[CANÅ’R Ri:SliARC1155.4(l41-4tHh. Scplcmbcr I?. IW]
Comparison of the Responses of Human Melanocytes with Different Melanin
Contents to Ultraviolet B Irradiation1
Diane Barker, Kathleen Dixon, Estela E. Medrano,2 Douglas Smalara, Sungbin Im, David Mitchell, George Babcock,
and Zalfa A. Abdel-Malek3
Departments of Dermatology ¡D.B, K. E. M,. O.S., S. !.. Z.A.AJ
ami Environmental Health [K.D.j. University of Cincinnali, Cincinnati, Ohio 45267-0592;
Carcinogenesis. The Universii\ of Texas M. 1). Anderson Cancer Center, Houston, TX ¡D,M. I; and Shrine r,\ Burns institute, Cincinnati, Ohio ¡G.H.¡
Department
of
include the generation of reactive oxygen intermediates, which cause
single-strand breaks and base damage in DNA (9). The frequency of
ABSTRACT
Melanin is thought to serve in photoprotection. To investigate this, we
have compared the responses of cultured human melanocytes derived
from different pigmentary phenotypes (skin types I-VI) to a single irra
diation with different doses of I A li light, ranging between 11.7 and 70.1
m.l/rm'. After I \ I! irradiation, heavily pigmented melanocytes had the
same percent survival but a greater capacity to resume proliferation than
their lightly pigmented counterparts. A significant increase in melanin
content was observed in heavily pigmented but not in lightly pigmented
melanocytes. Irradiation with 1X1! light blocked melanocytes, regardless
of their melanin content, in (!,, and induced the expression of the tumor
suppressor p53 protein within 4 h. This induction steadily increased up to
48 h in lightly pigmented melanocytes; however, in heavily pigmented
melanocytes, p53 level peaked at 24 h after DVB treatment and declined
thereafter. Additionally, DNA from lightly pigmented melanocytes con
tained significantly higher numbers of cyclobutane pyrimidine dimers
than did DNA from heavily pigmented melanocytes after irradiation with
increasing doses of INI! light. We speculate that the prolonged induction
of p53 in lightly pigmented melanocytes arrests them in ( ;, for a long time
period in order to repair extensive DNA damage. The above described
differences might partially explain the increased susceptibility of individ
uals with lightly pigmented skin compared to individuals with dark skin to
the photodamaging and photocarcinogenic effects of sun exposure.
INTRODUCTION
Excessive exposure to the sun is implicated in the etiology of skin
cancers (1-3). The UV light that reaches the surface of the earth is
mostly in the form of UVA (320-400 nm). Some is in the form of
UVB (290-320 nm). It is this latter spectrum of solar radiation that is
most active in inducing genotoxic and photocarcinogenic effects (3,
4). The depletion of the ozone layer, which mainly filters out UVB
and UVC rays, has become a major public health concern because it
is projected to increase the penetration of UVB rays through the
atmosphere of the earth, resulting in higher incidence of skin cancers,
including malignant melanoma, the most lethal (5). Because of these
concerns, we chose to investigate the effects of UVB light on, and to
elucidate the cellular and molecular changes induced in, human epi
dermal melanocytes.
The mechanisms by which UV light induces DNA damage and
mutations in humans is not well understood. Irradiation of mammalian
cells in culture with UV light results in inhibition of DNA replication
primarily due to the formation of cyclobutane pyrimidine dimers and
pyrimidine (6-4) pyrimidone photoproducts (6-8). Ultraviolet light
can also be lethal to mammalian cells by other mechanisms that
Received 3/15/45; accepted 7/IW5.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked titiverlisemenl in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by a grant from the Ohio Cancer Research Associates; National
Institute of Environmental Health Sciences Grants l ROI ES06882-OIA1 (Z.A.M.) and
5P3II ESOMW6-02: Shiseido Co.. Ltd. (Z.A.M.); and NIH National Institute on Aging
Grant AG(KI5°4-()2(E.E.M.).
2 Present address: Baylor College of Medicine. Huffington Center on Aging. One
Baylor Plaza. Houston, TX.
'To whom requests for reprints should be addressed, at Department of Immunology,
mutations induced by UV light seems to correlate with the amount of
DNA damage remaining at the time of DNA replication (10, II).
Evidence for this is provided by studies on cells from patients with the
genetic disease xcroderma pigmentosum; these cells are deficient in
repairing the UV induced DNA damage (12). Seventy % of patients
with this disease develop skin tumors at a median age of 8 years (13,
14). Further evidence is provided by studies using in vitro DNA
replication systems derived from human HeLa, monkey CV-1 cells,
and a shuttle plasmid, which concluded that removal of pyrimidine
dimers reverses the inhibition of DNA replication and diminishes the
mutation frequency (11).
In humans, one obvious effect of UV light is increased cutaneous
pigmentation. Melanin is the pigment that gives humans and other
mammals their distinctive skin or coat color. Melanin synthesis by
mammalian melanocytes is genetically controlled by at least three
genes that code for the regulatory enzymes tyrosinase. the rate limit
ing enzyme in the melanogenic pathway, and the two TRPs,4 TRP-1
and TRP-2 (15-18). In humans, there is a direct correlation between
constitutive skin pigmentation and the amount of the above three
melanogenic proteins expressed in melanocytes (19). A direct rela
tionship exists between the constitutive skin pigmentation and the
ability of individuals to synthesize melanin in response to UV light
(20). The ability of melanin to play a protective role is supported by
epidemiological and experimental evidence suggesting that individu
als with dark skin are less susceptible to the damaging effects of UV
light and have a lower incidence of skin cancers than do individuals
with fair skin (3, 21-24). It has been proposed by many that the main
function of melanin is to protect against the damaging effects of UV
radiation by quenching oxygen radicals and acting as a sunscreen
(25-27). It has been shown that more UV rays are transmitted through
the epidermis, and about five times more UV light reaches the upper
dermis, of Caucasian than black skin (26, 28, 29). However, the
question of whether melanin reduces the formation of photoproducts
in epidermal cells still needed to be further addressed.
It is now established that normal human melanocytes in culture
respond to UV light with increased melanin formation. Friedmann and
Gilchrest (30) were the first to report on the inhibition of proliferation
and increased melanogenesis of cultured human melanocytes after UV
irradiation. We have recently reported that human melanocytes re
spond to multiple irradiations with UVB light with increased mela
nogenesis and growth inhibition (31). It was shown that melanotic
melanocytes have a greater percent survival than do melanocytes with
low melanin contents after irradiation with UVA light (32). Highly
melanotic malignant melanoma cells were also found to be more
resistant than were amelanotic human melanoma cells to killing by
solar or UVB light (33). Moreover, the highly melanotic melanoma
cells are more resistant to the killing effect of 254 nm wavelength UV
light and contain less cyclobutane pyrimidine dimers and pyrimidine
1The abbreviations
University of Cincinnati. P.O. Box o705"2, Cincinnati. OH 452o7-()5u2.
used are: TRP, tyrosinase-related
protein. BrdUrd. bromode-
oxyuridine: PI. propidium iodide.
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RESPONSES OF HUMAN
MELANOCYTES
(6-4) pyrimidone photoproducls than do their less melanotic counter
parts (34). Although these results clearly demonstrate the responsive
ness of normal and malignant human melanocytes to UV light, com
parison of the responses of normal melanocytes with different
pigmentary phenotypes to treatment with UVB light has not been
documented.
It is known that UV irradiation results in the induction of several
genes that are involved in the regulation of cellular proliferation. One
of these genes is the tumor suppressor p53, which acts as a cell cycle
checkpoint, causing cells to be arrested in G, (35-38). This arrest is
thought to be essential to allow cells to repair the UV induced DNA
damage before resuming proliferation.
To elucidate the role of melanin in dictating the responses of
different pigmentary phenotypes to sun exposure, we have compared
the responses of several lightly pigmented and heavily pigmented
human mclanocyte strains to irradiation with increasing doses of UVB
light. Cell survival and resumption of proliferation, induction of p53
expression, melanin synthesis, and formation of cyclobutane pyrimidine dimers were compared among the above types of melanocytes
after a single treatment with UVB light. This study represents an
attempt to define some of the molecular mechanisms that account for
the differences in the responses of individuals with different skin
types to sun exposure, and ultimately in their susceptibility to develop
skin cancers.
MATERIALS
TO UV IRRADIATION
by Lee el al. (40). Melanocytes were harvested, rinsed with PBS, and counted.
Melanin was solubilized in 0.2 N NaOH (10'' cells/ml) and measured spectrophotometrically at an absorbance of 475 nm against a standard curve of known
concentrations of synthetic melanin (Sigma). Melanin content was expressed
as fig/10" cells.
Tyrosinase Activity. Tyrosinase activity was measured in .V//Husing a
modification of the charcoal absorption method of Pomerantz (41). On day 6
after plating, melanocytes received |'H]tyrosine (specific activity, 52 /xCi/
mmol; Amersham, Arlington Heights, IL) at a concentration of 0.5 /iCi/ml of
medium for 24 h. The medium was then collected, and the tyrosine hydroxylase activity of tyrosinase was measured by quantilating the amount of 'H,O
released to the culture medium as tyrosine is converted to i.-dihydroxyphenylalanine as described previously (42).
Cell Cycle Analysis. Melanocytes were plated in 100-mm dishes at a
density of 5 X 10s cells. Three days later, cells were irradiated with 0, 23.4,
35.0, or 46.7 mJ/cm2 of UVB light. Control untreated and UVB-irradiated
monolayer cultures were incubated 14 h after UVB irradiation with IO"5 M
BrdUrd for 60 min at 37°C.At the end of the incubation period, cells were
harvested, fixed, denatured, and stained with FITC-conjugated mouse mAb
against BrdUrd (20 fil/10" cells anti-BrdUrd-FITC; Becton Dickinson, San
Jose, CA) for l h at room temperature, as described in detail previously (31).
The cells were then washed twice, resuspended in PBS. and treated with 5
fig/ml propidium iodide (PI) and 1 mg/ml RNase A for 1 hour at room
temperature.
A negative control group consisting of cells that were not treated with
BrdUrd was included for both the control and the UVB-irradiated groups and
stained as above. Cells were analyzed by flow cytometry using an EPICS 753
flow cytometer (Coulter Cytometry. Coulter Corp., Hialcah, FL), as described
previously (43). FITC and PI were excited with the 488-nm line of an argon ion
AND METHODS
( nil m ini; of Human Melanocytes. Normal human mclanocyte cell strains
were established from individual neonatal foreskins (skin types I-VI) obtained
from the nursery of University Hospital after routine circumcision, as de
scribed previously (19). One neonatal tyrosinase-negative albino cell strain
was a generous gift from Dr. Raymond Boissy (Department of Dermatology,
University of Cincinnati). Melanocytes were maintained in a growth medium
consisting of MCDB 153 supplemented with 5% PCS, 1% penicillin-strepto
mycin, I ng/ml human transferrin, 5 fig/ml insulin. 1 /¿g/mla-tocopherol, and
the melanocyte-specific mitogens, 0.6 ng/ml human recombinant basic fibroblast growth factor, 8 nM 12-0-tetradecanoylphorbol-l3-acetate,
and 13 ng/ml
bovine pituitary extract (Clonetics) as described previously (39). All of the
above reagents were purchased from Sigma Chemical Co. (St. Louis. MO)
unless otherwise stated.
Irradiation of Melanocytes With Different Doses of UVB Light. The
UV source that we have used consists of a bank of FS-20 fluorescent UV
lamps manufactured by Weslinghouse, and has a continuous emission spec
trum wilh a peak at 313 nm. There is negligible emission in the UVC range,
with 75% of the emission in the UVB and 25% of the emission in the UVA
range of the spectrum. The source is equipped with a filter consisting of
calcium, zinc, and thallium phosphate phosphor in a special glass envelope.
Fluencc was measured wilh spcctro-line DM-2n5N. DM-300N. and DM-360N
meters.
To compare the responses of melanocytes with different melanin contents to
UVB light, we conducted dose-response experiments, whereby lightly pig
mented. darkly pigmented, and tyrosinase-negative albino melanocytes were
irradiated once with the following doses of UVB light: 0, 11.7, 23.4. 35.0. or
46.7 mJ/cm:. Melanocytes were plated at a density of 0.8 X IO5 cells/60-mm
dish. On day 3 after plating, the cultures were rinsed with sterile PBS, then
bathed in 1.5 ml PBS and irradiated. Immediately after irradiation. PBS was
replaced with fresh culture medium. Cells received fresh medium every other
day, and were counted 2, 4, and 6 days after irradiation (on days 5. 7, and 9
after plating), using a Coulter counter. Every experimental group, representing
each dose and a single time point, consisted of triplicate dishes. The data were
statistically analyzed using ANOVA that took into account skin type, time after
irradiation, and UVB dose. The viability of attached cells and cells floating in
the medium was determined 48 h after irradiation by calculating the percent of
viable cells that excluded Irypan blue dye.
Melanin Content. Constitutive and UVB-induced melanin contents were
compared on day 7 after plating. Melanin content was determined as described
laser. Fluorescence emission for FITC and PI was detected selectively by
collecting 530 ±15 and 575 ±12.5 nm, respectively. Electronic gates were
placed around the cells of interest using forward angle and 90°light scattering.
When analyzing PI. gates were also set on the peak versus integral PI signals
to eliminate doublets. The PI signals were analyzed using Multicycle (Phoenix
Software, San Diego, CA), and the bivariant histograms were analyzed using
Coulter XL software (Coulter Corp.).
Kinetics of Induction of p53 Protein. Melanocytes were seeded at a
density of 5-7 X IO5 cells/100-mm dish. Four days after plating, cells were
irradiated with 0 or 35.0 mJ/cnr, and 4, 8, 24, 32, or 48 h later were lysed in
RIPA buffer containing the phosphatase and protease inhibitors: 10 mM
Na,VO4, 200 mM phenylmethylsulfonyl fluoride, 10 /J.g/ml aprotinin. and 10
Hg/ml leupeptin (44).
Protein content per sample was determined using the Bio-Rad DC protein
assay. Equal amounts of protein (80 /xg/lane) were loaded on 7.5% SDS-PAGE
gels. After electrophoresis, the gels were transblotted onto nitrocellulose
membranes, which were blocked with 10% nonfat dry milk in 0.2% Twcen 20
in PBS. then incubated with the p53 mouse mAb Ab-6 (Oncogene Science,
Cambridge, MA), at a dilution of 1:100 overnight at 4°C.The membranes were
blocked again as described above and incubated with horseradish peroxidase
anti-mouse IgG (Amersham, Arlington Heights, IL) at a dilution of 1:15.(HH)
for l h at room temperature. The immunoreactivity was detected using the
enhanced chemiluminescence reaction (Amersham). followed by exposure of
the membranes to Kodak AR 5 X-ray films.
Formation of Cyclobutane Pyrimidine Dimers in Genomic DNA. Mela
nocytes from a lightly or a heavily pigmented melanocyte strain were plated at
a density of 7.5 x IO5 cells/100-mm dish (triplicate dishes/group). Three days
after plating, melanocytes were irradiated with 0, 23.4, 35.0, 46.7, or 70.1
mj/cm2 UVB lighl. The melanocytes were harvested immediately after irra
diation, and the DNA was extracted as follows: cell pellets were lysed in
3—4-mlsolution of 0.3 mg/ml proteinase K and 0.3% SDS overnight at 37°C.
Genomic
isoamyl
acetate
volume
ugation
DNA was then extracted
with an equal volume of chloroform-
alcohol (24:1, v/v). The extract was treated with 0.5 M ammonium
(2/5 of total volume), and the DNA was precipitated by adding 2.5
of absolute ethanol at -20°C overnight. This was followed by centrifat 10,000 rpm at 4°Cfor IO min. The DNA was finally resuspended in
1-tnl 10X TE buffer.
To quantitate the number of cyclobutane pyrimidine dimers induced in
lightly or heavily pigmented melanocytes, we used a RIA that has been
described previously (7, 45). The antiscrum was raised in rabbits against
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RESPONSES
heat-denatured
DNA, dissolved
in a triplet sensitizer
OF HUMAN MELANOCYTES
(acetophenone).
and
irradiated with high fluences of UVB light. These conditions produce a high
yield of cyclobutane dimers and a low yield of (6-4) photoproducts. The RIA
is a competitive inhibition assay in which approximately 2 fig of heatdenatured sample DNA are incubated with 10 pg of DNA (labeled to 2.5 X IO8
cpm/pig by nick translation with ['"PjdTTP) in a total volume of 1 ml 10 mw
TO UV IRRADIATION
Table 1 Constitutive ¡yrosinase activities and melanin contents of the cell strain.* used
in the UVB dose response experiments"
activityDPM/106cells
Group
A123Group
4757I57.WX)±
±
1426308,341
±64805,023
Tris (pH 7.8)-150 mM NaCI with 0.15% gelatin. Purified anti-cyclobutane
dimer IgG was added to the reaction mixture at a concentration that yields
30-60% binding to labeled ligand. After incubation overnight at 4°C,the
immune complex was precipitated with goal antirabbit immunoglobulin and
carrier serum from nonimmunized rabbits (Calbiochem). After centrifugation,
the pellet was dissolved in tissue solubilizer, mixed with acid Scintiverse, and
the *2P was quantified by liquid scintillation spectrometry. Under these con
ditions, antibody binding to an unlabeled competitor resulted in reduced
binding to radiolabeled ligand (i.e., inhibition). The percent inhibition of each
sample was extrapolated through a standard (dose-response) curve to deter
mine the cyclobutane dimer concentrations. Salmon sperm DNA irradiated at
a dose rate that yielded 2.45 cyclobutane dimers/10" daltons/J/sec was used as
a standard (46).
RESULTS
Constitutive Melanin Content of the Melanocyte Strains Used
in the UVB Dose-Response Experiments. To compare the dosedependent responses of melanocytes with different melanin contents
to UVB irradiation, we tested the responses of 3 darkly pigmented
(skin types IV-VI) and 4 lightly pigmented (skin types I-II) cell
strains. To verify that these two types of melanocyte strains differ
significantly in their constitutive pigmentation, we measured basal
tyrosinase activity and melanin content in each. Our results clearly
demonstrate that the heavily melanotic melanocytes from each of the
3 cell strains used expressed a higher tyrosinase activity and melanin
content than did any of the lightly pigmented cell strains included in
these experiments (Table 1).
Dose-dependent Effects of UVB Light on Cell Survival and
Proliferation. Melanocyte cultures established from different skin
types responded to a single irradiation with UVB light with a dosedependent decrease in proliferation and cell survival. These findings
contentHg/10"
cells23.519.637.54.25.35.07.9
SEM%,540
±
B1234Tvrosinasc
57420, ±
±66519,547
182
52824,482
±
±189Melanin
" Groups A and B refer to darkly pigmcnled and lightly pigmented cell strains, re
spectively. Melanocytes were plated at a density of 8 X 10' cells/60-mm dish and received
medium containing 0.5 /iCi/ml ¡~
Hjtyrosine on day 6 after plating. Twenty-four h later, the
conditioned medium was collected and assayed for tyrosinase activity, and the cell number in
each dish was determined using a Coulter counter. Mclanocytes from each group (triplicate
dishes/group) were collected and assayed for melanin content, as described in "Materials and
Methods."
are consistent with those of Friedman and Gilchrest (30) but differ
from the results of Libow et al. (47), which showed stimulation of
melanocyte proliferation by UV light. We observed that with increas
ing dose of UVB light there was an increase in the number of detached
cells. By trypan blue staining we determined that the floating cells
were dead, whereas those that remained attached to the plates were
viable (data not shown).
After irradiation with the high dose of 35 mJ/cm~ UVB light, we
observed that the heavily pigmented melanocytes resumed prolifera
tion, whereas the less melanotic melanocytes failed to proliferate even
8 days after irradiation (Fig. 1, a and b). This difference in the ability
to resume proliferation after UVB irradiation was not due to differ
ences in the proliferative rates of the heavily and lightly pigmented
melanocytes because both cell strains tested had comparable replica
tion rates (evident as similar control growth curves). The albino
melanocytes had the lowest survival and recovery rates at the highest
two doses of UVB light (Fig. \c). The percent of survival, determined
Ill
in
Fig. 1. Comparison of the responses of a heavily
pigmented («),lightly pigmented (h), and tyrosinase-negative albino melanocyte (<•)
strain to in
creasing doses of UV light. Melanocyte strains (/
and h had similar proliferative rates. Melanocytes
were plated onto 60-mm dishes at a density of
0.8 X IO5 cells (day 0) and irradiated on day 3 after
|
234567
plating with the following doses of UVB light:
control (O). 11.7 mJ/crrr (D), 23.4 mJ/cnr (A),
35.0 mJ/cm- (V), and 46.7 ml/cm2 (O). Cell num
bers were determined tin days 3. 5, 7, and *) after
plating. The decrease in cell numbers on day 5
represents cell killing, and the cell numbers on days
7 and 9 were used to determine recovery from
growth arrest and resumption of proliferation. Sim
ilar results were obtained using two other heavily
pigmented and three different lightly pigmented
cell strains.
Days After Plating
234567
Days After Plating
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RESPONSES OF HUMAN
Table 2 lime-dependent
MELANOCYTES
effects of UVK light on the cell cycle"
0,,-G,
Heavily pigmcnlcd mclanocytes
Control
23.4 mJ/cm2
35.0 mJ/cnr
46.7 mJ/cm~
We have compared the induction of p53 in heavily pigmented
versus lightly pigmented melanocytes after irradiation with 35.0 mil
cm2 UVB light. By Western blot analysis, we found that p53 expres
G,-M
80.0
88.7
90.3
92.3
13.8
2.6
1.6
1.9
6.1
8.8
8.1
5.8
Lightly pigmcntcd melanocyles
Control
23.4 mJ/cm2
35.11mJ/cm2
78.3
4.2
17.5
90.9
3.4
5.7
91.6
2.2
6.1
46.7 mJ/ctrr
93.2
1.9
4.8
" Melanocytes were plated at a density of 5 X 10" cells/l(K)-mm dish and were
irradiated once on day 3 after plating with 23.4, 35.0. or 46.7 ml/cm" UVB light. Fifteen
h after UVB exposure, melanocytes were harvested, double stained with anti-BrdUrdFITC and PI, and analyzed by flow cytometry.
11.7
(mJ/cm2)
23.4
(mJ/cm2)
sion was induced 4 h after UVB treatment in both types of melano
cytes. In the more melanotic melanocytes, induction of p53 peaked at
24 h and gradually declined 32 and 48 h after UVB exposure, whereas
in the lightly pigmented melanocytes, induction of p53 continued to
increase steadily up to 48 h after UVB treatment (Fig. 2, a and /;).
Formation of Cyclobutane Pyrimidine Dimers in Lightly and
Heavily Pigmented Melanocytes. We have used a RIA to quantitate
the number of cyclobutane pyrimidine dimers in genomic DNA pu
rified from lightly or heavily pigmented melanocytes after irradiation
with increasing doses of UVB light (23.4, 35.0, 46.7, or 70.1 mJ/cm2).
We have found that at all the UVB doses used, DNA from lightly
pigmented melanocytes contained more cyclobutane pyrimidine
dimers/107 dallons than their heavily pigmented counterparts (Fig. 3).
The formation of cyclobutane pyrimidine dimers correlated directly
with the doses of UVB light used to irradiate the melanocytes. These
results are similar to those reported previously by Kobayashi et al.
(34), who showed that more cyclobutane pyrimidine dimers and
pyrimidine (6-4) pyrimidone photoproducts are formed in UV-irradi-
Table 3 The tnelatittgentc response of heavily ami lightly pigmented melimocytes to a
single irradiation with UVB light"
Control
(Mg/106 cells)
TO UV IRRADIATION
35.0
(mj/cm2)
Heavily pigmentcd cell strains
47()b475h479bLightly
ated melanoma cell lines with a low melanin content than in cell lines
that are very melanotic.
pigmented cell strains
473477
DISCUSSION
493
6.83
5.84.727.7225.9245.566.418.91ND''
ND''23.2929.4348.275.458.37
In
5.36
49423.519.637.55.08.0
5.4828.5831.0155.384.576.436.97
most
' Melanocytes were plated at a density of 0.8 X H)5 cells/60-mm dish and irradiated
tion,
with 0. 11.7, 23.4, 35.0, or 46.7 mJ/cm2 UV light on day 3 after plating. Melanocytes were
collected and assayed for melanin content on day 7, 4 days after UV irradiation.
ND. not determined.
humans, a primary target for solar radiation is the epidermis. The
visible effect of sun exposure is increased cutaneous pigmenta
or "tanning," which results primarily from stimulation of mela-
nogenesis in epidermal melanocytes. Other effects of solar radiation,
particularly UVB light, include DNA damage and photocarcinogen-
on day 5 for the less melanotic cells, was comparable to that of the
deeply melanotic melanocytes after treatment with 11.7, 23.4, or 35.0
mJ/cnr UVB light. The former melanocytes, however, had a signif
icantly lower survival rate than the latter after irradiation with a dose
of 46.7 mJ/cm2 UV light (Fig. 1, a and b). The recovery rates
determined by the increase in cell number between days 5 and 9 were
comparable for both types of melanocytes at the lowest two doses of
UV light, but the rate of increase in cell number was considerably
higher for the heavily pigmented melanocytes after irradiation with 35
mJ/cm2 UV light. At the highest dose of UV light, neither type of
esis. Skin cancers are more common in individuals with lightly
pigmented skin than in those with dark skin. The molecular basis for
this difference, based on the responses of human melanocytes with
different melanin contents to irradiation with UVB light, is poorly
understood. The studies hereby presented were intended to investigate
the well accepted notion that melanin serves for photoprotection
against the damaging effects of sun exposure.
To investigate the role of melanin, we have compared the responses
of cultured human melanocytes derived from lightly pigmented (skin
types I-II) to those of melanocytes derived from heavily pigmented
melanocytes could resume proliferation.
Cell Cycle Profile following UVB Treatment. Flow cytometric
analysis of the cell cycle showed that melanocytes responded to UVB
irradiation with a dose-dependent accumulation in G, (Table 2). This
arrest in G, was evident in all melanocytc strains regardless of their
melanin content. A similar arrest of DNA synthesis has been observed
in HeLa cells after irradiation with 254 nm UV light and in human
fibroblasts after y-irradiation (11 48). After irradiation with 23.4
mj/crrr UVB light, the percent of melanocytes in G()-G, increased
from 78 to 90% in the lightly pigmented, and from 80 to 90% in the
heavily pigmented melanocytc group. These changes were accompa
nied by a reduction of the percent of cells in S phase, from 17.5 to 3%
in the former, and from 14 to 2% in the latter melanocytes. The
accumulation of cells in G, and the reduction of the number of cells
in S increased with higher doses of UVB light (Table 2).
Melanogenic Stimulation and Induction of p53 Expression by
UVB Light. In addition to the decrease in cell survival and prolifer
ation, human melanocytes responded to UVB treatment with an in
crease in melanin content. The stimulation of melanin formation after
a single irradiation was significant in the heavily pigmented but not in
the lightly pigmented melanocytes (Table 3).
4h
1
8h
24 h
32h
48h
8
10
Fig. 2. Time-dependent induction of p53 expression in lightly (<i) or heavily (/')
pigmented melanocytes after irradiation with UVB light. Melanocytes were plated onto
l(M)-mm dishes at an initial density of 6 X l(hs cells/dish and were either left unirradiated
or irradiated 72 h later with 35.0 mJ/cnr UVB light. Cells were lysed with RIPA buffer
containing phosphatase and protease inhibitors at 4, 8, 24, 32, and 48 h after irradiation.
The expression of p53 was determined by Western blotting as described in "Materials and
Methods." Lunes Ì,
3, 5, 7, and 9, control groups (-); and Lunes 2, 4, 6, 8, and 10.
irradiated groups [+ | at each time point tested. This experiment was repeated using three
different lightly pigmented and three different heavily pigmented melanocyle strains with
similar findings. Probing with a mAb against the kinase ERK-, (ERK2 NT; Upstate
Biotechnology, Lake Placid, NY) showed identical protein loading in all lanes (data not
shown).
4044
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RESPONSES
OF HUMAN MELANOCYTES
TO UV IRRADIATION
Western blot analysis, we could detect increased p53 protein expres
sion in both lightly and heavily pigmented melanocytes 4 h after UVB
irradiation. However, in the former, the increase in the induction of
p53 persists for a more prolonged time period than in the latter (Fig.
2, a and b). In the following manuscript, we describe in detail the
molecular events by which UVB light induces G, arrest (51). These
include the accumulation of p21Waf-1/sr>M/c'ip-1downstream from p53
induction, and the resulting inhibition of phosphorylation of the retinoblastoma protein (RB). The persistence of high levels of p53
protein in UVB-irradiated melanocytes with a low melanin content
might partially account for the inability of these cells to recover from
the UVB-induced growth arrest. It is possible that the prolonged arrest
observed in these melanocytes is due to the greater extent of DNA
damage, or the slower rate of repair in these cells relative to the
heavily pigmented melanocytes. Indeed, we have found that DNA
from lightly pigmented melanocytes contain more cyclobutane pyrim
idine dimers/107 daltons than DNA from heavily pigmented melano
23.4
35.0
46.7
UVB Dose (mJ/cm2)
70.1
Fig. 3. Dose-dependent formation of cyclohutane pyrimidinc dimers in heavily (•)and
lightly (•)pigmentcd mclanocytes after irradiation with increasing doses of UVB light.
Melanocytes were plated at a density of 7.5 X lOVlOO-mm dish and irradiated on day 3
after plating with 0, 23.4, 35.0, 46.7, and 70.1 mj/cnr UVB light. DNA was immediately
extracted after irradiation, and the number of cyclohutane pyrimidine dimers/107 dallons
was determined as described in "Materials and Methods." Points, average of duplicate
samples.
(skin types IV-VI) neonatal foreskins to a single irradiation with
different doses of UVB light. We chose to investigate this light
spectrum (295-320 nm) because it is more biologically active than
UVA in eliciting DNA damage (4). We found a dose-dependent
increase in cell killing after a single irradiation of melanocytes with
11.7, 23.4, 35.0, or 46.7 mJ/cm2 UVB light. The percent of cell killing
was not statistically different among melanocytes with different con
stitutive melanin contents (Fig. 1, a and b). We also observed that
UVB irradiation caused increased melanogenesis, particularly in the
heavily pigmented melanocytes (Tables 1 and 3). Our results are
consistent with those of Yohn er al. (32), who reported lower percent
survival of Caucasian melanocytes than Black melanocytes after treat
ment with UVA light, and with those of Kobayashi et al. (34), who
found that irradiation with a 254-nm light source caused more killing
of lightly pigmented in comparison to darkly pigmented melanoma
cells.
We also found that lightly pigmented melanocytes had less capacity
than heavily pigmented melanocytes to resume proliferation after
irradiation with UVB light, particularly with doses equivalent to 35.0
or 46.7 mJ/cm2, the highest two doses used in these experiments (Fig.
cytes (Fig. 3). The rates of removal of these photoproducts by mela
nocytes with different constitutive melanin contents are currently
being investigated.
In our study, we found that lightly pigmented melanocytes do not
respond to a single treatment with UVB light with a significant
increase in melanin formation (Table 3). However, previously we
showed that lightly as well as heavily pigmented melanocytes are
stimulated to synthesize melanin after multiple irradiations with UVB
light (31). This raises the question of whether the inability of the
lightly pigmented melanocytes to mount a rapid increase in melanin
formation further increases their susceptibility to DNA damage. A
novel but nevertheless interesting notion is that melanogenesis in
duced by UVB light correlates directly with DNA repair. This was
concluded from the observation that treatment of melanocytes with
the repair enzyme, T4 endonuclease V, resulted in a dose-dependent
increase in melanin content (49). This is consistent with the idea that
the increase in melanin associated with postinflammatory hyperpigmentation is assumed to rid the cell of the damaging products of the
inflammatory process (50). Whether melanogenesis correlates with
repair of cyclobutane pyrimidine dimers will be investigated by com
paring the rate of removal of these photoproducts in melanocytes with
different constitutive melanin contents.
Because of the drastic environmental changes that allow for more
penetration of UVB rays through the atmosphere of the earth and the
prediction that this would lead to increased incidence of skin cancers,
protection from the sun has become an important issue for skin cancer
prevention (5). However, the effectiveness of melanin as a photoprotective sunscreen is still debatable. Whether it is melanin alone or
additional cellular defense mechanisms, such as levels of glutathione, p53 induction, or the rate of DNA repair that are responsible
for the differences in the susceptibility of individuals with different
skin types to photoaging and skin cancers needs to be further
elucidated.
1, a and h). The differences in the responses of lightly and heavily
pigmented melanocytes were further substantiated by our findings that
melanocytes derived from a tyrosinase-negative albino patient had the
lowest survival and recovery rates after UVB irradiation (Fig. le).
These results suggested to us that heavily pigmented melanocytes can
recover from the growth-inhibitory effect of UVB irradiation, possibly
because they encounter less DNA damage and/or can repair their
DNA more efficiently than their lightly pigmented counterpart.
We observed that melanocytes, regardless of their melanin con
tents, were arrested in G, after UVB irradiation (Table 2). Our present
results are substantiated by our findings that UVB light induces the
expression of the product of the tumor suppressor gene p53, which is
known to block proliferation by arresting cells in G, (35-38). By
ACKNOWLEDGMENTS
We thank Dr. Ralph Buncher and Michael White for Iheir assistance in the
statistical analysis of the data. We also thank Dr. Raymond Boissy for providing
the albino melanocyte strains and Joan Griggs for her secretarial assistance.
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Comparison of the Responses of Human Melanocytes with
Different Melanin Contents to Ultraviolet B Irradiation
Diane Barker, Kathleen Dixon, Estela E. Medrano, et al.
Cancer Res 1995;55:4041-4046.
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