded into 25-cm2 flasks at 5 X lo6 cells/flask and exposed to MSH (2 X 10” M). At the times indicated, cells were processed for cAMP and assayed as described under “Experimental Procedures.” 0, MSH added at T = 0 and not removed; . . . . ., MSH removed at 10 min; ” - , MSH removed at 30 min; ., control.

tyrosinase in these cells, we measured the rate of synthesis of the enzyme by pulse-labeling cells with t3H]leucine and im- munoprecipitating the enzyme with a polyclonal antibody raised in rabbits against purified tyrosinase. A summary of the purification steps used to isolate tyrosinase for use in preparing an anti-tyrosinase serum is shown in Table 11. The use of ion exchange chromatography followed by a two-step polyacrylamide gel electrophoresis procedure resulted in a 5,000-fold purification with a recovery of 34%. Tyrosinase purified by this method has a molecular weight of approximately 65,500 (based on denaturing SDS-polyacrylamide gels) and an isoelectric point of 4.2. Based on these properties, the enzyme corresponds to the TI form of tyrosinase (19,20). The specificity of the antibody produced against purified tyrosinase was analyzed by gel diffusion, immunoelectrophoresis, immunoprecipitation of tyrosinase activity, and by SDS-polyacrylamide gel electrophoresis analysis of immunoprecipitated proteins from [3H]leucine-labeled melanoma cell cultures (Figs. 7 and 8). The antibody precipitates but does not inactivate the enzyme (Fig. 7, immunoelectrophoresis pat-


I I I

I 2 3 4

DAYS IN CULTURE FIG. 5. Kinetics of tyrosinase decay following MSH re-

moval. Cells (2 x 105/flask) were seeded into 25-cm2 flasks and incubated with or without MSH (2 X IO" M) for the times indicated. ['HITyrosine was also present in the medium. The medium was changed every 24 h and assayed for 3H20. Following exposure to MSH for the times indicated, cells were rinsed twice prior to further incubation in medium containing [3H]tyrosine. Values are the averages of six determinations f S.E. 0, 24-h exposure to MSH, ., 48-h exposure to MSH.

1 COROYCEPIN 1 AMANITIN I too -

BO - z 0 6 0 - k m z s X

4 0 -

20 -

0.1 0.5 1.0 0.5 1.0 5.0 IO

(Jig 1 ml 1 FIG. 6. Effects of cordycepin and a-amanitin on tyrosinase

stimulation by MSH. Cells (2 X lo5 cells/flask) were seeded into 25-cm2 flasks and exposed to the indicated concentration of inhibitor with or without MSH or 24 h. [3H]Tyrosine was present in the medium at a concentration of 1 pCi/ml. Flasks without either inhibitor or MSH served as controls. At the end of the incubation, medium was removed and assayed for 3H20 as described under "Experimental Procedures." 0, inhibitor alone; A, inhibitor + MSH. Values are the averages of six determinations f S.E.

tern). The antiserum produced is highly specific, recognizing predominantly only one protein of approximate M, = 65,500 as shown by SDS-gel electrophoresis and fluorography of immunoprecipitates prepared from radiolabeled melanoma cell protein extracts (Fig. 8). When pulse-labeling experiments

were carried out to determine tyrosinase synthesis rates, we found that during a 48-h exposure period, MSH stimulated the specific activity of tyrosinase 14-fold and had a small but reproducible stimulatory effect on the incorporation of t3H] leucine into trichloroacetic acid-precipitable protein (1.2- fold). MSH increased the synthesis rate of tyrosinase approximately 4-fold (the relative rate was increased 3-fold), an increase which did not correspond to the 14-fold increase in enzyme activity (Fig. 9). When we measured the rate of tyrosinase synthesis in cells treated for different times with MSH, a 2-%fold increase in the relative rate of tyrosinase synthesis over control values was seen in cells treated with hormone for 36,48, or 72 h while tyrosinase activity increased 7-, 14-, and 36-fold, respectively (Table 111). Thus, although MSH action requires both continued transcription and translation, it does not appear that the hormone is acting solely to increase the rate of synthesis of the enzyme. It is possible that MSH is acting to either: 1, increase the catalytic activity of a pre-existing population of inactive (or less active) tyrosinase molecules or 2, to reduce the degradation rate of the enzyme, or both. Continued transcription could be required for either mechanism.

To determine if MSH may function to increase the catalytic efficiency of tyrosinase, we carried out immunochemical titration experiments which were patterned after the principle discussed by Feigelson and Greengard (17). The principle of the method is based on the determination of equivalence points. If one adds increasing amounts of enzyme activity to a fixed amount of antiserum to form an immunoprecipitate, the point at which activity begins to appear in the supernatant represents the equivalence point. If two enzyme preparations differ in the amounts of inactive (or less catalytically active) enzyme, a difference in equivalence points will be observed because the antibody binds both forms of the enzyme and is saturated at lower activity by the enzyme preparation containing inactive molecules. Thus, if MSH promotes only enzyme synthesis (or reduced enzyme degradation) the equivalence points of tyrosinase from control and MSH-treated cultures will be the same. If, however, activation occurs in cells treated with MSH, the equivalence points of enzyme from control and MSH-treated cells will differ. When these experiments were carried out with cell extracts from control cultures and from cells treated for 48 h with MSH a shift in equivalence points was observed (Fig. 10). In a similar study this type of analysis was modified for use with ELISA protocols. The assay involves coating the wells of a microtiter plate with purified antigen and then adding a limiting amount of antiserum to each well plus or minus a known or unknown amount of competing antigen. Any soluble antigen will com- Pete with fixed antigen for binding to the antibody and, depending on the amount of soluble antigen, less antibody will be available for binding to the fixed antigen. If one adds cell extract to microtiter wells on the basis of tyrosinase activity, competitive curves obtained with enzyme preparations from control and MSH-treated cells will be the same if no activation of tyrosinase is occurring. If, however, MSH activates pre-existing tyrosinase, it will take more activity to prevent antibody from binding the plate when an enzyme preparation from MSH-treated cells is used (since fewer enzyme molecules are present per unit of enzyme activity). As shown in Fig. 11, we did observe a shift in the competition curves, suggesting that the catalytic activity of tyrosinase in untreated cells is less than that in MSH-treated cultures. This data is consistent with the possibility that the hormone may function, in part, to activate tyrosinase.

To determine if MSH may also act to stabilize tyrosinase,

4030 MSH Regulation of Tyrosinase TABLE I1

Summary of tyrosinase purification and recovery

Fraction Total Total protein activity Yield

Specific activity R

mg d h Z i t S ” milliunits/mg % Homogenate* 1017 166 0.163 100

-Fold

1000 X g supernatant 728 144 0.198 87 1

Triton X-100 trypsin digest 174 84 0.483 1.21

51 DEAE-Sephadex chromatography 12 86 7.17 52

2.95

0.07 44

Two-step polyacrylamide gel electrophoresis‘ 57 814 34 4996 A unit of tyrosinase is the amount of enzyme which metabolizes 1 pmol of tyrosine/min (measured as 3H20

Tyrosinase was prepared from 10 g of Cloudman S-91 mouse melanoma tumors raised in DBA/2 mice. Tyrosinase was electrophoresed first on a 10% T, 5% C discontinuous gel, the tyrosinase band cut out and re-

produced).

electrophoresed on a 10% T, 2.7% C SDS gel as described under “Experimental Procedures.”

A

A B C D E I “ ~-

C

B

205.000. 116,000. 97.400 *

66.000 -

45.000 -

29,000 -

D

FIG. 7. Purification of tyrosinase and characterization of a rabbit polyclonal anti-tyrosinase serum. Purification procedures for tyrosinase are described under “Experimental Procedures.” Panel A , a nondenaturing discontinuous polyacrylamide gel of tyrosinase a t various steps during purification. Lams A-C are stained with Coo- massie R-250; lanes D-F are stained with 0.2% L-DOPA followed by fixation with 10% trichloroacetic acid. Lanes A and D, 10 pg of purified tyrosinase; lunes B and E, tyrosinase partially purified by anion exchange chromatography (70 pg); lanes C and F, Triton X- 100/trypsin-treated melanoma extract (200 pg). Panel B, sodium dodecyl sulfate-gel electrophoresis pattern of purified tyrosinase. Purified tyrosinase (10 pg) was treated with SDS and P-mercaptoethanol as described under “Experimental Procedures” and t,hen electrophoresed on an SDS gel according to the method of Laemmli (12). The purified enzyme migrates just under the 66,000 molecular weight marker. Panel C, immunoelectrophoresis analysis of rabbit anti- tyrosinase antiserum. Run conditions were as described under “Ex- perimental Procedures.” A and C, partially purified tyrosinase from DEAE-Sephadex ion exchange chromatography; B and D, purified tyrosinase, A and B were stained with Coomassie R-250; C and D were stained with 0.2% L-DOPA in 0.1 M sodium phosphate buffer, pH 6.8, for 2 h a t 37 “C. Troughs contain anti-tyrosinase serum. Panel D, Ouchterlony double diffusion analysis of rabbit anti-mouse tyrosinase. Well contents were: 1 and 4, Triton X-lOO/trypsin-treated cell extract; 2 and 5, purified tyrosinase; 3 and 6, tyrosinase from DEAE-Sephadex chromatography. Rabbit anti-tyrosinase serum is in the center well. Gels were developed for 48 h, washed for 2 days in PBS, dried, and stained with Coomassie R-250.

t o o - 116.000 - r .

97.400 - 66.000 -

- 70

A

:: IO 20 30 40 50 60 70

ANTISERUM ADDED (id)

FIG. 8. Immunoprecipitation of mouse melanoma tyrosinase with rabbit anti-tyrosinase serum. Reaction mixtures contained 4 milliunits of partially purified tyrosinase from DEAE-Sephadex chromatography, the indicated increasing amounts of antiserum and immunoprecipitation buffer (40 mM PBS, 0.1% BSA, 0.1% SDS, and 0.5% Triton X-100) to bring the volume to 300 pl. Mixtures were incubated for 45 min a t 37 “C and then at 4 “C overnight. Samples were then centrifuged at 15,000 X g, tyrosinase activity determined in the supernatant (0) and protein measured in the precipitate (A). Inset, immunoprecipitation analysis of pulse-labeled tyrosinase. Three 75-cm2 culture flasks were seeded with 5 X lo5 cells/flask and pulsed with [3H]leucine, homogenized, and immunoprecipitated as described under “Experimental Procedures.” The immunoprecipitates were boiled in SDS treatment buffer (see text) and electrophoresed on SDS-polyacrylamide gels. The gels were stained with Coomassie R-250, soaked in Enlightening (New England Nuclear DuPont) for 30 min, and fluorographed on Kodak X-Omat film for 2 days. Tyro- sinase bands are detected just below the 66,000 molecular weight marker in duplicate samples.

we measured the degradation rate of pre-labeled tyrosinase in control and MSH-treated cultures (Fig. 12). The half-life of the enzyme was found to be approximately 18-19 h in the absence or presence of MSH. Thus, a t least for the 48-h exposure period used in this study, MSH does not stabilize tyrosinase. Finally, we carried out studies to determine if MSH may promote the appearance of a cytosol “factor” which may be involved in the activation process. When a cytosol preparation from MSH-treated cells was incubated with untreated cell homogenates for 4 h and the homogenates then assayed for tyrosinase activity, we observed an increase (1.6- fold) in enzyme levels (Table IV).

DISCUSSION

Various aspects of the MSH-mediated induction of tyrosinase activity in Cloudman S-91 mouse melanoma cell cultures have been previously reported. MSH apparently exerts its effects through cyclic AMP since either theophylline or di-

MSH Regulation of Tyrosinase 403 1 “.

W v) 4

v) 0 z

- 0 0 4

f

- 0

-003 E 4

- 0 0 2 2 W

I- 4

W -001

ac

CONTROL MSn

FIG. 9. Effect of MSH on the rate of tyrosinase synthesis in

seeded with 2 X lo6 cells/flask and 10 treated with MSH (2 X melanoma cell cultures. Twenty culture flasks (150 cm2) were

M) for 48 h. Cells were then pulsed for 3 h with 10 pCi/ml of 13H] leucine. Following this pulse, cells were homogenized and tyrosinase immunoprecipitated. The specific activity of tyrosinase (units/mg of protein), the incorporation of [3H]leucine into protein, and the rate of tyrosinase synthesis (disintegrations/min of tyrosinase/mg of protein) were determined as described under “Experimental Procedures.” Values are the averages of four determinations f S.E. This experiment was repeated five times with similar results. P, protein.

TABLE 111 MSH induction of tyrosinase synthesis and activity in melanoma cell

cultures Melanoma cells (2 X 106/flask) were seeded into 20 150-cm2 flasks

and allowed to attach overnight. Ten flasks were treated with 2 X M MSH for the times indicated and then all flasks were pulsed

with [3H]leucine for 3 h. Labeled tyrosinase was immunoprecipitated and quantitated as described under “Experimental Procedures.” Each time point experiment was repeated at least three times with similar results.

Additions Time Tyrosinase % of activity total protein

”

h microunitslmg P Control 36 6.0 0.012 MSH 36 42.6 (7.1X) 0.026 (2.2X) Control MSH

48 10.3 0.011 48 140.0 (13.6X) 0.035 (3.3X)

Control 72 6.1 0.010 MSH 72 220.1 (36X) 0.028 (2.8X)

butyryl CAMP will stimulate tyrosinase activity (2, 3). Both prostaglandin El and cholera toxin stimulate adenylate cyclase and increase tyrosinase activity in these cells (4, 21). In the present study we have shown that in order for any significant tyrosinase response to be generated, cells must be continuously exposed to MSH for a minimum of 2 h. It is of interest that a 10-h exposure produces levels of tyrosinase which nearly equal those observed after a 24-h incubation with MSH. It is possible that continued exposure of melanoma cells to MSH for 10 h results in the complete formation of the hormonally induced cellular pathway required for increased tyrosinase activity. Once this threshold is reached, near-maximal enzyme stimulation is attained for the remainder of a 24-h period even in the absence of hormone. Our finding that either cordycepin or a-amanitin will block the MSH stimulation of tyrosinase activity implicates a role for transcription in mediating the hormonal response and sup- ports previous evidence from our laboratory which suggested that both continued RNA and protein synthesis were neces- sary for MSH action ( 7 ) . While these results would suggest that MSH is likely inducing tyrosinase synthesis, the data

c l o o t I

TYROSINASE ADDED (uU) FIG. 10. Immunochemical titration of tyrosinase from con-

trol and MSH-treated cultures. Increasing amounts of tyrosinase activity from untreated cells (0) or from cultures exposed to MSH (2 X 10”) for 72 h (U) were added to 1 pl of rabbit anti-tyrosinase serum as described under “Experimental Procedures.” Following sedimen- tation of the immunoprecipitates, tyrosinase activity remaining in the supernatant was determined by the tyrosine hydroxylase assay method. Values are the averages of triplicates & S.E.

T I , , , 1

’O)

TYROSINASE ACTIVITY,unilr (x 10’) 1.0

FIG. 11. Competitive ELISA analysis of immunologically active tyrosinase from control and MSH-treated cell cultures. Eight flasks (150 cmZ) were seeded with 2 X lo6 cells/flask and four flasks treated with MSH (2 X IO-? M) for 40 h. Cells were then removed from the flasks and cell extracts prepared as described under “Experimental Procedures.” Cell extracts from control (0) and MSH- treated cultures (A) were serially diluted through microtiter wells containing absorbed tyrosinase (1 pg/ml). Rabbit anti-tyrosinase (1:2000) serum was then added and plates incubated for 3 h at room temperature. The wells were washed and exposed to peroxidase- labeled goat anti-rabbit IgG for 3 h followed by incubation with substrate as described under “Experimental Procedures.” Values are the averages of triplicate assays. BIB, represents the percentage of peroxidase-labeled goat anti-rabbit IgG bound in the presence ( E ) or absence (Eo) of competing tyrosinase extract.

presented in this paper has shown, surprisingly, that while there is an increase in tyrosinase synthesis in cells treated with MSH, the increase is insufficient to account for the large increase (up to 90-fold in 6 days) in tyrosinase activity. Thus, MSH must also act through a cellular pathway which requires continued RNA and protein synthesis but which does not involve increased enzyme synthesis. In contrast to our findings, Halaban et al. (9) have recently shown that tyrosinase synthesis increases approximately 4-fold in melanoma cells treated with MSH for 4 days and that the specific activity of the enzyme also increases about 4-fold. This finding has led

4032 MSH Regulation of Tyrosinase A

b- 0 K n

f !z >- >

t o 4

0 0 a a LL 0 ap

W In a I In 0 0: t I-

z t 2

>

b- o U

0 4 0:

LL 0

0

ac

- 60 -

4 0 -

20 -

0 24 48 72

T IME ( h o u r s )

€3 1

** CON-t1,2 19.2 h

H M S H - tIn 18. I h

- 40 -

20 -

I I I I I I 0 12 24 36 48

T I M E ( h o u r s ) FIG. 12. Analysis of decay of [3H]leucine-labeled tyrosinase

and soluble protein in control and MSH-treated cells. Mela- noma cells were pulsed for 8 h in medium containing [3H]leucine and then incubated in complete medium in the presence (m) or absence (0) of MSH (2 X M). At the times indicated, cells were removed, homogenized, and tyrosinase immunoprecipitated as described under "Experimental Procedures." An aliquot of each extract was precipitated with 10% trichloroacetic acid to determine the degradation rate of total radiolabeled protein (panel A ) . Tyrosinase degradation rates are shown in panel B. Degradation is expressed as a percentage of radioactivity remaining as a function of time. Values are the averages of four experiments t S.E. Half-life values were determined from the slope of the line which was derived by linear regression analysis. The correlation coefficient ( r value) for all linear plots was -0.990 or greater.

to the suggestion that the level of tyrosinase activity correlates closely with the abundance of the enzyme. Although we have shown here that MSH promotes an increase in tyrosinase synthesis, we find a discrepancy between synthesis and activity. Furthermore, while Halaban et al. (9) observe only a 4- fold increase in tyrosinase activity in cells exposed to MSH for 4 days, we find that tyrosinase levels will increase over 50-fold during this length of exposure to MSH. This difference in the level of induction may be due to differences in the basal activity of tyrosinase in the cells used for the studies. Our studies suggest that, at least in mouse melanoma cell cultures with low basal tyrosinase activity, an important site for MSH action may be at the level of activation. Results from immunotitration experiments to determine equivalence points of tyrosinase from control and MSH-treated cells also suggest that an activation process may play a role in the MSH regulation of tyrosinase activity. A model for the activation

TABLE IV Activation of tyrosinase in melanoma cell homogenates by cytosol

from MSH-treated cells Sample Tyrosinase activity

pml 3 ~ ~ 0 / h Control homogenate" (100 pl) 68.4 +- 12.0b MSH ~yt0s01' (50 pl) 28.1 t 2.8 Control homogenate (100 pl) + 153.2 f 8.0

MSH cytosol (50 pl) ' lo7 melanoma cells were sonicated for 60 s in 2 ml of PBS, pH

7.2, and incubated in the presence or absence of cytosol from MSH- treated cells for 4 h at 37 "C. [3H]Tyrosine (2 pCi/ml) in 0.1 M sodium phosphate, pH 6.8, containing 0.1 mM tyrosine and 0.1 mM L-DOPA was then added for 2 h and the amount of 3Hz0 formed during that period was determined as described under "Experimental Proce- dures."

Values are the averages of triplicate assays +- S.E. Cells (3 X lo6 cells/flask) were seeded into 150-cm2 flasks and

treated with 2 X lo-' M MSH for 48 h. Cells were then removed, sonicated in PBS, pH 7.2, and centrifuged at 50,000 X g for 20 min at 4 "C. The supernatant (cytosol) was then added to the homogenates as indicated.

of tyrosinase in melanoma cells has been presented by Korner and Pawelek (22). In this model, the stimulation of CAMP- dependent protein kinase by MSH leads to the phorphoryla- tion and inactivation of a tyrosinase inhibitor. No requirement for either transcription or translation is indicated in this model. However, our studies (7) and reports from other laboratories have shown that both RNA and protein synthesis are required for the MSH-induced increase in tyrosinase not only in melanoma cells but also in normal melanocytes (23, 24). Furthermore, we have been unable to demonstrate any effect of CAMP-dependent protein kinase on tyrosinase activity in cell homogenates (data not shown). Halaban et al. (9) have reported that tyrosinase itself is not phosphorylated, and thus, CAMP-dependent protein kinase does not apparently have any direct effect on the enzyme. Although our results are not consistent with the previously reported CAMP- dependent protein kinase model for tyrosinase activation, our studies do suggest that melanoma cells contain inactive (or at least catalytically less active) tyrosinase molecules which can undergo an "activation" event leading to increased catalytic efficiency. Because of the stringent requirements for both RNA and protein synthesis, if MSH is promoting the activation of tyrosinase, it must be doing so through a transcrip- tionally dependent process. The hormone may act to: 1, promote the synthesis of a tyrosinase activator protein or 2, to inactivate a tyrosinase inhibitor. Both of these possible pathways could require continued transcription and translation. The activator molecule produced could act directly on an inactive population of tyrosinase or could act at the level of enzyme processing such as glycosylation, intracellular transport, or insertion into the membrane matrix of the melanosome. Several papers have appeared which suggest that tyrosinase activity may be controlled by activation of pre- existing enzyme, possibly by the selective removal of a tyrosinase inhibitor. Evidence for the presence of tyrosinase inhibitors in both melanoma cells (8, 25-27) and in normal melanocytes (28) has been reported. Studies by Lee et al. (29) which examined the subcellular distribution of tyrosinase in mouse melanoma following MSH administration, suggested that de nouo synthesis of the enzyme was not responsible for the increase in tyrosinase activity and that MSH may act to remove a tyrosinase inhibitor. Further studies by this group demonstrated that an increase in tyrosinase activity would occur in cell homogenates treated with MSH (30). The ques- tion of inactive uersus active forms of tyrosinase in melano-


cytes has been addressed in our study by using immunotitration analysis of equivalence points and the results suggest that tyrosinase does exist in different catalytically active states. The presence of immunologically active but enzymat- ically inactive tyrosinase in an albino fowl has been recently reported and, interestingly, the inactive form of the enzyme showed no difference in migration pattern on two-dimensional gel electrophoresis when compared to active tyrosinase (31). Other studies have shown that tyrosinase activity in mouse melanoma cells increased more than 10-fold in cells treated with ionophores and that this response was not inhibited by cycloheximide (32). Taken together, these studies suggest that tyrosinase activity in mammalian pigment cells may be regu- lated concurrently by molecular pathways which alter enzyme synthesis rates as well as the catalytic activity of pre-existing molecules. From the results of the degradation studies carried out on control and MSH-treated cells it does not appear that the hormone is stabilizing tyrosinase. The experiments, however, examined MSH effects for up to 48 h and if MSH exerts a slower stabilizing effect on the enzyme we may not have been able to detect it. Further studies need to be conducted to examine longer term effects of MSH on tyrosinase stability. Altered degradation rates have been shown to markedly influ- ence enzyme levels in several cell systems (33-35). Halaban et al. (36) have examined both synthesis and decay rates of tyrosinase in a variety of normal and malignant human melanocytes and have found that cells which differ markedly in their basal level of tyrosinase activity also degrade tyrosinase at different rates. Wong and Pawelek (8) have suggested that tyrosinase activity decays at the same rate whether or not cells are treated with MSH. In their studies, tyrosinase decay rates were determined by inhibiting cellular protein synthesis with cycloheximide and monitoring the loss of enzyme activity over time. We have used the more stringent analysis of measuring the decay of radio-labeled tyrosinase by immunoprecipitation of pulse-labeled enzyme and our results are in general agreement with their findings. Thus, although in some melanocytes and melanoma cells differences in decay rates contribute significantly to the cellular level of tyrosinase, it appears that altered decay rates do not play a significant role in MSH action. Finally, it is likely that, in addition to stimulating tyrosinase activity, MSH may exert an effect on melanin synthesis at a post-tyrosinase point in the pathway (37). Studies are now in progress to more clearly assess the roles of synthesis and activation in the MSH regulation of tyrosinase.

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