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Induction of tyrosinase in human melanoma cells by l -tyrosine phosphate and cytochalasin D
EXPERIMENTAL
CELL
RESEARCH
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Induction of Tyrosinase in Human Melanoma Cells by L-Tyrosine Phosphate and Cytochalasin D ALISON J. WINDER AND HENRY HARRIS Sir William
Dunn
School of Pathology,
University
of Oxford, South Parks Road, Oxford, OX1 3RE, United Kingdom
mRNAs [ 111. In addition, cytochalasin is thought to affect gene expression at a post-transcriptional level [12, 131. Melanoma cells were treated with cytochalasin D to determine whether increased pigmentation correlates with increased tyrosinase activity and whether there is an effect on the expression of the tyrosinase gene. L-Tyrosine has been shown to induce pigmentation in hamster, mouse, and human melanoma cell lines [6,14, 151. Slominski et al. [6] have demonstrated that induction of pigmentation in hamster melanoma cells is not simply due to increased availability of substrate, but also to increased tyrosinase activity and a greater number of melanosomes per cell. The induction of melanin synthesis by L-tyrosine in these cells follows a pathway unrelated to cyclic AMP, cyclic GMP, or inositol triphosphate and is inhibited by cycloheximide, indicating that protein synthesis is required [6, 161. We have treated melanoma cells with L-tyrosine phosphate in an attempt to confirm the effect of tyrosine on tyrosinase activity and to determine whether there is also an effect on the amount of tyrosinase mRNA. LTyrosine phosphate was used in preference to L-tyrosine because of its greater solubility. L-Tyrosine phosphate is not transported into melanoma cells [ 171 but is thought to be hydrolyzed to L-tyrosine by cell-surfaceassociated phosphatases [ 181 and acts as a tyrosine precursor on intraperitoneal administration to mice [19]. Furthermore, phosphorylated isomers of L-dopa, another melanin precursor, have been shown to induce increased tyrosinase activity in mouse melanoma cells [20]. Two variants of the human melanoma cell line RVH 421 [21] were used in this study. The line nRVH is of relatively low passage (P = 55-65), whereas the line oRVH is of relatively high passage (P > 120). Both lines, nRVH and oRVH, were derived in our laboratory from the original RVH 421 parent. nRVH cells have a more differentiated phenotype than oRVH cells: they are more dendritic and have a higher basal level of pigmentation. Both variants are relatively unpigmented when seeded at low density, but pigmentation increases when the cells reach confluence. Variations in tyrosinase activity and melanin synthesis with cell density have been
Pigmentation of RVH 421 human melanoma cells is induced when cell division is inhibited by cytochalasin D or L-tyrosine phosphate. Increased pigmentation correlates with increased tyrosinase activity when this is monitored over a time-course. Parallel measurements show that the amount of tyrosinase mRNA correlates with enzyme activity in cells growing without these additives. In contrast, in the presence of cytochalasin D or L-tyrosine phosphate, the increase in amount of tyrosinase mRNA is not sufficient to account for the increase in enzyme activity, indicating that these compounds act mainly at a post-transcriptional level. 0 1992 Academic Press, Inc.
INTRODUCTION Induction of melanin synthesis in cultured melanoma cells generally correlates with inhibition of cell growth [l, 21. Tyrosinase (EC 1.14.18.1) is a key enzyme in melanin synthesis. It catalyses both the critical initial step, the hydroxylation of L-tyrosine to L-dopa (tyrosine hydroxylase activity), and the second step, the oxidation of L-dopa to dopaquinone (dopa oxidase activity). Pigmentation is generally found to correlate with tyrosinase activity [3-61. However, it has been shown that melanin synthesis can be regulated further down the pathway [7]. Both protein factors and metal ions are thought to be involved in post-tyrosinase regulation [8]. We have studied the regulation of tyrosinase in human melanoma cells treated with compounds that induce pigmentation in order to gain insight into their mechanisms of action. We have screened a large number of compounds for their ability to induce pigmentation and inhibit growth of melanoma cells in u&o. In the present paper we describe the effects of two of these, cytochalasin D and L-tyrosine phosphate. Cytochalasin D binds to actin filaments and disrupts the cytoskeleton, inhibiting cell division and altering cell morphology [9, lo]. The nucleus may respond to signals relating to the structural organization of the cytoskeleton, and its disruption by cytochalasin D has been shown to cause changes in the synthesis of specific 0014-4827/92 $3.00 Copyright 0 1992 hy Academic Press, Inc. All rights of reproduction in any form reserved.
248
INDUCTION
OF TYROSINASE
observed in several melanoma cell lines but the underlying mechanisms are not understood [3, 22, 231. Measurements of cell growth, pigmentation, tyrosinase activity, and amount of tyrosinase mRNA were made over a time-course for both control and treated cells. The human tyrosinase cDNA Pme134 [24] was used as a hybridization probe for detection of tyrosinase mRNA. Expression studies using a homologous cDNA have confirmed that this sequence encodes tyrosinase [25]. MATERIALS
AND
METHODS
Materials. Agarose (ultra-pure DNA grade) was purchased from Bio-Rad (Hemel Hempstead, UK). Ammonium acetate and gold-label ethanol were from Aldrich (Gillingham, UK). Ficoll was from Pharmacia (Uppsala, Sweden). Cytochalasin D, Mops, O-phospho-Ltyrosine, polyvinylpyrrolidone, salmon testes DNA, and Triton X-100 were obtained from Sigma (Poole, UK). Phosphate-buffered saline A (PBSA) tablets were from Oxoid (Basingstoke, UK), trypsin was from GIBCO (Paisley, UK), and trypan blue was from Flow Labs (Irvine, UK). Antibiotics for cell culture were benzyl penicillin from Glaxo (Greenford, UK), kanamycin acid sulfate from Winthrop (Guildford, UK), and streptomycin sulfate from Evans (Horsham, UK). All other reagents were of analytical grade. Leibovitz L-15 medium (GIBCO) was suppleStandard medium. mented with 10% (v/v) fetal calf serum (Imperial Laboratories, Andover, UK), 2 mAf~-glutamine (GIBCO), and antibiotics (0.1 mg ml-’ kanamycin, 0.05 mg ml-’ benzyl penicillin, and 0.05 mg ml-’ streptomycin) . Cytochalasin D was made up as a 0.05 mg Cytochulasin D medium. ml-’ solution in ethanol and 0.8 ml added per 100 ml standard medium, giving a final concentration of 0.4 pg ml-‘. As a control, 0.8 ml ethanol was added to standard medium. L-15 medium was supplemented L-Tyrosine phosphate medium. with antibiotics, then 0-phospho+tyrosine (14.5 mg/lOO ml) was dissolved in this medium by incubation at 37°C for 1 h. The pH was adjusted to 7.2 with 1 M sodium hydroxide solution and the medium was then sterilized by filtration through a 0.22~pm filter (Millipore). The medium was supplemented with 10% (v/v) fetal calf serum and 2 mM L-glutamine, giving a final concentration of 5 mM L-tyrosine phosphate and effectively increasing the L-tyrosine concentration fourfold. Cell culture. RVH 421 human melanoma cells growing in standard medium were harvested by incubation in PBSA/EDTA (PBSA containing 0.02% (w/v) EDTA; 37’C for 5 min), counted using a haemocytometer (Weber, UK), then seeded in 5 ml standard medium in 25 cm2 Nunc tissue culture flasks (GIBCO) at a density of 2 x lo6 cells per flask. The cells were incubated for 18-24 h and the medium was then replaced with fresh standard medium (controls) or experimental medium. Medium was not changed further during the experiment. Representative flasks were used to prepare enzyme or RNA at 2448 h intervals over the course of the experiment. Cell extract preparation, growth curves, and assessment of pigmentation. Cells were harvested by incubation in PBSA/EDTA/ trypsin (PBSA/EDTA containing 0.125% (w/v) trypsin; 37°C for 5 min), the culture medium being pooled with the cells. Flasks were rinsed twice with PBSA and the washings also pooled. Cells were pelleted by centrifugation (3OOg for 5 min at room temperature) and the color of the pellet noted. Pigmentation was scored subjectively on an arbitrary scale from 1 to 8, reflecting the range of pellet color from white to very dark brown. Cells were resuspended in ice-cold PBSA and an aliquot was mixed with an equal volume of trypan blue (0.5% (w/v) in 0.85% (v/v) saline) and counted using a hemocytometer: viable cells exclude the dye. Cells were pelleted (3OOg for 5 min) then
IN HUMAN
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resuspended in four volumes of enzyme buffer (20 m&f potassium phosphate pH 7.5,0.5% (v/v) Triton X-100). Lysates were sonicated for 30 s (M.S.E. Ultrasonic Disintegrator model 7100, setting 5 pm) then microcentrifuged (9OOOgfor 15 min at 4°C). The centrifugation step was repeated with the resulting supernatant, and the final supernatant was dialyzed against 20 mh4 potassium phosphate, pH 7.5 (1 liter for 4 h then 4 liters overnight at 4°C) then frozen at -20°C before use in assays. Enzyme assays. Tyrosine hydroxylase activity was determined using the ‘*CO, assay [26], and dopa oxidase activity measured using the MBTH assay [26]. Protein concentrations were determined by the method of Bradford [27], using bovine serum albumin as a reference standard, and used to calculate specific activities. Total cytoplasmic RNA was prepared accordRNA preparation. ing to the method of Perbal [28]. RNA was precipitated with isopropanol and stored at -70°C then resuspended in RNase-free water. Northern blots. RNA samples were denatured with formaldehyde and formamide then electrophoresed through 1.5% (w/v) agarose gels containing 2.3 M formaldehyde in 40 mM Mops, pH 7,10 mM sodium acetate, and 1 mM EDTA. Gels were blotted to nitrocellulose (Schleicher and Schiiell, Germany) overnight in 15X SSC, 1 M ammonium acetate (1X SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7). Nitrocellulose filters were baked at 80°C for 2 h then prehybridized in 3~ SSC, 3~ Denhardt’s, 50% (v/v) formamide, 500 pg ml-’ denatured salmon testes DNA at 42°C for 2-4 h (1X Denhardt’s is 0.02% (v/v) ficoll, 0.02% (w/v) BSA, 0.02% (w/v) polyvinylpyrrolidone). Blots were hybridized in the same solution at 42°C overnight with the 1.6kb insert of plasmid Pme134 [24] labeled with s2P by nick translation. The final wash was in 0.5X SSC + 0.1% (w/v) SDS at 65°C. Hybridizing RNA was quantitated by densitometric scanning of autoradiographs using an LKB Ultroscan laser densitometer. Equal amounts of intact RNA were run in each lane as shown by ethidium bromide staining of equivalent samples Nn in parallel. All errors are given at the level of 95% confiStatistical analysis. dence. Mean values are compared using Student’s t test [29].
RESULTS
Treatment
of nRVH
Cells with Cytochalasin
D
Cell growth and pigmentation. On treatment with 0.4 pg ml-’ cytochalasin D, nRVH cells undergo pronounced morphological changes within a few hours due to disruption of the cytoskeleton (data not shown). The cells appear much bigger than untreated cells as viewed in two dimensions under the light microscope, and many cells round up. Treatment with cytochalasin D completely inhibits growth of nRVH cells (Fig. la), but the majority of cells remain viable for the duration of the experiment. Pigmentation of treated cells increases at an earlier stage of the time-course, and to a higher level, than in untreated controls (Fig. lb). Cytochalasin D is added to the culture medium in ethanol, but addition of ethanol alone has no effect relative to the control on any of the parameters studied in this experiment (data not shown). Tyrosine hydroxylase and dopa oxiduse actiuities. Both activities of tyrosinase are induced to a higher maximum level in cytochalasin D-treated than in untreated cells. Between Days 1 and 5, tyrosine hydroxylase activity increases 2.7-fold in untreated, and 7.0-fold
250
WINDER
AND
HARRIS
‘-b
1
.I 0
.I
.,
1
.
2
, 4
3
.,
. 5
, 6
.,
0 7
.,',',.,',',', 1
0
2
3
Time (days)
4
5
6
7
Time (days)
FIG. 1. Effect of cytochalasin D on cell growth and pigmentation. nRVH cells were cultured in standard medium (0) or cytochalasin D medium (0). (a) Growth curves: values are the mean viable counts for three flasks k 1 SEM. (b) Pigmentation: values are estimated from observation of pellets from three flasks.
in treated cells (Fig. 2a). Dopa oxidase activity increases 2.6-fold in untreated, and 7.1-fold in treated cells (Fig. 2b). Total cytoplasmic Amount of tyrosinase mRNA. RNA was isolated from treated and control cells approximately 4 h after preparing enzyme, and equal amounts of RNA were run in each lane of gels used to prepare Northern blots. Screening with the cDNA Pme134 shows two hybridizing transcripts: a major transcript of 2.0 kb and a minor transcript of approximately 4 kb (Fig. 3). Laser densitometer scans show that the amount of major transcript in untreated cells increases 2.4-fold between Days 1 and 3, then remains constant (Fig. 4a). In contrast, the amount of minor transcript in untreated cells increases 3.3-fold between Days 1 and 3, and by Day 6 decreases to approximately 60% of its maximum level (Fig. 4b). Thus, the kinetics of induction of major and minor transcripts are different. In treated cells, the amount of major transcript increases 3.7-fold between Days 1 and 3, then decreases to approximately 70% of this maximum value by Day 6 (Fig. 4a). The amount of minor transcript increases 5.1-
0:.
,
0
1
.
,
2
.
I
3
.
,
4
Time (days) FIG. 2. cytochalasin
.
I.,
5
fold between Days 1 and 3, then decreases to approximately 35% of this maximum by Day 6 (Fig. 4b). Treatment of oRVH Cells with 5 mM L-Tyrosine Phosphate Addition of 5 mM LCell growth and pigmentation. tyrosine phosphate to the culture medium decreases the growth rate and saturation density of oRVH cells (Fig. 5a). Pigmentation increases at an earlier stage of the time-course and to a higher level in treated compared with untreated cells (Fig. 5b). Note that the cells used in this experiment were relatively highly pigmented when seeded. In Tyrosine hydroxylase and dopa oxidase activities. control and treated oRVH cells, a decrease in both enzyme activities is observed between Days 1 and 3, a consequence of the cells used having relatively high enzyme activities when seeded (Fig. 6). Over the remaining time-course, both activities are induced to a higher maximum level in treated than in untreated cells. Between Days 3 and 5, tyrosine hydroxylase activity increases 1.7-fold in untreated, and 3.2-fold in treated
,
6
7
0:
.,.,.,‘,‘,‘,‘I
0
1
2
3
4
5
6
7
Time (days)
Effect of cytochalasin D on tyrosine hydroxylase and dopa oxidase activities. nRVH cells were cultured in standard medium (0) or D medium (0). (a) Tyrosine hydroxylase specific activity; (b) dopa oxidase specific activity. Values are the means for two samples.
INDUCTION
Day
3
I1
1 4
OF TYROSINASE
1
IN HUMAN
5
FIG. 3. Tyrosinase mRNA in untreated cells. Northern blot of RNA samples prepared over the time-course from nRVH cells cultured in standard medium and hybridized to the cDNA Pme134. Each lane contains 8 pg total cytoplasmic RNA. Small and large arrowheads indicate minor and major transcripts, respectively; markers are ribosomal RNAs.
cells (Fig. 6a). Dopa oxidase activity increases 2.3-fold in untreated and 3.2-fold in treated cells between Days 3 and 5 (Fig. 6b). Kinetics of induction Amount of tyrosinuse mRNA. of the major transcript in control and treated samples are virtually identical (Fig. 7), the amount increases approximately 2.5-fold between Days 2 and 4 then decreases to its initial level by Day 6. Because of a weak signal on Northern blots, the kinetics of induction of the minor transcript could not be determined accurately. DISCUSSION
When human melanoma cells are treated with cytochalasin D or L-tyrosine phosphate, increased melanin synthesis correlates with inhibition of cell growth. Disruption of the cytoskeleton by cytochalasin D prevents cell division, but it is not clear whether induction of melanin synthesis has an inhibitory effect itself.
0
0
.,.,.,.,.,.,., 1
3
4
Time (days)
FIG. 4.
5
6
7
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When the cells are treated with L-tyrosine phosphate, however, it appears that induction of melanin synthesis and inhibition of cell division are directly related. One possible explanation for the correlation between increased melanin synthesis and inhibition of cell growth is the production of toxic melanin precursors [30, 311. Alternatively, or in addition, decreased cell growth may reflect induction of a more differentiated phenotype. To gain insight into the mechanisms of action of cytochalasin D and L-tyrosine phosphate, their effect on tyrosinase activity was monitored. In both cases there was a correlation between increased pigmentation and increased tyrosinase activity (both tyrosine hydroxylase and dopa oxidase). This does not rule out the possibility that treatment with either compound might affect posttyrosinase steps, but increased activity of the enzyme catalyzing the critical initial step is likely to affect pigmentation. The only deviation from the correlation between pigmentation and tyrosinase activity is seen at the end of each time-course when there is a slight decrease in enzyme activity. This is possibly due to inhibition of tyrosinase by melanin deposited within melanosomes. The amount of tyrosinase mRNA was studied to determine whether it correlates with enzyme activity. The tyrosinase cDNA Pme134 hybridizes to two transcripts in both nRVH and oRVH cells: a major transcript of 2.0 kb and a minor transcript of approximately 4 kb. (For comparison, Kwon et al. [24] reported that Pme134 hybridizes to one 2.4-kb mRNA in normal and malignant human melanocytes; Bouchard et al. [25] detected a major transcript of 2.4 kb and a much less abundant transcript of 4.7 kb in human melanoma cells.) The relationship between the major and minor transcripts detected in RVH 421 cells is not yet known. They may represent alternatively spliced transcripts of one precursor RNA or transcripts of different genes. The former hypothesis
0.0 2
MELANOMA
. 0
, 1
.
, 2
.
, 3
.
, 4
.
, 5
.
, 6
.
, 7
Time (days)
Effect of cytochalasin D on amount of tyrosinase mRNA. Comparison of amount of tyrosinase transcripts in nRVH cells cultured in standard medium (0) or cytochalasin D medium (0). (a) Major transcript; (b) minor transcript. Relative amounts of RNAs hybridizing to Pme134 were estimated by scanning autoradiographs with a laser densitometer. Differences between intensities of autoradiographs were corrected for by positive controls. Values are the means for two or three samples.
252
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“‘!a
1 0
HARRIS l
a b
.,.,.,',.,',',., 12
0 3
4
5
6
7
6
,:’
.,.,.,.,.,.,.,., 12
0
3
Time (days)
4
5
6
7
8
Time (days)
FIG. 5. Effect of L-tyrosine phosphate on cell growth and pigmentation. oRVH cells were cultured in standard medium (0) or L-tyrosine phosphate medium (6). (a) Growth curves: values are the mean viable counts for three flasks f 1 SEM. (b) Pigmentation: values are estimated from observation of pellets from three flasks.
increases in amount of tyrosinase mRNA are due to increased mRNA synthesis or decreased degradation. Further studies are needed to clarify this point. The increase in tyrosinase activity that cannot be accounted for by an increase in mRNA is probably due to a post-transcriptional mechanism. Activation of preexisting enzyme by removal of inhibitors, addition of activators, or by altered post-translational modification, as well as decreased enzyme degradation, are all mechanisms that have been previously postulated to be involved in regulation of tyrosinase [35-371. Cytochalasin D may take effect through another post-transcriptional mechanism. Nearly 100% of polysomes active in protein synthesis are normally associated with the cytoskeleton, and this association is thought to compartmentalize mRNAs within the cytoplasm [12,13]. Treatment of cells with cytochalasin D alters the subcellular organization of the cytoskeleton and disrupts the association of polysomes, releasing specific mRNAs preferentially. The released transcripts are not translated [12,13]. It is conceivable that there may thus be an effect on the translation of tyrosinase mRNA. Further work is
seems more likely since alternatively spliced tyrosinase transcripts have been reported in the mouse [32]. Tyrosinase-related proteins have been described [33, 341, but the degree of homology is low and such transcripts would not hybridize to the cDNA Pme134 in the protocol described. In studying the correlation between tyrosinase activity and amount of mRNA, we have assumed that the more abundant 2.0-kb transcript encodes active tyrosinase. In both experiments, the increase in amount of tyrosinase mRNA preceded the increase in enzyme activity. The increase in amount of the major tyrosinase transcript can account for the increase in tyrosinase activity of untreated cells (Table 1). When the cells are treated with cytochalasin D there is a slight increase in the amount of mRNA above the control level (Fig. 4a), but not sufficient to account for the increase in enzyme activity (Table 1). In the case of treatment with L-tyrosine phosphate, the amount of tyrosinase mRNA in control and treated cells is not significantly different (Fig. 7), but enzyme activity is induced to a higher level in treated cells (Table 1). We do not know whether the
1
4 b
o;.,.,.,.,.,.,.,., 0 12
0:. 3
4
5
6
7
Time (days) FIG. 6. Effect of L-tyrosine phosphate on tyrosine hydroxylase (0) or L-tyrosine phosphate medium (6). (a) Tyrosine hydroxylase three samples + 1 SEM.
8
0
, . 12
,
.
, 3
.
, 4
.
, 5
.
, 6
.
, 7
.
t 8
Time (days) and dopa oxidase activities. oRVH cells were cultured in standard medium specific activity; (b) dopa oxidase specific activity. Values are the means for
INDUCTION
OF TYROSINASE
needed to elucidate the exact mechanisms of action of cytochalasin D and L-tyrosine phosphate. Other studies in which tyrosinase activity and mRNA have been monitored in parallel have shown that enzyme activity generally correlates with the amount of tyrosinase mRNA in melanoma cells [4,38-401, but discrepancies have been noted. Specifically, Hoganson et al. [38] reported that the amount of tyrosinase mRNA did not always correlate with enzyme activity in melanocyte stimulating hormone-treated mouse melanoma cells, the ratio of mRNA to enzyme activity increasing in hormone-treated cells. Rauth et al. [4] described a discrepancy between tyrosinase activity and mRNA level in hamster melanoma cells: treatment with either 300 pM cyclic AMP or 0.4 pM bromodeoxyuridine reduced enzyme activity to the same level, but on treatment with cyclic AMP the amount of mRNA decreased proportionately, whereas on treatment with bromodeoxyuridine tyrosinase mRNA was no longer detectable. These discrepancies support the concept that tyrosinase activity is not regulated simply by the abundance of its mRNA. In the two experiments described in this report, tyrosine hydroxylase and dopa oxidase activities were induced co-ordinately in both nRVH and oRVH cells (Figs. 2 and 6). When the two studies are compared, however, it is apparent that the two lines have similar tyrosine hydroxylase activities, but high-passage oRVH cells have significantly lower dopa oxidase activity. This is observed consistently so there appears to have been selective loss of dopa oxidase activity on passage of RVH 421 cells. One possible explanation is that there is differential regulation of the two activities. A second is that a mutation in the tyrosinase gene that decreases dopa oxidase activity but does not affect tyrosine hydroxylase activity may have occurred. A third is that one or both of the activities may be encoded by more than
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TABLE
1
Comparison of the Induction of the Tyrosine Hydroxylase and Dopa Oxidase Activities of Tyrosinase and Tyrosinase mRNA Ratio”
Treatment Experiment 1 Control Cytochalasin D Experiment 2 Control r.-Tyrosine phosphate
Tyrosine hydroxylase activity
Dopa oxidase activity
Tyrosinase mRNA
2.7 7.0
2.6 7.1
2.4 3.7
1.7 3.2
2.3 3.2
2.3 2.8
a Experiment 1: Ratio of enzyme activities on Day 5 relative to Day 1; amount of mRNA on Day 3 relative to Day 1. Experiment 2: Ratio of enzyme activities on Day 5 relative to Day 3; amount of mRNA on Day 4 relative to Day 2.
one gene, and mutation or altered regulation of these multiple tyrosinase genes may result in the altered ratio of tyrosine hydroxylase and dopa oxidase activities. It has been suggested that there is more than one enzyme with tyrosinase activity in the mouse [41]. This work was supported by the Cancer Research Campaign. A.J.W. was in receipt of a Medical Research Council research studentship and a scholarship from St. Hugh’s College, Oxford, funded by the Dee Corporation. We acknowledge Dr. B. S. Kwon for the gift of plasmid Pme134. We also thank Mrs. K. W. Brownsill for technical assistance and Dr. J. M. Cooper for helpful discussions.
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Induction of Tyrosinase in Human Melanoma Cells by L-Tyrosine Phosphate and Cytochalasin D ALISON J. WINDER AND HENRY HARRIS Sir William
Dunn
School of Pathology,
University
of Oxford, South Parks Road, Oxford, OX1 3RE, United Kingdom
mRNAs [ 111. In addition, cytochalasin is thought to affect gene expression at a post-transcriptional level [12, 131. Melanoma cells were treated with cytochalasin D to determine whether increased pigmentation correlates with increased tyrosinase activity and whether there is an effect on the expression of the tyrosinase gene. L-Tyrosine has been shown to induce pigmentation in hamster, mouse, and human melanoma cell lines [6,14, 151. Slominski et al. [6] have demonstrated that induction of pigmentation in hamster melanoma cells is not simply due to increased availability of substrate, but also to increased tyrosinase activity and a greater number of melanosomes per cell. The induction of melanin synthesis by L-tyrosine in these cells follows a pathway unrelated to cyclic AMP, cyclic GMP, or inositol triphosphate and is inhibited by cycloheximide, indicating that protein synthesis is required [6, 161. We have treated melanoma cells with L-tyrosine phosphate in an attempt to confirm the effect of tyrosine on tyrosinase activity and to determine whether there is also an effect on the amount of tyrosinase mRNA. LTyrosine phosphate was used in preference to L-tyrosine because of its greater solubility. L-Tyrosine phosphate is not transported into melanoma cells [ 171 but is thought to be hydrolyzed to L-tyrosine by cell-surfaceassociated phosphatases [ 181 and acts as a tyrosine precursor on intraperitoneal administration to mice [19]. Furthermore, phosphorylated isomers of L-dopa, another melanin precursor, have been shown to induce increased tyrosinase activity in mouse melanoma cells [20]. Two variants of the human melanoma cell line RVH 421 [21] were used in this study. The line nRVH is of relatively low passage (P = 55-65), whereas the line oRVH is of relatively high passage (P > 120). Both lines, nRVH and oRVH, were derived in our laboratory from the original RVH 421 parent. nRVH cells have a more differentiated phenotype than oRVH cells: they are more dendritic and have a higher basal level of pigmentation. Both variants are relatively unpigmented when seeded at low density, but pigmentation increases when the cells reach confluence. Variations in tyrosinase activity and melanin synthesis with cell density have been
Pigmentation of RVH 421 human melanoma cells is induced when cell division is inhibited by cytochalasin D or L-tyrosine phosphate. Increased pigmentation correlates with increased tyrosinase activity when this is monitored over a time-course. Parallel measurements show that the amount of tyrosinase mRNA correlates with enzyme activity in cells growing without these additives. In contrast, in the presence of cytochalasin D or L-tyrosine phosphate, the increase in amount of tyrosinase mRNA is not sufficient to account for the increase in enzyme activity, indicating that these compounds act mainly at a post-transcriptional level. 0 1992 Academic Press, Inc.
INTRODUCTION Induction of melanin synthesis in cultured melanoma cells generally correlates with inhibition of cell growth [l, 21. Tyrosinase (EC 1.14.18.1) is a key enzyme in melanin synthesis. It catalyses both the critical initial step, the hydroxylation of L-tyrosine to L-dopa (tyrosine hydroxylase activity), and the second step, the oxidation of L-dopa to dopaquinone (dopa oxidase activity). Pigmentation is generally found to correlate with tyrosinase activity [3-61. However, it has been shown that melanin synthesis can be regulated further down the pathway [7]. Both protein factors and metal ions are thought to be involved in post-tyrosinase regulation [8]. We have studied the regulation of tyrosinase in human melanoma cells treated with compounds that induce pigmentation in order to gain insight into their mechanisms of action. We have screened a large number of compounds for their ability to induce pigmentation and inhibit growth of melanoma cells in u&o. In the present paper we describe the effects of two of these, cytochalasin D and L-tyrosine phosphate. Cytochalasin D binds to actin filaments and disrupts the cytoskeleton, inhibiting cell division and altering cell morphology [9, lo]. The nucleus may respond to signals relating to the structural organization of the cytoskeleton, and its disruption by cytochalasin D has been shown to cause changes in the synthesis of specific 0014-4827/92 $3.00 Copyright 0 1992 hy Academic Press, Inc. All rights of reproduction in any form reserved.
248
INDUCTION
OF TYROSINASE
observed in several melanoma cell lines but the underlying mechanisms are not understood [3, 22, 231. Measurements of cell growth, pigmentation, tyrosinase activity, and amount of tyrosinase mRNA were made over a time-course for both control and treated cells. The human tyrosinase cDNA Pme134 [24] was used as a hybridization probe for detection of tyrosinase mRNA. Expression studies using a homologous cDNA have confirmed that this sequence encodes tyrosinase [25]. MATERIALS
AND
METHODS
Materials. Agarose (ultra-pure DNA grade) was purchased from Bio-Rad (Hemel Hempstead, UK). Ammonium acetate and gold-label ethanol were from Aldrich (Gillingham, UK). Ficoll was from Pharmacia (Uppsala, Sweden). Cytochalasin D, Mops, O-phospho-Ltyrosine, polyvinylpyrrolidone, salmon testes DNA, and Triton X-100 were obtained from Sigma (Poole, UK). Phosphate-buffered saline A (PBSA) tablets were from Oxoid (Basingstoke, UK), trypsin was from GIBCO (Paisley, UK), and trypan blue was from Flow Labs (Irvine, UK). Antibiotics for cell culture were benzyl penicillin from Glaxo (Greenford, UK), kanamycin acid sulfate from Winthrop (Guildford, UK), and streptomycin sulfate from Evans (Horsham, UK). All other reagents were of analytical grade. Leibovitz L-15 medium (GIBCO) was suppleStandard medium. mented with 10% (v/v) fetal calf serum (Imperial Laboratories, Andover, UK), 2 mAf~-glutamine (GIBCO), and antibiotics (0.1 mg ml-’ kanamycin, 0.05 mg ml-’ benzyl penicillin, and 0.05 mg ml-’ streptomycin) . Cytochalasin D was made up as a 0.05 mg Cytochulasin D medium. ml-’ solution in ethanol and 0.8 ml added per 100 ml standard medium, giving a final concentration of 0.4 pg ml-‘. As a control, 0.8 ml ethanol was added to standard medium. L-15 medium was supplemented L-Tyrosine phosphate medium. with antibiotics, then 0-phospho+tyrosine (14.5 mg/lOO ml) was dissolved in this medium by incubation at 37°C for 1 h. The pH was adjusted to 7.2 with 1 M sodium hydroxide solution and the medium was then sterilized by filtration through a 0.22~pm filter (Millipore). The medium was supplemented with 10% (v/v) fetal calf serum and 2 mM L-glutamine, giving a final concentration of 5 mM L-tyrosine phosphate and effectively increasing the L-tyrosine concentration fourfold. Cell culture. RVH 421 human melanoma cells growing in standard medium were harvested by incubation in PBSA/EDTA (PBSA containing 0.02% (w/v) EDTA; 37’C for 5 min), counted using a haemocytometer (Weber, UK), then seeded in 5 ml standard medium in 25 cm2 Nunc tissue culture flasks (GIBCO) at a density of 2 x lo6 cells per flask. The cells were incubated for 18-24 h and the medium was then replaced with fresh standard medium (controls) or experimental medium. Medium was not changed further during the experiment. Representative flasks were used to prepare enzyme or RNA at 2448 h intervals over the course of the experiment. Cell extract preparation, growth curves, and assessment of pigmentation. Cells were harvested by incubation in PBSA/EDTA/ trypsin (PBSA/EDTA containing 0.125% (w/v) trypsin; 37°C for 5 min), the culture medium being pooled with the cells. Flasks were rinsed twice with PBSA and the washings also pooled. Cells were pelleted by centrifugation (3OOg for 5 min at room temperature) and the color of the pellet noted. Pigmentation was scored subjectively on an arbitrary scale from 1 to 8, reflecting the range of pellet color from white to very dark brown. Cells were resuspended in ice-cold PBSA and an aliquot was mixed with an equal volume of trypan blue (0.5% (w/v) in 0.85% (v/v) saline) and counted using a hemocytometer: viable cells exclude the dye. Cells were pelleted (3OOg for 5 min) then
IN HUMAN
MELANOMA
249
CELLS
resuspended in four volumes of enzyme buffer (20 m&f potassium phosphate pH 7.5,0.5% (v/v) Triton X-100). Lysates were sonicated for 30 s (M.S.E. Ultrasonic Disintegrator model 7100, setting 5 pm) then microcentrifuged (9OOOgfor 15 min at 4°C). The centrifugation step was repeated with the resulting supernatant, and the final supernatant was dialyzed against 20 mh4 potassium phosphate, pH 7.5 (1 liter for 4 h then 4 liters overnight at 4°C) then frozen at -20°C before use in assays. Enzyme assays. Tyrosine hydroxylase activity was determined using the ‘*CO, assay [26], and dopa oxidase activity measured using the MBTH assay [26]. Protein concentrations were determined by the method of Bradford [27], using bovine serum albumin as a reference standard, and used to calculate specific activities. Total cytoplasmic RNA was prepared accordRNA preparation. ing to the method of Perbal [28]. RNA was precipitated with isopropanol and stored at -70°C then resuspended in RNase-free water. Northern blots. RNA samples were denatured with formaldehyde and formamide then electrophoresed through 1.5% (w/v) agarose gels containing 2.3 M formaldehyde in 40 mM Mops, pH 7,10 mM sodium acetate, and 1 mM EDTA. Gels were blotted to nitrocellulose (Schleicher and Schiiell, Germany) overnight in 15X SSC, 1 M ammonium acetate (1X SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7). Nitrocellulose filters were baked at 80°C for 2 h then prehybridized in 3~ SSC, 3~ Denhardt’s, 50% (v/v) formamide, 500 pg ml-’ denatured salmon testes DNA at 42°C for 2-4 h (1X Denhardt’s is 0.02% (v/v) ficoll, 0.02% (w/v) BSA, 0.02% (w/v) polyvinylpyrrolidone). Blots were hybridized in the same solution at 42°C overnight with the 1.6kb insert of plasmid Pme134 [24] labeled with s2P by nick translation. The final wash was in 0.5X SSC + 0.1% (w/v) SDS at 65°C. Hybridizing RNA was quantitated by densitometric scanning of autoradiographs using an LKB Ultroscan laser densitometer. Equal amounts of intact RNA were run in each lane as shown by ethidium bromide staining of equivalent samples Nn in parallel. All errors are given at the level of 95% confiStatistical analysis. dence. Mean values are compared using Student’s t test [29].
RESULTS
Treatment
of nRVH
Cells with Cytochalasin
D
Cell growth and pigmentation. On treatment with 0.4 pg ml-’ cytochalasin D, nRVH cells undergo pronounced morphological changes within a few hours due to disruption of the cytoskeleton (data not shown). The cells appear much bigger than untreated cells as viewed in two dimensions under the light microscope, and many cells round up. Treatment with cytochalasin D completely inhibits growth of nRVH cells (Fig. la), but the majority of cells remain viable for the duration of the experiment. Pigmentation of treated cells increases at an earlier stage of the time-course, and to a higher level, than in untreated controls (Fig. lb). Cytochalasin D is added to the culture medium in ethanol, but addition of ethanol alone has no effect relative to the control on any of the parameters studied in this experiment (data not shown). Tyrosine hydroxylase and dopa oxiduse actiuities. Both activities of tyrosinase are induced to a higher maximum level in cytochalasin D-treated than in untreated cells. Between Days 1 and 5, tyrosine hydroxylase activity increases 2.7-fold in untreated, and 7.0-fold
250
WINDER
AND
HARRIS
‘-b
1
.I 0
.I
.,
1
.
2
, 4
3
.,
. 5
, 6
.,
0 7
.,',',.,',',', 1
0
2
3
Time (days)
4
5
6
7
Time (days)
FIG. 1. Effect of cytochalasin D on cell growth and pigmentation. nRVH cells were cultured in standard medium (0) or cytochalasin D medium (0). (a) Growth curves: values are the mean viable counts for three flasks k 1 SEM. (b) Pigmentation: values are estimated from observation of pellets from three flasks.
in treated cells (Fig. 2a). Dopa oxidase activity increases 2.6-fold in untreated, and 7.1-fold in treated cells (Fig. 2b). Total cytoplasmic Amount of tyrosinase mRNA. RNA was isolated from treated and control cells approximately 4 h after preparing enzyme, and equal amounts of RNA were run in each lane of gels used to prepare Northern blots. Screening with the cDNA Pme134 shows two hybridizing transcripts: a major transcript of 2.0 kb and a minor transcript of approximately 4 kb (Fig. 3). Laser densitometer scans show that the amount of major transcript in untreated cells increases 2.4-fold between Days 1 and 3, then remains constant (Fig. 4a). In contrast, the amount of minor transcript in untreated cells increases 3.3-fold between Days 1 and 3, and by Day 6 decreases to approximately 60% of its maximum level (Fig. 4b). Thus, the kinetics of induction of major and minor transcripts are different. In treated cells, the amount of major transcript increases 3.7-fold between Days 1 and 3, then decreases to approximately 70% of this maximum value by Day 6 (Fig. 4a). The amount of minor transcript increases 5.1-
0:.
,
0
1
.
,
2
.
I
3
.
,
4
Time (days) FIG. 2. cytochalasin
.
I.,
5
fold between Days 1 and 3, then decreases to approximately 35% of this maximum by Day 6 (Fig. 4b). Treatment of oRVH Cells with 5 mM L-Tyrosine Phosphate Addition of 5 mM LCell growth and pigmentation. tyrosine phosphate to the culture medium decreases the growth rate and saturation density of oRVH cells (Fig. 5a). Pigmentation increases at an earlier stage of the time-course and to a higher level in treated compared with untreated cells (Fig. 5b). Note that the cells used in this experiment were relatively highly pigmented when seeded. In Tyrosine hydroxylase and dopa oxidase activities. control and treated oRVH cells, a decrease in both enzyme activities is observed between Days 1 and 3, a consequence of the cells used having relatively high enzyme activities when seeded (Fig. 6). Over the remaining time-course, both activities are induced to a higher maximum level in treated than in untreated cells. Between Days 3 and 5, tyrosine hydroxylase activity increases 1.7-fold in untreated, and 3.2-fold in treated
,
6
7
0:
.,.,.,‘,‘,‘,‘I
0
1
2
3
4
5
6
7
Time (days)
Effect of cytochalasin D on tyrosine hydroxylase and dopa oxidase activities. nRVH cells were cultured in standard medium (0) or D medium (0). (a) Tyrosine hydroxylase specific activity; (b) dopa oxidase specific activity. Values are the means for two samples.
INDUCTION
Day
3
I1
1 4
OF TYROSINASE
1
IN HUMAN
5
FIG. 3. Tyrosinase mRNA in untreated cells. Northern blot of RNA samples prepared over the time-course from nRVH cells cultured in standard medium and hybridized to the cDNA Pme134. Each lane contains 8 pg total cytoplasmic RNA. Small and large arrowheads indicate minor and major transcripts, respectively; markers are ribosomal RNAs.
cells (Fig. 6a). Dopa oxidase activity increases 2.3-fold in untreated and 3.2-fold in treated cells between Days 3 and 5 (Fig. 6b). Kinetics of induction Amount of tyrosinuse mRNA. of the major transcript in control and treated samples are virtually identical (Fig. 7), the amount increases approximately 2.5-fold between Days 2 and 4 then decreases to its initial level by Day 6. Because of a weak signal on Northern blots, the kinetics of induction of the minor transcript could not be determined accurately. DISCUSSION
When human melanoma cells are treated with cytochalasin D or L-tyrosine phosphate, increased melanin synthesis correlates with inhibition of cell growth. Disruption of the cytoskeleton by cytochalasin D prevents cell division, but it is not clear whether induction of melanin synthesis has an inhibitory effect itself.
0
0
.,.,.,.,.,.,., 1
3
4
Time (days)
FIG. 4.
5
6
7
251
CELLS
When the cells are treated with L-tyrosine phosphate, however, it appears that induction of melanin synthesis and inhibition of cell division are directly related. One possible explanation for the correlation between increased melanin synthesis and inhibition of cell growth is the production of toxic melanin precursors [30, 311. Alternatively, or in addition, decreased cell growth may reflect induction of a more differentiated phenotype. To gain insight into the mechanisms of action of cytochalasin D and L-tyrosine phosphate, their effect on tyrosinase activity was monitored. In both cases there was a correlation between increased pigmentation and increased tyrosinase activity (both tyrosine hydroxylase and dopa oxidase). This does not rule out the possibility that treatment with either compound might affect posttyrosinase steps, but increased activity of the enzyme catalyzing the critical initial step is likely to affect pigmentation. The only deviation from the correlation between pigmentation and tyrosinase activity is seen at the end of each time-course when there is a slight decrease in enzyme activity. This is possibly due to inhibition of tyrosinase by melanin deposited within melanosomes. The amount of tyrosinase mRNA was studied to determine whether it correlates with enzyme activity. The tyrosinase cDNA Pme134 hybridizes to two transcripts in both nRVH and oRVH cells: a major transcript of 2.0 kb and a minor transcript of approximately 4 kb. (For comparison, Kwon et al. [24] reported that Pme134 hybridizes to one 2.4-kb mRNA in normal and malignant human melanocytes; Bouchard et al. [25] detected a major transcript of 2.4 kb and a much less abundant transcript of 4.7 kb in human melanoma cells.) The relationship between the major and minor transcripts detected in RVH 421 cells is not yet known. They may represent alternatively spliced transcripts of one precursor RNA or transcripts of different genes. The former hypothesis
0.0 2
MELANOMA
. 0
, 1
.
, 2
.
, 3
.
, 4
.
, 5
.
, 6
.
, 7
Time (days)
Effect of cytochalasin D on amount of tyrosinase mRNA. Comparison of amount of tyrosinase transcripts in nRVH cells cultured in standard medium (0) or cytochalasin D medium (0). (a) Major transcript; (b) minor transcript. Relative amounts of RNAs hybridizing to Pme134 were estimated by scanning autoradiographs with a laser densitometer. Differences between intensities of autoradiographs were corrected for by positive controls. Values are the means for two or three samples.
252
WINDER
AND
“‘!a
1 0
HARRIS l
a b
.,.,.,',.,',',., 12
0 3
4
5
6
7
6
,:’
.,.,.,.,.,.,.,., 12
0
3
Time (days)
4
5
6
7
8
Time (days)
FIG. 5. Effect of L-tyrosine phosphate on cell growth and pigmentation. oRVH cells were cultured in standard medium (0) or L-tyrosine phosphate medium (6). (a) Growth curves: values are the mean viable counts for three flasks f 1 SEM. (b) Pigmentation: values are estimated from observation of pellets from three flasks.
increases in amount of tyrosinase mRNA are due to increased mRNA synthesis or decreased degradation. Further studies are needed to clarify this point. The increase in tyrosinase activity that cannot be accounted for by an increase in mRNA is probably due to a post-transcriptional mechanism. Activation of preexisting enzyme by removal of inhibitors, addition of activators, or by altered post-translational modification, as well as decreased enzyme degradation, are all mechanisms that have been previously postulated to be involved in regulation of tyrosinase [35-371. Cytochalasin D may take effect through another post-transcriptional mechanism. Nearly 100% of polysomes active in protein synthesis are normally associated with the cytoskeleton, and this association is thought to compartmentalize mRNAs within the cytoplasm [12,13]. Treatment of cells with cytochalasin D alters the subcellular organization of the cytoskeleton and disrupts the association of polysomes, releasing specific mRNAs preferentially. The released transcripts are not translated [12,13]. It is conceivable that there may thus be an effect on the translation of tyrosinase mRNA. Further work is
seems more likely since alternatively spliced tyrosinase transcripts have been reported in the mouse [32]. Tyrosinase-related proteins have been described [33, 341, but the degree of homology is low and such transcripts would not hybridize to the cDNA Pme134 in the protocol described. In studying the correlation between tyrosinase activity and amount of mRNA, we have assumed that the more abundant 2.0-kb transcript encodes active tyrosinase. In both experiments, the increase in amount of tyrosinase mRNA preceded the increase in enzyme activity. The increase in amount of the major tyrosinase transcript can account for the increase in tyrosinase activity of untreated cells (Table 1). When the cells are treated with cytochalasin D there is a slight increase in the amount of mRNA above the control level (Fig. 4a), but not sufficient to account for the increase in enzyme activity (Table 1). In the case of treatment with L-tyrosine phosphate, the amount of tyrosinase mRNA in control and treated cells is not significantly different (Fig. 7), but enzyme activity is induced to a higher level in treated cells (Table 1). We do not know whether the
1
4 b
o;.,.,.,.,.,.,.,., 0 12
0:. 3
4
5
6
7
Time (days) FIG. 6. Effect of L-tyrosine phosphate on tyrosine hydroxylase (0) or L-tyrosine phosphate medium (6). (a) Tyrosine hydroxylase three samples + 1 SEM.
8
0
, . 12
,
.
, 3
.
, 4
.
, 5
.
, 6
.
, 7
.
t 8
Time (days) and dopa oxidase activities. oRVH cells were cultured in standard medium specific activity; (b) dopa oxidase specific activity. Values are the means for
INDUCTION
OF TYROSINASE
needed to elucidate the exact mechanisms of action of cytochalasin D and L-tyrosine phosphate. Other studies in which tyrosinase activity and mRNA have been monitored in parallel have shown that enzyme activity generally correlates with the amount of tyrosinase mRNA in melanoma cells [4,38-401, but discrepancies have been noted. Specifically, Hoganson et al. [38] reported that the amount of tyrosinase mRNA did not always correlate with enzyme activity in melanocyte stimulating hormone-treated mouse melanoma cells, the ratio of mRNA to enzyme activity increasing in hormone-treated cells. Rauth et al. [4] described a discrepancy between tyrosinase activity and mRNA level in hamster melanoma cells: treatment with either 300 pM cyclic AMP or 0.4 pM bromodeoxyuridine reduced enzyme activity to the same level, but on treatment with cyclic AMP the amount of mRNA decreased proportionately, whereas on treatment with bromodeoxyuridine tyrosinase mRNA was no longer detectable. These discrepancies support the concept that tyrosinase activity is not regulated simply by the abundance of its mRNA. In the two experiments described in this report, tyrosine hydroxylase and dopa oxidase activities were induced co-ordinately in both nRVH and oRVH cells (Figs. 2 and 6). When the two studies are compared, however, it is apparent that the two lines have similar tyrosine hydroxylase activities, but high-passage oRVH cells have significantly lower dopa oxidase activity. This is observed consistently so there appears to have been selective loss of dopa oxidase activity on passage of RVH 421 cells. One possible explanation is that there is differential regulation of the two activities. A second is that a mutation in the tyrosinase gene that decreases dopa oxidase activity but does not affect tyrosine hydroxylase activity may have occurred. A third is that one or both of the activities may be encoded by more than
IN HUMAN
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TABLE
1
Comparison of the Induction of the Tyrosine Hydroxylase and Dopa Oxidase Activities of Tyrosinase and Tyrosinase mRNA Ratio”
Treatment Experiment 1 Control Cytochalasin D Experiment 2 Control r.-Tyrosine phosphate
Tyrosine hydroxylase activity
Dopa oxidase activity
Tyrosinase mRNA
2.7 7.0
2.6 7.1
2.4 3.7
1.7 3.2
2.3 3.2
2.3 2.8
a Experiment 1: Ratio of enzyme activities on Day 5 relative to Day 1; amount of mRNA on Day 3 relative to Day 1. Experiment 2: Ratio of enzyme activities on Day 5 relative to Day 3; amount of mRNA on Day 4 relative to Day 2.
one gene, and mutation or altered regulation of these multiple tyrosinase genes may result in the altered ratio of tyrosine hydroxylase and dopa oxidase activities. It has been suggested that there is more than one enzyme with tyrosinase activity in the mouse [41]. This work was supported by the Cancer Research Campaign. A.J.W. was in receipt of a Medical Research Council research studentship and a scholarship from St. Hugh’s College, Oxford, funded by the Dee Corporation. We acknowledge Dr. B. S. Kwon for the gift of plasmid Pme134. We also thank Mrs. K. W. Brownsill for technical assistance and Dr. J. M. Cooper for helpful discussions.
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