From the Department of Dermatology, Yale University
School of Medicine, New Haven, Connecticut 06520 and the
¶ Department of Biochemistry and Molecular Biology, Program in
Molecular and Cellular Biology, University of Massachusetts, Amherst,
Massachusetts 01003
Received for publication, September 22, 2000, and in revised form, December 1, 2000
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ABSTRACT |
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Tyrosinase is essential for pigmentation
and is a source of tumor-derived antigenic peptides and cellular immune
response. Wild type tyrosinase in melanoma cells and certain albino
mutants in untransformed melanocytes are targeted to proteolytic
degradation by the 26 S proteasome due to retention of the misfolded
protein in the endoplasmic reticulum and its subsequent
retranslocation to the cytosol. Here, we demonstrate that the
substrates DOPA and tyrosine induced in melanoma cells a transition of
misfolded wild type tyrosinase to the native form that is resistant to
proteolysis, competent to exit the endoplasmic reticulum, and able to
produce melanin. Because the enzymatic activity of tyrosinase is
induced by DOPA, we propose that proper folding of the wild type
protein, just like mutant forms, is tightly linked to its catalytic
state. Loss of pigmentation, therefore, in tyrosinase-positive melanoma cells is a consequence of tumor-induced metabolic changes that suppress
tyrosinase activity and DOPA production within these cells.
Loss of pigmentation in human melanoma cells is common in advanced
and metastatic lesions due to suppression of melanocyte-specific proteins. Down-regulation at the transcriptional level is likely to
involve inactivation of microphthalmia, the transcription factor controlling pigment-specific genes such as tyrosinase, the key enzyme
in melanin synthesis (1, 2). However, post-transcriptional processes
cause the amelanotic phenotype of melanoma cells expressing wild type
tyrosinase. In these cells tyrosinase is synthesized at levels similar
to those of pigmented normal melanocytes but is retained in the
endoplasmic reticulum (ER)1
as an early 70-kDa glycoform that is subsequently degraded by the
proteasome (3). Retention of tyrosinase in the ER is also characteristic of mutant tyrosinase proteins associated with
oculocutaneous albinism type 1 (4, 5). Individuals with this
recessively inherited condition have a normal number of melanocytes in
the skin that are defective in melanin production. Thus, in melanoma cells, wild type tyrosinase behaves like a mutant protein in that it is
not processed to the 80-kDa mature form, and its level in the
melanosomes, the site of melanin synthesis, is not sufficient to induce
pigmentation. These observations explain not only the loss of
pigmentation but also suggest a mechanism by which tyrosinase peptides
are produced for antigen presentation at the cell surface (see for
example Refs. 6 and 7).
Tyrosinase (monophenol, L-dopa:oxygen oxidoreductase; EC
1.14.18.1) is a copper-containing enzyme that catalyzes the oxidation of tyrosine and DOPA to DOPAquinone, the rate-limiting reactions in
melanin synthesis (8). Because the catalytic activity of tyrosinase is
dependent on DOPA (8-10), we explored the possibility that DOPA also
elicits a conformational change favorable for exit of tyrosinase from
the ER to the Golgi. Here we show that the release of wild type
tyrosinase from the ER was enhanced in melanoma cells by treatment with
the cofactor DOPA and the substrate tyrosine. DOPA/tyrosine promoted
the accumulation of proteolytically resistant and Golgi-processed
tyrosinase, as confirmed by biochemical analyses and immunofluorescence microscopy.
Cell Culture--
Normal human melanocytes were cultured from
newborn foreskins in Ham's F-10 medium supplemented with glutamine (2 mM), penicillin-streptomycin (100 units/ml), and 7% fetal
bovine serum (all from Life Technologies, Inc.), termed basal
medium, that was further enriched with several ingredients
required for normal melanocyte proliferation. They included 85 nM 12-O-tetradecanoylphorbol-13-acetate (TPA),
0.1 mM 3-isobutyl-1-methyl xanthine (IBMX), 2.5 nM cholera toxin, 1 µM
Na3VO4, and 0.1 mM dbcAMP, all from
Sigma (11) designated TICVA. The highly pigmented state of normal
melanocytes was stable regardless of passage number or changes in the
composition of the external growth factors.
Human metastatic melanoma cells YUGEN8, 501 mel, 888 mel, YUSAC2, and
YUSIT1 (all expressing wild type tyrosinase) were maintained in Ham's
F-10 basal medium as described (11). Experiments were performed with
the basal medium and unmodified OptiMEM (Life Technologies, Inc.) plus
2% fetal bovine serum or tyrosine-free minimal essential medium
or RPMI medium. Experimental medium was supplemented with 50 µM freshly prepared DOPA (stock solution 1 mM
in PBS) or tyrosine (Life Technologies, Inc.; at the indicated
concentration) and adjusted to pH 7.4-7.7 prior to use. The
concentration of tyrosine in the unmodified Ham's F-10 and OptiMEM
media was 10 and 200 µM, respectively. To test the
effect of tyrosine elimination, cells were starved in tyrosine-free
medium for 2-4 h prior to any further manipulations. The effect of
external tyrosinase activity was explored by adding cell extract of
normal human melanocytes prepared in a solution containing 0.5% CHAPS,
50 mM HEPES, and 200 mM NaCl, pH 7.5. Tyrosinase activity in the melanocyte cell extract was determined as
described below.
Western Blot Analysis, Precipitation, and Antibodies--
CHAPS
lysis buffer (2% CHAPS in 50 mM HEPES and 200 mM NaCl, pH 7.5) containing protease inhibitors
(CompleteTM protease inhibitor mixture; Roche
Molecular Biochemicals, Indianapolis, IN) was used to lyse cells and to
wash bead-bound precipitated material as described (3). Immunoblotting
analyses of whole cell lysates (40 µg protein/lane, as measured by
the Bio-Rad protein assay reagent), or affinity purified glycoproteins
using wheat germ agglutinin (WGA) bound to beads (lectin from
triticum vulgaris; Sigma;) were performed following standard
procedures or the manufacturer's instructions. Tyrosinase was detected
with mouse monoclonal antibody T311 (12), and equal protein loading was
verified by staining the gels with Coomassie Brilliant Blue after
transfer of the proteins to membranes and by subsequent immunoblotting
with anti-actin antibodies. Densities of tyrosinase bands on the x-ray
films were determined using an NIH image analyzer, selecting all
visible bands, as described in the manual.
Carbohydrate Cleavage--
Cell lysates (200 µg of protein in
~100 µl of CHAPS lysis buffer) were incubated with 50-70-µl
slurry of WGA bound to beads and shaking for 2 h at 8 °C.
Bead-bound proteins were hydrolyzed with endoglycosidase H (Endo H)
according to the manufacturer's instruction (Roche Molecular
Biochemicals). Reactions were stopped with sample buffer at 95 °C
for 5 min, and proteins were subjected to Western blotting as above.
Immunofluorescence Microscopy--
Melanoma cells were grown on
chamber slides in unmodified Ham's F-10 medium at pH 7.6. Cells were
incubated with and without 50 µM DOPA in Ham's F-10
medium for 4 h. Attached cells were washed with PBS, fixed in 4%
formaldehyde/PBS, and permeabilized with 0.1% Triton X-100/PBS. The
permeabilized and fixed cells were then incubated with polyclonal
antibodies against tyrosinase (goat C-19; Santa Cruz Biotechnology
Inc., Santa Cruz, CA) or calnexin (rabbit; StressGen Biotechnologies
Corp., Victoria, BC, Canada) to localize the ER and then with
fluorescein anti-goat conjugates (Santa Cruz Biotechnology Inc.) or
rhodamine anti-rabbit conjugates (Molecular Probes, Eugene, OR), all
diluted in 0.1% bovine serum albumin/PBS. Indirect immunofluorescence
was visualized with an inverted Bio-Rad MRC-600 laser confocal
microscope system. Images were processed with Bio-Rad Confocal
Assistant software.
Trypsin Digestion--
Whole cell lysates (4 µg protein/µl
in 2% CHAPS lysis buffer without protease inhibitors) were digested
with trypsin (TPCK (N-tosyl-L-phenylalanine chloromethyl
ketone)-treated) at the indicated concentration at 27 °C for 10 or
15 min. Digestion was stopped by the addition of an equal volume of 3×
SDS sample buffer and heating for 1 min at 95 °C. Reaction products
(40 µg/lane) were subjected to Western analysis with anti-tyrosinase
mAb followed by immunoblotting with polyclonal antibodies against actin
(Sigma) as a control.
Metabolic Labeling--
Pulse-chase experiments were performed
as described (4). Briefly, cells were pulse-labeled (15 min) with
[35S]Met/Cys (0.7 mCi/ml; EasyTag, PerkinElmer Life
Sciences) in methionine/cysteine-free RPMI medium (200 µM tyrosine; Life Technologies, Inc.) and either
collected immediately or after 4 h of chase incubation with
nonradioactive medium. DOPA (50 µM) was added to half of the samples during the pulse, chase, or both incubation times. Cell
extracts (2-10 × 107 cpm in trichloroacetic
acid-precipitable material) were treated with Zysorbin (fixed and
killed Staphylococcus aureus; Zymed Laboratories Inc., San Francisco, CA) to remove nonspecific binding proteins and then subjected to immunoprecipitation with mouse monoclonal T311
anti-tyrosinase mAb. Following extensive washing with radioimmune precipitation buffer, half of the precipitated products were digested with Endo H overnight followed by SDS polyacrylamide gel
electrophoreses fractionation and autoradiography. Densities of
radioactive tyrosinase bands on the x-ray films were determined using a
Molecular Dynamics PhosphorImager.
Tyrosinase Activity--
Total cell lysates were prepared in 2%
CHAPS buffer plus protease inhibitors as above, and tyrosinase assays
were performed with L-tyrosine-[3,5-3H]
(PerkinElmer Life Sciences) as a substrate (13-15). Briefly, reaction
mixtures (200-µl final volume), contained 50-150 µg of cell
extract protein, 50 µM L-tyrosine, and 50 µM L-DOPA (in triplicates or duplicates, as
indicated) and 1 µCi/assay
L-tyrosine-[3,5-3H]. After 15-120 min (as
indicated) of incubation at 37 °C, reactions were stopped with 200 µl of charcoal slurry, passed through 350-µl packed Dowex columns,
and radioactivity of the eluate in scintillation fluid was measured
with a scintillation counter. One unit of tyrosinase was defined as the
amount of enzyme that catalyzed the oxidation of 1 mmol of tyrosine in
1 min. Standard errors were in the range of 15% of total counts.
DOPA-induced Tyrosinase Maturation--
The amelanotic
YUGEN8 human melanoma cells that originated from a pigmented
metastatic tumor were first tested for the effect of DOPA, because they
displayed unstable pigmentation and variability in the levels of ER-
and Golgi-processed tyrosinase proteins (3). The cells were incubated
in medium supplemented with DOPA for increasing periods of time. Visual
examination of the harvested and sedimented cell pellets revealed
increased pigmentation after 4 h of DOPA treatment (data not shown).
Western blotting with anti-tyrosinase antibodies of melanoma whole cell
lysates, or affinity fractionated glycoproteins digested with Endo H,
confirmed previous observations (3). Under conditions that allowed
tyrosinase to become fully mature in normal human melanocytes (Ref. 3;
see also Fig. 3B, lane 9), the steady-state tyrosinase in the amelanotic YUGEN8 melanoma cells was mainly a
distinct ~70-kDa species sensitive to Endo H digestion, indicative of
an immature ER glycoform (Fig.
1A, lanes 1 and
7, empty arrow). However, within 2 h of DOPA
treatment, mature Endo H-resistant glycoforms accumulated (Fig.
1A, lanes 2 and 8). Most of the enzyme was fully processed and Endo H-resistant after 8 h of treatment (Fig. 1A, lanes 4, 5, 10,
and 11). In addition, DOPA caused an increase in the
abundance of the 70-kDa species, as well as the 6D-kDa forms,
distinctly visible after 2 and 4 h of incubation, respectively (Fig. 1A, lanes 2 and 3,
bands marked with empty arrow and
arrowhead, respectively). Tyrosinase of 60 kDa was
previously identified as the deglucosylated protein that accumulated
after treatment with the proteasome inhibitors MG132 or lactacystin but
not with the lysosomal inhibitors E64
(L-trans-epoxysuccinic acid) and
NH4Cl (3). Therefore, DOPA stabilized the ER
glycoform of tyrosinase, as well as protein that had been diverted from the ER to the cytoplasm for degradation.
DOPA-induced tyrosinase maturation and stability was not restricted to
YUGEN8 melanoma cells but was also observed in other cell lines
originating from pigmented or nonpigmented metastatic tumors (16) (Fig.
1B, compare lane 1 to lane 2, and data
not shown). Quantitative RT polymerase chain reaction analysis of tyrosinase mRNA showed no increase in message levels (Fig.
1B, compare lane 5 to lane 6),
suggesting that the changes occurred post-transcriptionally. Taken
together, the increased abundance of tyrosinase species of 70 and 60 kDa and the accumulation of Endo H-resistant glycoforms indicated that
DOPA induced a conformational change that conferred resistance to
endogenous proteolytic degradation and competency to exit from the ER
to the Golgi.
DOPA Treatment Enhanced Tyrosinase Export from the ER to Distal
Sites--
Subcellular localization by indirect confocal
immunofluorescent microscopy confirmed the biochemical analyses.
Tyrosinase in amelanotic YUGEN8 melanoma cells was displayed in a
perinuclear reticular pattern coincident with the ER marker calnexin
(Fig. 2, panels marked
Tyrosine Is Required for DOPA-enhanced Maturation--
As the
experiments described above were performed in Ham's F-10 or OptiMEM
medium containing tyrosine (10 and 200 µM, respectively), DOPA supplementation may have served to enhance tyrosinase maturation in cooperation with tyrosine. Indeed, the requirement for substrate in
this process was confirmed by exclusion of tyrosine from the medium.
There was no induction of DOPA-mediated tyrosinase maturation in
tyrosine-free minimal essential medium (Fig.
3A, compare lanes 1 and 2), indicating that the substrate and cofactor of
tyrosinase are both critical for inducing export-competent tyrosinase
species.
We then explored the possibility that tyrosine by itself can induce
maturation, because DOPA is generated by an autocatalytic process
during the initial phase of the tyrosinase reaction (8-10). In these
experiments, accumulation of endogenously produced DOPA was favored by
supplementing the medium with excess tyrosine and maintaining high
ratio of melanoma cells to medium (such as ~1.5 × 105 cells/cm2/ml). The results showed that
enriching the medium with additional tyrosine to a final concentration
of 400 µM was sufficient to induce maturation of YUGEN8
tyrosinase within 2 h (Fig. 3A, compare lane
3 to lanes 5 and 6). Accumulation of
tyrosinase reaction products sufficient to stabilize tyrosinase and
induce maturation was dependent on the concentration of extracellular
tyrosine, the level of residual tyrosinase within the melanoma cells,
the density of the cells, the volume of the medium, and the duration of
the incubation (Fig. 3A, lanes 3-6;
B, lanes 1-8; C, lanes 1-3, and data not shown).
The likely enzymatic source of intracellular DOPA in the
tyrosine-supplemented medium is tyrosinase and not the dopaminergic enzyme tyrosine hydroxylase (TH). These melanoma cells did not express
TH mRNA (tested by RT polymerase chain reaction with TH-specific primers; data not shown), and endogenous TH protein was not detected by
Western blotting with anti-TH antibodies (sc-7847; Santa Cruz Biotechnology Inc.; data not shown). Furthermore, ectopic expression of L-DOPA-producing TH rendered constitutively active by
truncation of the N-terminal regulatory domain (termed tHTH; see Ref.
17) did not induce any changes in tyrosinase processing in 501 mel amelanotic melanoma cells (data not shown). In contrast, the effect of
DOPA and tyrosine on maturation was enhanced by exogenous tyrosinase from normal human melanocytes (40 microunits/ml) supplied to the medium
as whole cell extract (Fig. 3C, compare lanes 3 and 4). Altogether, these results showed that aberrant ER
retention of tyrosinase in melanoma cells could be corrected by the
concerted effect of the cofactor DOPA and the substrate tyrosine.
Stabilization of Tyrosinase--
DOPA and tyrosine enhanced the
formation of tyrosinase that was more resistant to endogenous
proteolytic degradation, as seen by accumulation of the 70-kDa early
glycoforms and the apparent deglucosylated 60-kDa species (Fig.
1A, lanes 2 and 3). Therefore, we
further explored structural differences between the 70-kDa and fully
processed protein by probing their resistance to trypsin digestion
using tyrosinase from normal human melanocytes as a control. The
results showed that the Golgi-processed enzyme produced in normal human
melanocytes was resistant to up to 200 µg of trypsin, whereas the
faster migrating 70-kDa ER form was trypsin-sensitive (Fig.
4, lanes 1-8). In melanoma
cells that solely accumulated the 70-kDa protein, TYR was highly
sensitive to proteolytic digestion as it was completely digested by 50 µg of trypsin in 10 min (Fig. 4, lanes 9-14). Incubation
of 501 mel melanoma cells with 400 µM tyrosine for 5 h induced the accumulation of processed tyrosinase protein that was
trypsin-resistant up to 200 µg/ml of trypsin (Fig. 4, lanes
15-20). In contrast, high levels of tyrosine in the medium did
not confer trypsin resistance to actin (data not shown). These results
suggest that the ER-retained tyrosinase is a trypsin-sensitive
conformer, compared with the trypsin-resistant Golgi-processed species
whose production was enhanced by the presence of high concentrations of
tyrosine in the medium.
Pulse-chase metabolic labeling experiments were carried out to
determine the stage at which DOPA stabilized the ER-retained tyrosinase. As shown in Fig. 5, newly
synthesized tyrosinase remained an ER-retained and Endo H-sensitive
conformer in the absence of DOPA (Fig. 5, lanes 1,
2, 5, and 6). The addition of DOPA
during the 4-h chase period was sufficient to stabilize the protein
into an exit-competent conformation and induced maturation into an Endo-H resistant form (Fig. 5, lanes 7 and 8).
Including DOPA during the pulse and chase periods further enhanced its
stability and maturation (Fig. 5, lanes 9 and
10). These results indicated that DOPA stabilization of
tyrosinase occurred both co- and post-translationally.
Low Tyrosinase Activity as the Cause for DOPA Depletion--
The
data suggested that DOPA is in low abundance in melanoma cells probably
because of inactive tyrosinase. Because normal melanocytes always
possessed Golgi-processed tyrosinase even in the low tyrosine Ham's
F-10 medium, we tested whether the mitogenic ingredients (TICVA) added
to maintain growth and known to stimulate intracellular levels of cAMP
(cholera toxin and IBMX), or protein kinase C (the phorbol ester TPA),
can also promote maturation. However, no changes in tyrosinase
processing or activity were observed in the melanoma cells in response
to TPA, IBMX, or TICVA (data not shown). Conversely, normal melanocytes
expressed normal tyrosinase glycoforms and remained highly pigmented
when grown in the presence of peptide growth factors, such as basic
fibroblast growth factor, hepatocyte growth factor, and
endothelin (without TICVA) (18, 19).
Therefore, we tested whether the ER form of tyrosinase from melanoma
cells can be activated by DOPA and tyrosine after cell lysis.
Tyrosinase activity in whole cell lysates from normal melanocytes that
represented largely the mature form and from YUGEN8 and 501 mel
melanoma cells expressing low levels of proteins mostly of the immature
Endo H-sensitive forms was measured in the presence of DOPA (Fig.
6A; see also Ref. 3). Compared
with normal melanocytes, extracts from melanoma cells possessed low
levels but nevertheless measurable tyrosinase activity (Fig.
6B). This activity is likely a result of the post-lysis
activation of the immature protein, because standardizing the activity
to tyrosinase band density displayed by the Western blot (Fig.
6A, numbers indicated on the bottom)
eliminated the differences between the three samples (Fig. 6C). Thus, DOPA in the in vitro assay clearly
activated not only fully matured but also immature tyrosinase
species.
Malignant transformation of normal melanocytes to melanoma cells
is associated with down-regulation of pigmentation. In previous studies, we showed that tumor-specific retention of tyrosinase in the
ER is the cause of the amelanotic phenotype in tyrosinase-positive melanoma cells (3). Thus, wild type tyrosinase in these cells displays
a phenotype similar to inactive mutant tyrosinase proteins common in
oculocutaneous albinism type 1 (4, 5). In this report, we provide
evidence that DOPA and tyrosine, the cofactor and substrates of
tyrosinase, are required for wild type tyrosinase maturation in
melanoma cells. DOPA and tyrosine stabilize the ER-retained protein and
permit its transport through the secretory pathway in these amelanotic
melanoma cells.
The two lectin ER molecular chaperones, calnexin and calreticulin,
appear to be responsible for the ER retention of incompletely or
improperly folded tyrosinase (3, 4, 20, 21). These chaperones bind and
retain proteins possessing monoglucosylated glycans (22, 23). The
retention of a protein is the result of continued reglucosylation of
the protein by the UDP-glucose:glycoprotein glucosyltransferase, an ER
sensor that reglucosylates glycoproteins containing misfolded or mobile
regions (24). Therefore, DOPA/tyrosine binding must stabilize
tyrosinase so that it is no longer a substrate for reglucosylation and
is therefore subsequently released from the ER.
DOPA/tyrosine can be added to the growing list of chemical chaperones
that stabilize target proteins, including otherwise inactive mutants.
For example, drug substrates or modulators allowed mutant forms of the
human ATP-dependent drug pump, P-glycoprotein, to attain an
ER export-competent configuration (25, 26). In this case,
core-glycosylated, trypsin-sensitive, and ER-retained mutants of human
P-glycoprotein were converted to the mature trypsin-resistant form by
synthesis in the presence of a drug substrate. Likewise, nonpeptidic
antagonists increased cell surface expression and rescued the function
of mutant V2 vassopressin receptor (27). In another case, small
synthetic molecules promoted the stability of wild type p53 and allowed
mutant p53 to maintain an active conformation (28). Although the
ER-retention defect of mouse albino mutant tyrosinase C85S (4, 5) could
not be corrected by incubating the melanocytes with DOPA/tyrosine (data
not shown), it is possible that other less severe albino mutations,
such as those causing temperature-sensitivity, may be rescued by
DOPA/tyrosine treatment.
For over 70 years it has been known that DOPA activates tyrosinase (8,
29). However, its mechanism of activation has been elucidated only
recently by Riley and colleagues (9, 30, 31). These investigators
demonstrated that, unlike tyrosine, DOPA (and other catechols) can be
oxidized to the corresponding quinone by tyrosinase with oxidized
copper atoms (without bound dioxygen), thus reducing the copper
atoms in the active site and enabling the generation of the active,
oxygen-bound form. It is tempting to speculate that only the
oxygen-bound active enzyme is competent to exit the ER, and conditions
that block its formation, such as mutations common in albinism or
tumor-induced metabolic changes, can result in ER retention and
subsequent degradation.
Promotion of tyrosinase folding required the specific interaction with
DOPA. Glycerol, a chemical chaperone shown to stabilize immature,
core-glycosylated, mutant cystic fibrosis transmembrane conductance
regulator molecules that are normally degraded rapidly (32), was
not effective in inducing tyrosinase
maturation.2 The likely
cellular source of DOPA is tyrosinase. Incubation of melanoma cells in
high levels of tyrosine, in the absence of supplemented DOPA, also
caused release from the ER to the Golgi. We suggest that autocatalytic
activation by tyrosine and subsequent build up of DOPA induced proper
folding of ER tyrosinase glycoforms. Taken together, the data support a
scenario in which the generation of exit-competent early glycoforms is
dependent on autocatalytic activation of tyrosinase within the ER.
Because autocatalytic activation is a slow process (8-10), these
observations may explain the relative long delay in tyrosinase release
from the ER as compared with the homologous melanogenic glycoprotein
TRP1 (3, 33). Thus, tyrosinase may exert a self-regulatory mechanism
ensuring continuous trafficking of properly folded protein only under
conditions favorable for activity and melanin production.
If this is indeed the mechanism, then albino tyrosinase mutations that
produce inactive protein should be retained in the ER, and copper
binding to tyrosinase must first occur in the ER. Thus far, three loss
of function TYR albino mutants have been found to accumulate in the ER
(4, 5), and a copper transporter has been localized to the ER membrane
(34). Furthermore, it is possible that copper loading is required
across the vesicular network, as the Menkes copper transporter that has
been localized to the trans-Golgi (35) is required for
tyrosinase enzymatic activity in fibroblasts (36). Further studies will
be needed to characterize the cellular localization of other albino TYR mutations and to identify the proteins responsible for copper loading
and their distribution within the secretory pathway.
This activation hypothesis suggests that inactivation of tyrosinase
enzymatic activity by tumor-induced changes in the microenvironment is
the likely cause for ER retention. It is possible that the culprit is
altered metabolic processes, such as high rates of glucose uptake and
glycolysis commonly displayed in tumors (37). Cancer cells are able to
overproduce lactic acid aerobically probably because of activation of
lactate dehydrogenase-A (38). Accumulation of such metabolic acids can
cause DOPA protonation and tyrosinase inactivation (9). The appearance
of amelanotic clones in advanced primary and metastatic pigmented
tumors suggests that down-regulation of tyrosinase is a frequent
consequence of these metabolic changes in vivo, which is
also reflected in the cultured cells.
Our data shed light on several observations published in the past two
decades using melanoma cells from other species. Down-regulation of
tyrosinase activity and conversion from highly melanotic to amelanotic
cells was observed in Cloudman S91 melanoma cells grown in
phenylalanine-supplemented, tyrosine-free medium (14, 39). Likewise,
the supply of extracellular tyrosine and DOPA was critical for
tyrosinase in Bomirski Ab amelanotic hamster melanoma cells. Tyrosine
and DOPA increased the intracellular levels of tyrosinase activity and
immunoreactive protein (40) and may have also affected the synthesis of
additional components in melanogenesis (41). The combined experience
with Bomirski Ab amelanotic hamster melanoma cells enticed Slominski
and Paus (42) to suggest that tyrosine and DOPA act as hormone-like
bioregulators in mammals.
The level of tyrosinase protein within melanoma cells is consequential
not only for melanin production but also for melanoma cell immunity.
Tyrosinase peptides are recognized by class I-restricted, melanoma-specific tumor-infiltrating lymphocytes (see for example Refs.
6 and 43). Large resources are dedicated to boosting the immune
reaction of patients to this tumor with tumor-infiltrating lymphocyte
vaccines (44). Continuous presentation of the relevant epitope
integrated into the vaccine is critical for a robust immune response.
As production of tyrosinase peptides is dependent on the level of
tyrosinase protein and its proteasomal degradation products, it is
conceivable that dopaminergic agents can be used as a means to
synchronize high production of tyrosinase peptides to optimize the
response of melanoma patients to tumor-infiltrating lymphocytes or
dendritic cell vaccines.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
DOPA-induced Golgi processing and increased
tyrosinase levels. A, YUGEN8 melanoma cells were
incubated for increasing duration in unmodified OptiMEM medium (5 ml of
200 µM tyrosine), in the presence or absence of 50 µM DOPA. The pH value was maintained at 7.4. Western
blotting was performed with whole cell lysates (WCL) or with
WGA precipitated proteins that were incubated overnight with or without
Endo H. The open and closed arrows designate the
70-kDa ER form and the 60-kDa deglycosylated form of tyrosinase,
respectively. B, tyrosinase processing in 501 mel cells in
response to DOPA. Cells were incubated with 50 µM DOPA in
OptiMEM medium and harvested for Western blot analysis with
anti-tyrosinase mAb followed by blotting with anti-actin (lanes
1 and 2). Tyrosinase is marked with a
bracket. Alternatively, the same batch of treated cells were
harvested and processed for mRNA analysis using quantitative RT
polymerase chain reaction and primers for human actin (lanes
3 and 4) or human TYR (lanes 5 and 6). M,
size markers 1 and 0.5 kb.
DOPA, compare TYR and CNX to
Merge). In contrast, after 4 h of incubation with DOPA,
tyrosinase was distributed outside of the ER and Golgi, as observed by
the abundant appearance of spots at distal sites that did not merge
with calnexin staining (Fig. 2, panels marked
+DOPA, compare TYR and CNX to
Merge). Therefore, DOPA treatment permitted tyrosinase to
become transport-competent.
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Fig. 2.
DOPA induces ER export of tyrosinase to
distal sites. Immunofluorescence confocal microscopy of
YUGEN8 melanoma cells grown in Ham's F-10 medium before and after
treatment with 50 µM DOPA for 4 h is shown. The
left panels stained with green fluorescein
represent TYR detected with polyclonal anti-tyrosinase antibodies. The
middle panels stained with red rhodamine show the
ER resident calnexin (CNX), and the right panels
display merged images. Tyrosinase colocalization with calnexin is
indicated in yellow in the merge. The bars
represent 1 µm.
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Fig. 3.
Tyrosine is required to induce maturation of
tyrosinase. Western blots with anti-tyrosinase mAb T311 of whole
cell lysates derived from melanoma cells seeded at high densities
(~1 × 106 cells) in 25-cm2 flasks and
incubated for short periods in medium manipulated to affect endogenous
tyrosinase activity, compared with normal melanocytes, are shown.
A, YUGEN8 melanoma cells were deprived of tyrosine for
4 h. Cultures were incubated with fresh tyrosine-free medium
without or with DOPA for an additional 4 h (lanes 1 and
2, respectively). Alternatively, cultures of YUGEN8 melanoma
cells were incubated in 2.5 ml of unmodified OptiMEM medium (200 µM tyrosine; lane 3) or medium that was
supplemented to 400 µM tyrosine final concentration and
harvested at different time intervals (lanes 3-6).
B, melanoma cells were incubated in 3 ml of unmodified (200 µM tyrosine; lanes 1, 3,
5, and 7) or modified (400 µM
tyrosine; lanes 2, 4, 6, and
8) OptiMEM medium for 16 h. Tyrosinase from normal
human melanocytes (NM) grown in Ham's F-10 medium (10 µM tyrosine) is shown in lane 9. SIT,
YUSIT1; SAC, YUSAC2; L, 200 µM tyrosine; H, 400 µM tyrosine. C, maturation of tyrosinase is
manipulated by the concentrations of accumulated extracellular
tyrosinase reaction products. YUGEN8 melanoma cells were incubated
overnight in the presence of DOPA (20 µM) in 5 ml of
unmodified Ham's F-10 medium (lane 1), 2.5 or 10 ml of
unmodified OptiMEM medium (lanes 2 and 3,
respectively), or 10 ml of OptiMEM medium supplemented with cell
extract (60 µg/ml) from normal human melanocytes containing 40 microunits/ml TYR activity (lane 4). The membrane was probed
consecutively first with anti-tyrosinase
( -TYR) and then with anti-actin polyclonal
rabbit antibodies (
-actin).
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Fig. 4.
Melanoma ER-70-kDa tyrosinase is sensitive to
trypsin digestion. Tyrosinase trypsin digests of cell extracts
from normal melanocytes and 501 mel cells grown continuously in
unmodified Ham's F-10 medium (10 µM tyrosine;
lanes 1-14) and 501 mel cells incubated in modified OptiMEM
medium (400 µM tyrosine) for 5 h (lanes
15-20) are shown. Trypsin digestion was for 10 (lanes
1-4 and 9-20) or 15 min (lanes 5-8).
Digestion products were subjected to Western blotting with T311
tyrosinase mAb.
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Fig. 5.
DOPA-induced proper folding of newly
synthesized and pre-existing tyrosinase early glycoforms. Melanoma
cells YUGEN8 were pulsed with [35S]Met/Cys in
RPMI medium for 15 min in the absence or presence of 50 µM DOPA and harvested immediately or after a 4-h chase in
nonradioactive RPMI media in the absence or presence of 50 µM DOPA. The RPMI medium contained 200 µM
tyrosine. After tyrosinase precipitation with anti-tyrosinase
antibodies, half of the bound immunoprecipitates were subjected to Endo
H digestion.
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Fig. 6.
Tyrosinase activity in normal
versus malignant melanocytes. A,
immunoblot (T311 mAb) of whole cell lysates showing tyrosinase species
present at the time the extracts from normal melanocytes
(NM), YUGEN8 (GEN), and 501 mel (501)
melanoma cells were used to measure DOPA-catalyzed tyrosinase activity.
The number under each lane represents arbitrary units of
band density of all bands within each lane assessed with an
NIH image analyzer. B, tyrosinase activity. A histogram
showing tyrosinase activity measured in the presence of DOPA expressed
as microunits/mg total protein is shown. Data are averages of
triplicate values. Each tyrosinase assay was repeated at least once
with similar results. C, histogram showing tyrosinase
activity of each cell type (B) normalized to the respective
band density displayed in A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Lloyd Old (Memorial Sloan- Kettering Cancer Center, New York, NY) for anti-tyrosinase mAb, Karen O'Malley (Washington University Medical School, Saint Louis, MO) for the tHTH expressing plasmid, and Patrick A. Riley (University College London, London, United Kingdom) for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service Grants AR39848 and CA44542 (to R. H.) and AR41942 (to R. E. Tigelaar, Program Investigator, Yale Skin Diseases Research Center), grants from The Medical Foundation and Edward Mallinckrodt, Jr. Foundation, and USPHS Grant CA79864 (to D. N. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Dermatology, Yale University School of Medicine, P. O. Box 208059, 15 York St., HRT 610, New Haven, CT 06520-8059. Tel.: 203-785-4352; Fax: 203-785-7234; E-mail: ruth.halaban@yale.edu.
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M008703200
2 Unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; TYR, tyrosinase; WGA, wheat germ agglutinin; TPA, 12-O-tetradecanoylphorbol-13-acetate; IBMX, 3-isobutyl-1-methyl xanthine; PBS, phosphatebuffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; Endo H, endoglycosidase H; TH, tyrosine hydroxylase; mAb, monoclonal antibody.
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