Tyrosinase-related proteins suppress tyrosinase-mediated cell death of melanocytes and melanoma cells

Experimental Cell Research 298 (2004) 317 – 328 www.elsevier.com/locate/yexcr

Tyrosinase-related proteins suppress tyrosinase-mediated cell death of melanocytes and melanoma cells Hesamaddin Hejazy Rad, a Toshiharu Yamashita, a Hai-Ying Jin, a Kuninori Hirosaki, a Kazumasa Wakamatsu, b Shosuke Ito, b and Kowichi Jimbow a,* a

Department of Dermatology, School of Medicine, Sapporo Medical University, Chuo, Sapporo 060-8543, Japan Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi 470-1192, Japan

b

Received 4 September 2003, revised version received 22 March 2004 Available online 9 June 2004

Abstract The synthesis of melanin intermediates through tyrosinase (TYR) involves the production of cytotoxic free radicals. By using recombinant adenoviruses that express TYR, tyrosinase-related protein 1 (TYRP1) or DOPAchrome tautomerase (DCT), we analyzed the biological function of these proteins with regard to melanin production and the growth of melanocytes, fibroblasts, melanoma cells and nonmelanoma cancer cells. High-level expression of TYR produced newly synthesized melanin and induced cell death in all of these cells. However, when TYRP1 or DCT was coexpressed with TYR in melanocytes and melanoma cells, TYR-mediated cell death was clearly decreased. This decrease was not observed in nonmelanocytic cells. Western blot analysis and measurement of enzyme activity revealed that the expression of TYRP1 or DCT had little effect on the amount or activity of cointroduced TYR in either the melanocytic or nonmelanocytic cells. In cells expressing both TYR and TYRP1 or TYR and DCT, the total amount of melanin and/or eumelanin increased substantially more than that in cells expressing TYR alone. On the other hand, the level of pheomelanin was similar in these three cell types. These findings suggest that TYRP1 and DCT play an important role in suppressing TYR-mediated cytotoxicity in melanocytic cells without decreasing TYR expression and/or activity. These biological activities of TYRP1 and DCT may work through the interaction with TYR in melanosomal compartment. D 2004 Elsevier Inc. All rights reserved. Keywords: Melanoma cell lines; Cell death; Tyrosinase; Tyrosinase-related proteins; Melanogenesis; Eumelanin; Pheomelanin

Introduction Melanosomal proteins are divided into two major groups. Melanogenic proteins, i.e., tyrosinase (TYR) and its related proteins (TYRPs), TYRP1 and TYRP2/DCT(DOPAchrome tautomerase), catalyze the biochemical steps of melanin biosynthesis. Members of another group of melanosomal proteins, i.e., gp100, Pmel17, Rab7 and Rab27, have roles in retaining melanosomal structures and/or transporting melanogenic proteins or melanin pigments [1,2]. Human TYR, composed of 548 amino acids with a deduced molecular weight of 62.6 kilodalton (kDa) [3], possesses an enzyme activity critical to the melanogenic * Corresponding author. Department of Dermatology, School of Medicine, Sapporo Medical University, South 1, West 16, Chuo, Sapporo 060-8543, Japan. Fax: +81-11-613-3739. E-mail address: [email protected] (K. Jimbow). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.04.045

pathway [4]. This protein catalyzes conversions of tyrosine to dopa and subsequently to DOPAquinone, a process that is the initial and rate-limiting step of melanin production [5]. TYRP1, encoded by mouse brown locus [6– 8], is the second member of the TYR gene family. Murine TYRP1 possesses an oxidative activity to convert DHICA (5,6dihydroxyindole-2-carboxylic acid) to a carboxylated indole-quinone at the final step of eumelanin production [9]. TYRP-2/DCT, a mouse slaty locus, is cloned from a mouse cDNA library and its product is identified as the third member of TYR family protein [10,11]. DCT isomerizes a eumelanin intermediate, DOPAchrome to DHICA rather than to DHI (5,6-dihydroxyindole) [11,12]. DOPAchrome is spontaneously converted to DHI. DCT thus regulates the proportion of carboxylated subunits in the melanin biopolymer. The melanogenic pathway in melanocytes occurs within a discrete cytoplasmic compartment, the melanosome,

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probably because of the inherent cytotoxicity of melanin intermediates. Several studies have suggested that melanin precursors, DOPA, DHI and DHICA are toxic to cells, and that, of these, DHI is the most effective in suppressing the growth of cells [13,14]. Because TYR produces melanin precursors in the absence of TYRP1 or DCT in cells, the exogenous expression of TYR may cause severe cytotoxicity to nonmelanocytic cells in which no melanosomal compartment is present [15]. TYRP1 may play a protective role against the cytotoxicity of melanin intermediates, e.g., light, which is a cytotoxic mutation [16]. Slaty light (TYRP-2/DCT) mutation may also be cytotoxic because DCT is essential for eumelanogenesis [14]. We have previously shown that cotransfection of TYR and TYRP1 cDNAs prevents the cell death of melanocytes, which is related to TYRmediated production of melanin intermediates [17]. Because TYR-mediated melanin production is known to be toxic to cells, we have been interested in characterizing the cytotoxicity of TYR and possible anticytotoxic effect of TYRP1 and DCT in various types of cells. By using adenovirus-mediated gene transfer, we studied the biological and biochemical activities of these proteins in melanocytic and nonmelanocytic cells and compared the biological interactions between them. We show here that, despite the increase of melanin synthesis, TYRP1 and DCT can suppress TYR-mediated cell death in melanocytic cells, whereas in nonmelanocytic cells they cannot. The possible biological and biochemical roles of TYRP1 and DCT against TYR-mediated cell death in melanocytic cells are discussed.

Materials and methods Cells and cell culture The following human cell strains and cell lines were used in this study. MRC5 and CTN are untransformed human fibroblast cell strains derived from Caucasian embryonic lung and Japanese subcutaneous tissues, respectively. SK-mel-23, 70 W and G361 are melanotic melanoma cell lines, and SK-mel-24 and SK-mel-118 are amelanotic melanoma ones. HeLa and SiHa are HPV18 DNA- and HPV16 DNA-containing cervical carcinoma cell lines, respectively. SaOS2, T98G and NCI-H1229 are osteosarcoma, glioblastoma and lung carcinoma cell lines, respectively. The 293 cell is an adenovirus type 5 DNAtransformed human neuronal cell line [18], which is used for the growth of recombinant adenovirus. Melanocyte strains were purchased from BioWhittaker Inc. (Walkerville, USA) and cultured according to the provider’s instruction. Other cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (Gibco-BRL, Tokyo, Japan), penicillin G and streptomycin.

Recombinant adenovirus Recombinant adenoviruses were constructed as described previously [19 – 21]. Briefly, cDNAs of human TYR, human TYRP1 and human DCT were prepared from pcDNA-HT [14], pRHOHTa´ [22] and pHDT8 [23] by cleaving them with EcoRI, SalI + XbaI and EcoRI, respectively. After the cDNA fragments were blunted by T4 polymerase, they were inserted into the HindIII site, which had also been blunted by T4 polymerase, of pAdBglII, downstream of the human cytomegalovirus early promoter [19]. Parts of the cDNAs inserted into pAdBglII were verified by nucleotide sequencing. The resulting pAd-TYR, pAd-TYRP1 and pAd-DCT (each 2.0 Ag) were separately cotransfected into 293 cells (3  105/well of 24-well plate) with pJM17 (2.0 Ag) [24] by using SuperFect (Qiagen K.K., Tokyo, Japan). Recombinant adenovirus was cloned from 293 cell culture, which showed a cytopathic effect characteristic of adenovirus infection. Cloning of recombinant adenoviruses and titration of viral stocks were carried out in 293 cells as described [20]. Ad-LacZ is the recombinant adenovirus that expresses bacterial h-galactosidase [20]. Ad-p53 [20], Ad-p63 [25,26] and Ad-E1A [20,27] are recombinant adenoviruses carrying human p53, p63 (p51A) and adenovirus type 5 E1A 12S cDNAs, respectively. Western blotting Expression of TYR and TYRP1 was detected by Western blotting as described previously [27]. Firstly, 5  105 – 106 cells were infected with Ad-TYR and/or AdTYRP1 at a multiplicity of infection (moi) of 20 plaqueforming units (pfu)/cell and cultured for 60 h. After cells had been collected and lysed in 10 mM KCl, 1.5 mM MgCl2, 10 mM Tris (pH 7.4), 0.5% SDS, and 1 mM PMSF, they were sonicated for 10 s using a Branson sonicator. Protein concentration in the samples was determined by using a BCA protein assay kit (Pierce, Rockford, IL). Samples containing 5.0 Ag protein were electrophoresed in 5 – 20% gradient gels with SDS/polyacrylamide gel electrophoresis (PAGE) (Ready Gel, BioRad Laboratories, Tokyo, Japan) at 100 V for 80 min. The separated proteins were transferred onto nitrocellulose membranes (Protran, Schleicher & Schuell GmbH, Dassel, Germany) by electroblotting at 100 V for 60 min. The membranes were probed with the primary antibodies as follows: 1 Ag/ml of mouse monoclonal antibody against TYR (Novocastra, Newcastle upon Tyne, UK) or 1/2000diluted rabbit antiserum against TYRP1 [21]. The blot was then washed with PBS-T (0.1% Tween 20 in phosphatebuffered saline [PBS]) and incubated with antimouse IgGHRP (Santa Cruz Biotechnology, Santa Cruz, CA). The specific complexes were detected by ECL chemiluminescence reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK).

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Flow cytometry

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For flow cytometric analysis, adherent and floating cells were collected together and washed in ice-cold PBS. Cells were dehydrated in 75% cold ethanol and stored on ice for 2 h. Then, cells were rehydrated in cold PBS and treated with RNaseA (50 Ag/ml) (Sigma Aldrich Japan, Tokyo, Japan) at 37jC for 30 min. After incubation, cells were rinsed twice in ice-cold PBS and resuspended in 2.0 ml PBS containing 50 Ag/ml of propidium iodide (Sigma Aldrich Japan) at 4jC for 2 h. Cell debris and fixation artifacts were gated out, and sub-G1, G1, S and G2/M populations were quantified with a FACScan Cell Sorter (Nippon Becton Dickinson, Tokyo, Japan) using the CELL QUEST program.

teinyl-L-dopa was determined by HPLC with electrochemical determination (ECD) at 500 mV. DCT activity was measured according to the method described by Aroca et al. [29]. One hundred microliters of 2 mM NaIO4 was added to 100 Al of 1 mM L-dopa prepared in 50 mM phosphate buffer, pH 6.5, including 0.1 mM EDTA. Then, 0.5 mM DOPAchrome was prepared after 5 – 10 s. Immediately, 50 Al of 0.5 mM DOPAchrome was added to 50 Al of cell homogenate (cells were homogenized in 200 Al of 50 mM phosphate buffer, pH 6.5, including 0.1 mM EDTA) and mixed for 8 min at 37jC. After diluting the reaction mixture fivefold and after centrifugation, aliquots of 10 Al were directly subjected to HPLC (flow rate: 0.7 ml/min, column temperature: 35jC) with ECD at 600 mV. The peak of DHICA appeared at the retention time of 15.0 min.

Tyrosinase and DOPAchrome tautomerase activity

Measurement of total melanin

TYR activity was determined by measurement of the quantity of 5-S-L-cysteinyl-L-dopa formed in the presence of D,L-dopa and L-cysteine [28]. The amount of 5-S-L-cys-

After 106 cells had been seeded in 10-cm dishes and cultured for 24 h, they were infected with a recombinant adenovirus at a total moi of 20 pfu/cell and cultured for

Fig. 1. Effect of TYR and/or TYRP1 expression on the growth of cells. After 2  105 cells were seeded in 6-cm dishes and cultured for 24 h, cells were infected with Ad-LacZ (20 pfu/cell), Ad-TYR (10 pfu/cell) plus Ad-LacZ (10 pfu/cell), Ad-TYRP1 (10 pfu/cell) plus Ad-LacZ (10 pfu/cell) or Ad-TYR (10 pfu/cell) plus Ad-TYRP1 (10 pfu/cell). In G361 cells, Ad-LacZ was replaced by DMEM with 1% serum, because the cells are sensitive to Ad-LacZ infection. On the 7th day after infection, cells were detached and numbers of cells unstained by trypan blue were counted by hemocytometer. Mean F SD were determined from three dishes per infection.

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another 60 h. Cells were detached and collected in Eppendorf tubes. Then, cells were dissolved in 1 ml of alkaline solution (1 M NaOH in 10% DMSO) and incubated at 80jC for 2 h. After centrifugation at 500  g for 10 min, the protein concentration and melanin in the remaining supernatant were measured. The amount of melanin was determined with a spectrophotometer at 420 nm using synthetic melanin (Sigma Aldrich Japan) as a standard. Measurement of eumelanin and pheomelanin First, 1  106 to 2  106 cells in 10-cm dishes were infected with Ad-LacZ, Ad-TYR, Ad-TYRP1, Ad-TYR plus Ad-TYRP1 or Ad-TYR plus Ad-DCT at a total moi of 20 pfu/cell and maintained for 60 h. Then, infected cells were collected and frozen at 80jC until use. Measurements of eumelanin and pheomelanin by chemical degradation and HPLC analyses were performed as described

elsewhere [30,31]. Briefly, 400 Al of Milli-Q water was added to 1  106 to 2  106 cells, and sample homogenates (100 Al) were degraded by KMnO4 oxidation in duplicate and hydriodic acid (HI) reduction. The oxidated products were analyzed using a JASCO HPLC system [30,31] with a mobile phase of 0.1 M potassium-phosphate buffer (pH 2.1)/methanol, 99:1 (v/v). Absorbance of the eluent was monitored at 269 nm. The amount of eumelanin present could be determined by multiplying the amount of pyrrole2,3,5-tricarboxylic acid (PTCA) by a conversion factor of 160. HI reduction was performed by heating 100 Al of the sample homogenate in 30 Al of 30% H3PO2 and 500 Al of 57% HI at 130jC for 20 h. For the determination of 4-amino3-hydroxyphenylalanine (4-AHP), samples resolved in 200 Al of 0.1 M HCl were subjected to HPLC with a mobile phase of 0.1 M sodium citrate buffer (pH 3.0) containing 1 mM sodium octanesulfonate and 0.1 mM Na2EDTA/methanol at a ratio of 96:4. The analyses were performed at 35jC

Fig. 2. Flow cytometric analysis of TYR-expressing MRC5 cells. MRC5 fibroblasts were infected with recombinant adenovirus for 60 h or cultured in the presence of anti-CD95 antibody for 6 h. Adherent and floating cells were collected by centrifugation and resuspended in ice-cold 75% ethanol. Cells were then treated with propidium iodide and processed for FACScan analysis as described in Materials and methods.

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at a flow rate of 0.7 ml/min and an oxidation potential of +500 mV vs. an Hg/Hg2Cl2 reference electrode. The amount of pheomelanin was calculated by multiplying the amount of 4-AHP by a conversion factor of 9.

Results High-level expression of TYR induces cell death When cells were infected with Ad-LacZ at the moi of 20 pfu/cell, more than 90% of them expressed h-galactosidase, the exception being SK-mel-23, only about 60% of which expressed this protein [32]. For CTN, SaOS2, T98G and NCI-H1229 cells, h-galactosidase-expressing cells were also detected at >90% when infected with Ad-LacZ at the moi of 20 pfu/cell (data not shown). We examined the effect

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of TYR expression on the growth of cells, including cell strains of melanocytes and fibroblasts, and cell lines of melanoma and nonmelanoma human cancers. Fig. 1 summarizes the number of cells on the 7th day after Ad-TYR infection. With Ad-TYR infection of both nonmelanocytic cells (MRC5, CTN, HeLa, SiHa, T98G, SaOS2 and NCIH1229) and melanocytic cells (melanocytes, SK-mel-23, SK-mel-24, SK-mel-118, 70 W and G361), the level of degradation and detachment from the dish floor was observed with the rate of approximately 75– 90% of that with Ad-LacZ or mock infection (Fig. 1). To determine whether the TYR-mediated cell death was caused by an apoptotic mechanism, we analyzed DNA of Ad-TYR-infected cells by flow cytometry. An anti-CD95 antibody stimulates Fas, a receptor known to trigger apoptosis, was used as a positive control for induction of apoptosis. Apoptosis was measured by the percentage of

Fig. 3. Cell cycle fractions of virus-infected cells. After cells were infected with recombinant adenoviruses for 60 h, adherent and floating cells were collected and processed for flow cytometry as described in Materials and methods. A combination of adenovirus E1A (Ad-E1A) plus p53 (Ad-p53), a strong inducer of apoptosis, was used as a positive control.

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cells stained with propidium iodide in the subdiploid (i.e. sub-G1) range. MRC5 fibroblasts treated with the antiCD95 antibody contained a large amount of the sub-G1 fraction, but Ad-TYR-infected ones contained only a small amount, comparable to Ad-LacZ- and Ad-TYRP1-infected cells (Fig. 2). Fig. 3 shows a representative result of cell cycle analyses of cells infected with Ad-LacZ, Ad-TYR, Ad-TYRP1, Ad-DCT and Ad-p53 plus Ad-E1A (a positive control of apoptosis). MRC5, SiHa, SK-mel-23, SK-mel24 and SK-mel-118 did not show any increase in sub-G1 fractions of Ad-TYR-infected cells (Fig. 3). Moreover, AdTYR-infected MRC5, SiHa, SK-mel-23, SK-mel-24 and SK-mel-118 cells did not generate a fragmented DNA ladder in agarose gel electrophoresis (data not shown). Interestingly, TYR expression in p53-null SaOS2 cells produced an increased sub-G1 fraction, which was comparable to that in cells expressing p53 and adenovirus E1A (Fig. 3). We concluded that TYR-mediated cell death of p53-containing melanoma and nonmelanoma cells was caused by a nonapoptotic mechanism. Effect of TYRP1 and DCT on the TYR-mediated cell death We then studied the effect of TYRP1 and DCT on the TYR-mediated cell death. TYRP1 and DCT by themselves had little effect on the cell growth, because the number of cells infected with Ad-TYRP1 or Ad-DCT was comparable to those infected with Ad-LacZ (Figs. 1 and 4). As described above, Ad-TYR infection resulted in significant cell degradation in all cell types. However, when Ad-TYRP1 was coinfected with Ad-TYR, melanocytic cells (melanocytes, SK-mel-23, SK-mel-24, SK-mel-118, 70 W and G361), but

not nonmelanocytic cells (MRC5, CTN, HeLa, SiHa, SaOS2, T98G and H1229), were able to survive and grow continuously (Fig. 1). A similar result was obtained by the coinfection of Ad-TYR and Ad-DCT in the same series of melanocytic and nonmelanocytic cells (Fig. 4). Thus, both TYRP1 and DCT can suppress the cytotoxic effect of TYR in melanocytic cells, but they are inert in nonmelanocytic cells. To test whether the anticytotoxicity of TYRP1 and DCT was specific to TYR-mediated cell death, we compared the growth of cells infected with Ad-p63 alone and Ad-p63 plus Ad-TYRP1 (Fig. 5). p63, also known as p51A, p73L or ket, is a member of the p53 family, and it mediates apoptosis in various types of cancer cells [25,26,33]. As shown in Fig. 5, p63 induced cell death in MRC5, SiHa, SaOS2, SK-mel-23, SK-mel-24 and SK-mel-118 cells. However, unlike TYRmediated cell death (Figs. 1 and 4), the p63-mediated one was not suppressed by TYRP1 in several types of the cells tested (Fig. 5). Cells infected with Ad-DCT could not be rescued from p63-mediated cell death (data not shown). Moreover, cell death caused by a DNA-damaging agent, 4nitroquinoline-1-oxide, was not suppressed by TYRP1 or DCT in MRC5, SiHa, SaOS2, SK-mel-23, SK-mel-24 and SK-mel-118 cells (data not shown). These findings suggest that the anticytotoxic activity of TYRP1 and DCT is restricted to TYR-mediated cell death. TYRP1 and DCT do not decrease TYR expression or melanin synthesis It is possible that TYRP1 and DCT suppress cytotoxicity of TYR by decreasing the expression of TYR or melanin production, because melanin intermediates produced by

Fig. 4. DCT can also suppress TYR-mediated cell death. After 2  105 cells were seeded in 6-cm dishes and cultured for 24 h, they were infected with AdLacZ, Ad-TYR plus Ad-LacZ, Ad-DCT plus Ad-LacZ, or Ad-TYR plus Ad-DCT as described in the legend of Fig. 1. In G361 cells, Ad-LacZ was replaced by DMEM with 1% serum, because the cells are sensitive to Ad-LacZ infection [32]. On the 7th day after infection, cells were detached and the numbers of trypan blue-negative ones were counted. Mean F SD was determined from three dishes per infection.

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Fig. 5. Growth of cells infected with Ad-p63 and/or Ad-TYRP1. After 2  105 cells were seeded in 6-cm dishes and cultured for 24 h, they were infected with Ad-LacZ, Ad-p63 plus Ad-LacZ, Ad-TYRP1 plus Ad-LacZ, or Ad-p63 plus Ad-TYRP1 as described in the legend of Fig. 1. On the 7th day after infection, cells were detached and the numbers of trypan bluenegative ones were counted. Mean F SD was determined from three dishes per infection.

TYR are considered to be a source of cytotoxicity [13,14]. To test this possibility, we firstly analyzed the level of TYR protein in cells infected with Ad-TYR, Ad-TYRP1 and Ad-

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TYR plus Ad-TYRP1. Fig. 6 shows the results of Western blotting, which detected TYR and TYRP1 in the virusinfected melanocytic and nonmelanocytic cells. Ad-TYRand Ad-TYRP1-infected cells produced TYR and TYRP1 bands at approximately 75 and 70 kDa, respectively. The amount of TYR appeared to be the same in cells expressing TYR alone and those expressing TYR plus TYRP1 (Fig. 6). Measurement of enzyme activities in cells infected with AdTYR with or without Ad-DCT revealed that DCT expression did not significantly decrease the expression of TYR activity (Table 1). We then measured the amounts of melanin in nonmelanocytic cells as well as melanotic and nonmelanotic melanoma cells expressing TYR and/or TYRP1 (Fig. 7). TYR expression clearly increased melanin production in MRC5, SaOS2, SK-mel-24 and SK-mel-118, and slightly increased it in SiHa, while TYRP1 alone was not melanogenic to all of them (Fig. 7). Because SK-mel-23 cells showed no increase of melanin content after infection with Ad-TYR or Ad-TYR plus Ad-TYRP1 on the 3rd day, we measured melanin on the 1st, 2nd, 3rd, 5th and 7th days after virus infection (Fig. 8). Unlike in MRC5, SiHa and SK-mel-24 cells, the increase of melanin in SK-mel-23 cells became detectable later than the 4th day after Ad-TYR infection (Fig. 8B). To measure eumelanin and pheomelanin, infected cells were degraded by KMnO4 and hydriodic acid. They were then followed by HPLC analysis to determine amounts of PTCA (a specific marker of eumelanin) and 4-AHP (a specific marker of pheomelanin). The increase in PTCA

Fig. 6. Detection of TYR and TYRP1 by Western blotting. Cells infected with recombinant adenovirus (20 pfu/cell) for 60 h were lysed and samples containing 5.0 Ag of protein were electrophoresed in 5 – 20% SDS/PAGE at 100 V for 60 min. Antibodies against TYR and TRP-1 were reacted and specific bands were detected by chemiluminescence as described in Materials and methods.

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Table 1 DOPAchrome tautomerase and tyrosinase activity in TYR- and DCTexpressing cells 5-S-L-cysteinylL-dopa

DHICA [nmol/(min 106 cells)] MRC5

SiHa

SaOS2

SK-mel-23

SK-mel-24

SK-mel-118

Ad-LacZ Ad-TYR Ad-DCT Ad-TYR + Ad-LacZ Ad-TYR Ad-DCT Ad-TYR + Ad-LacZ Ad-TYR Ad-DCT Ad-TYR + Ad-LacZ Ad-TYR Ad-DCT Ad-TYR + Ad-LacZ Ad-TYR Ad-DCT Ad-TYR + Ad-LacZ Ad-TYR Ad-DCT Ad-TYR +

Ad-DCT

Ad-DCT

Ad-DCT

Ad-DCT

Ad-DCT

Ad-DCT

0.97 0.48 6.80 2.01 0.14 0.18 4.73 0.91 0.26 0.32 11.1 8.23 2.73 5.38 3.44 4.48 0.19 0.26 3.24 2.79 0.16 0.17 0.31 0.32

1

[nmol/(min 106 cells)]

1

0.06 131.0 0.10 135 0.07 172.0 0.08 156.0 0.11 437.0 0.21 381.0 7.71 48.3 12.0 41.0 0.02 258.0 0.04 264.0 0.02 3.81 0.01 1.67

The enzyme activity for DCT and tyrosinase is expressed as the content of DHICA and 5-S-L-cysteinyl-L-dopa formed, respectively.

reflects increased DCT activity, resulting in an increase of DHICA content in eumelanin. It is also well known that the production of pheomelanin does not require any activities of DCT [34]. In MRC5, SiHa and SK-mel-24 cells, coexpression of TYRP1 and/or DCT with TYR increased the amount

of PTCA compared to that of TYR expression alone (Fig. 9A), while it did not increase any levels of 4-AHP (Fig. 9B). As described above, SK-mel-23 cells produced almost the same amounts of eumelanin (Fig. 8B) total melanin (Fig. 7) at the point of measurement (60 h after virus infection) in cells infected with Ad-TYR, Ad-TYR plus Ad-TYRP1 and Ad-TYR plus Ad-DCT. Enhancement of eumelanin production (PTCA/4-AHP ratio) by DCT was higher than that of TYRP1 in MRC5 and SK-mel-24 cells (Table 2), suggesting that DCT was more effective for eumelanin synthesis than TYRP1. These results suggested that TYRP1 and DCT did not suppress the cytotoxicity of TYR by decreasing the TYR-mediated production of total melanin or pheomelanin.

Discussion TYR, TYRP1 and DCT are melanogenic proteins with structural similarities such as signal sequences followed by cysteine-rich and copper-binding regions. However, they possess enzyme activities that function at the different steps of melanin biosynthesis [11,12]. In the present study, we showed that TYR, TYRP1 and DCT exhibit distinct biological activities, i.e., effects on cell growth, cell survival and cell death. Extending our previous reports on COS7 cells and rat glioma cells [15,17], we showed here that adenovirus-mediated expression of TYR induces cell death in various types of cells, including melanotic and amelanotic melanoma cell lines. Melanocytes and melanotic melanoma cell lines SK-mel-23, 70 W and G361 expressed not only TYR but also TYRP1 and DCT. However, the amounts of endogenous TYRP1 and DCT in these melanocytic cells may be insufficient for protection from the cytotoxicity caused by exogenous expression of TYR, because a large amount of melanin was produced by virus-mediated expres-

Fig. 7. Total melanin content in Ad-TYR- and/or Ad-TYRP1-infected cells. Cells were infected with recombinant adenovirus for 60 h. After cells had been collected in microcentrifuge tubes, they were dissolved in alkaline solution (1 M NaOH in 10% DMSO) and incubated at 80jC for 2 h. The relative amount of melanin was determined by using synthetic melanin as a standard and indicated as the numerical value (mg) at 420 nm per miligram protein.

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Fig. 8. Time courses of cell number and melanin content after virus infection. After 2  105 (A) or 106 (B) cells were seeded and cultured for 24 h, they were infected with Ad-LacZ, Ad-TYR, Ad-TYRP1 or Ad-TYR plus Ad-TYRP1. Amounts of inoculated virus were adjusted to 20 pfu/cell per dish as described in the legends of Figs. 1, 4 and 5). The number of cells (A) and melanin content (B) were monitored on the 1st, 2nd, 3rd, 5th and 7th days after virus infection.

sion of TYR (Table 1; Fig. 6), and because a part of newly synthesized melanin may be leaned into the cytoplasm. Flow cytometric analysis and agarose gel electrophoresis (data not shown) revealed that TYR-mediated cell death was not associated with increased sub-G1 populations or DNA fragmentation. Only the p53-null cancer cell line, SaOS2, underwent apoptosis induced by TYR expression and melanin production (Fig. 3). It seems that p53 expression determines the process of cell death, i.e., apoptosis or necrosis. Further molecular study of TYR-mediated apoptosis in p53-null cells is required, including investigation of the involvement of caspase family activation. Johnson and Jackson [16] reported that a single missense mutation of TYRP1 results in an increased risk of cytotoxicity in mouse melanocytes. Also, in a cultured system of murine melanocytes, TYRP1 mutations resulted in the

attenuation of cell proliferation rates and DNA synthesis [35]. A mouse DCT mutant has a missense mutation at the position of 486 (Gly486Arg), which results in mislocation and functional loss of this protein [36]. DCT is essential for eumelanogenesis [14], which may decrease the risk of the cytotoxic effect of pheomelanin. Herein, we described a novel finding with regards to biological role of TYRP1 and DCT; anticytotoxic activities of TYRP1 and DCT against TYR were significantly different in melanocytic and nonmelanocytic cells. The anticytotoxicity of TYRP1 and DCT against TYR was not caused by the down-regulation of TYR or the resulting melanin production, because Western blot analysis, TYR activity assay and measurement of melanin detected no significant decrease in the TYR protein level, its enzyme activity or melanin production. Similar to previous reports [37 – 41], coexpression of TYRP1 with

Fig. 9. Amounts of (A) eumelanin (PTCA) and (B) pheomelanin (4-AHP) in TYR, TYRP1- and/or DCT-expressing cells. After 5  105 (MRC5) or 106 (SiHa, SK-mel-23 and SK-mel-24) cells were seeded in 10-cm dishes and cultured for 24 h, they were infected with Ad-LacZ (column 1), Ad-TYR (column 2), AdTYRP1 (column 3), Ad-DCT (column 4), Ad-TYR plus Ad-TYRP1 (column 5) or Ad-TYR plus Ad-DCT (column 6) and maintained for 60 h. Amounts of inoculated virus were adjusted to 20 pfu/cell per dish as described in the legend of Fig. 1. Infected cells were chemically degraded and analyzed by HPLC as described in Materials and methods. The values on the y axis are (A) PTCA (ng/106 cells) and (B) 4-AHP (ng/106 cells).

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Table 2 Effect of TYRP1 and DCT on TYR-mediated eumelanin synthesis Cell

Infected virus

MRC5

Ad-TYR Ad-TYR Ad-TYR Ad-TYR Ad-TYR Ad-TYR Ad-TYR Ad-TYR Ad-TYR Ad-TYR Ad-TYR Ad-TYR

SiHa

SK-mel-23

SK-mel-24

+ Ad-TYRP1 + Ad-DCT + Ad-TYRP1 + Ad- DCT + Ad-TYRP1 + Ad- DCT + Ad-TYRP1 + Ad- DCT

Resulting PTCA/AHPa

Effect of TYRP1 or DCTb

10.9/6688 23.5/7649 26.5/8865 44.2/1895 31.6/2157 90.4/1767 231/2165 281/2022 222/1711 11.0/4036 36.1/3130 55.4/2308

1.0 1.9 2.6 1.0 0.7 2.2 1.0 1.3 1.2 1.0 4.4 8.9

(0.0016) (0.0031) (0.0042) (0.023) (0.015) (0.051) (0.11) (0.14) (0.13) (0.0027) (0.012) (0.024)

a

Figures are means of three dishes from Figs. 8A,B. PTCA/AHP value of cells infected with Ad-TYR + Ad-TYRP1 or AdTYR + Ad-DCT/PTCA/AHP value of cells infected with Ad-TYR. b

TYR somewhat increased melanin production in MRC5, SaOS2 and SK-mel-24 cells compared to the single expression of TYR (Fig. 7). Different from mouse TYR, human TYR is reported to possess DHICA oxidase activity [42]. This activity of human TYR may be responsible for the enhanced production of eumelanin in MRC5 and SK-mel-24 cell lines (Fig. 9). Thus, it is of interest to compare eumelanin production among cells expressing TYR, TYR + TYRP1, TYR + DCT and TYR + TRP1 + DCT. Because photoactivated pheomelanin has been shown to be mutagenic in bacterium and drosophila [43], pheomelanin and its precursors can be cytotoxic in mammalian cells [44,45]. We measured and compared the total amounts of eumelanin and pheomelanin produced in cells that express TYR with or without TYRP1 or DCT. Eumelanin production as measured by PTCA was increased in MRC5, SiHa and SK-mel-24 cells. TYR-mediated production of eumelanin was particularly enhanced by combined infection of TYRP1 or DCT in SK-mel-24 cells by 4.4- or 8.8-fold, respectively (Table 2). The amounts of pheomelanin in cells expressing both TYR and TYRP1 or TYR and DCT were almost the same (MRC5, SiHa and SK-mel-23) or slightly decreased (SK-mel-24) compared to those in cells expressing TYR alone (Fig. 9). Meanwhile, neither pheomelanin nor eumelanin in SK-mel-23 cells expressing both TYR and TYRP1 or TYR and DCT increased, when compared to those of SKmel-23 cells expressing TYR alone (Fig. 9). Unlike MRC5, SiHa and SK-mel-24, SK-mel-23 cells showed increased melanin production later than the 4th day after Ad-TYR plus Ad-TYRP1 infection (Fig. 8B). It is likely that, as with other cells, the amount of eumelanin in SK-mel-23 cells infected with Ad-TYR and Ad-DCT would become greater than that with Ad-TYR alone when measured later than the 4th day. There is no basis to assume that SK-mel-23 cells underwent cell death within 3 days (Fig. 8A). It is, however, possible that the melanin precursors could be the source of cytotoxicity in the highly pigmented SK-mel-23 cells.

It has been indicated that TYRP1 and DCT form a stable complex with TYR in the melanosomal compartment, in which the leakage of cytotoxic melanin intermediates in the cytoplasm of melanocytes may be prevented [40,46,47]. TRP1 has been shown recently to be exclusively transported to melanosomal compartment [48]. In contrast, TYR can be transported to both melanosomes and lysosomes in melanocytic and nonmelanocytic cells, respectively [21,47]. It was indicated that TYRP1 and DCT decrease the leakage of toxic melanin intermediates in melanocytes by forming a stable complex with TYR in melanocytic cells [39,40,46,47]. In fact, we found in our recent immunohistochemistry study that exogenously introduced TYR and TYRP1 are incorporated together into melanosomal compartment in amelanotic melanoma cells (SK-mel-24) [21]. We also proposed previously that TYRP1, by interacting with Lamp1, which is present in the same melanosomal compartment, participates in scavenging cytotoxic melanin intermediates generated by TYR [17]. In addition, Manga et al. [41] reported recently that murine TYRP1 stabilizes TYR but decreases TYR activity, while DCT increases TYR activity by stabilizing the TYR protein. TYRP1 has been proposed to possess DHICA oxidase activity [9] that is confirmed in murine cells but not in human melanocytes [49]. Human TRP1 was reported to have some tyrosine hydroxylase activity and possibly to modulate TYR activity [50]. However, DCT, which similarly suppressed TYR-mediated cell death in melanocytic cells, did not affect significantly the coexpressed TYR activity (Table 1). In nonmelanocytic cells that do not possess any melanosomal compartments, TYRP1 or DCT introduced exogenously would be simply degraded within the cytoplasm or lysosomes. Thus exogenously introduced TYRP1 or DCT in nonmelanocytic cells would not have any biological role nor form any stable complex with TYR; hence, they are unable to function as the scavenger for TYR-mediated cytotoxicity. Acknowledgments This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We thank Dr. S. Shibahara of Tohoku University for providing us with the recombinant plasmids separately containing human TRP1 and DCT cDNA. References [1] G. Prota, Melanin and Melanogenesis, Academic Press, New York, 1992, pp. 1 – 290. [2] K. Jimbow, W.C. Quevedo Jr., G. Prota, T.B. Fitzpatrick, Biology of Melanocytes, in: I.M. Freedberg, A.Z. Eisen, K. Wolff, K.F. Austin, L.A. Goldsmith, S.I. Katz, T.B. Fitzpatrick (Eds.), Dermatology in General Medicine, fifth ed., McGraw-Hill, New York, 1999, pp. 192 – 222. [3] B.S. Kwon, A.K. Haq, S.H. Pomerantz, R. Halaban, Isolation and sequence of a cDNA clone for human tyrosinase that maps at the

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