Open Journal of Pathology, 2012, 2, 113-119
Published Online October 2012 (http://www.SciRP.org/journal/ojpathology)
http://dx.doi.org/10.4236/ojpathology.2012.24021
1
Distinct Subcellular Localization of GSK-3β in Melanocytic
Nevi: Implications in Melanocyte Senescence*
Jonathan L. Curry1#, Carlos A. Torres-Cabala1, Carla L. Warneke2, Peter Zhang1, Victor G. Prieto1
1Departments of Pathology and Dermatology, The University of Texas MD Anderson Cancer Center, Houston, USA; 2Departments
of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, USA.
Received July 25th, 2012; revised August 21st, 2012; accepted September 3rd, 2012
ABSTRACT
Melanocytic nevi are a transient in vivo proliferation of melanocytes that after time undergo cellular senescence. Most
nevi harbor B-Raf mutations, which appear to activate cellular mechanisms of senescence in melanocytes. Glycogen
synthase kinase 3β (GSK-3β), a critical downstream effector of the AKT signaling pathway, is involved in the devel-
opment of melanoma and has been associated with senescence in melanocytes. Our immunohistochemical and im-
munofluorescence studies revealed distinct, perinuclear, dot-like reactivity of GSK-3β in melanocytic nevi. Furthermore,
our tissue microarray analysis demonstrated significant perinuclear dot-like sublocalization of GSK-3β in melanocytic
nevi compared with the amount of GSK-3β observed in melanoma (P < 0.0019). In summary, the subcellular localiza-
tion of GSK-3β in human nevi may contribute to senescence in melanocytes.
Keywords: GSK-3β; Nevi; Melanoma; Senescence
1. Introduction
Cutaneous melanoma, a common skin malignancy with
high morbidity and mortality rates, is the second most
common form of cancer in women aged 20 - 29 years,
and its incidence continues to increase [1,2]. Although
melanoma may be surgically curable with early detection,
this disease typically becomes resistant to chemotherapy
and radiotherapy and progresses unpredictably to more
advanced stages [3]. Melanomas may arise in pre-exist-
ing nevi or de novo and therefore have a variable clinical
presentation. Lesions which are asymmetric, with notched
borders, irregular color, diameter greater than 6.0 mm,
and evolve over time or “ABCDE” criteria are clinical
worrisome features of melanoma requiring biopsy ex-
amination [4]. As lesions evolve over time, pigmentation
may become more irregular, lesions may develop nod-
ules as tumor grows vertically into the dermis or appear
white or regressed in some areas [4,5].
Melanoma typically affects the elderly and middle age
to young adults; however, it can occur at any age includ-
ing children. [6] Melanoma has a higher predilection for
fe- males until the age of 40 years and by 75 years of age
melanoma is 3 times higher in males than females.
Melanoma can occur throughout the body on chronic sun
exposed sites (head and neck), intermittent sun exposed
sites (trunk, legs, and arms) and on body sites with min-
imal sun exposure (feet and sunugual regions). Mela-
noma is a heterogeneous disease, arising most frequently
through oncogenic mutations in B-raf, N-ra s, and Kit and
with variable exposure to ultraviolet light, depending on
the clinical subtype [7].
Cutaneous melanoma is treated by surgical excision.
Current recommendations are 0.5 cm margins for mela-
noma in situ, 1.0 cm margins for tumors less than 1.0
mm in Breslow thickness (BT), and 2.0 cm surgical mar-
gins for tumors of intermediate thickness (BT = 1 - 4 mm)
and high risk tumors (BT > 4.0 mm) [8-10]. Lymphatic
mapping with sentinel lymph node biopsy is recom-
mended in patients with tumors greater than 1.0 mm in
thickness. Sentinel node positive patients will undergo
further lymph node dissection to determine the extent of
tumor burden in the regional lymph nodes and for staging
[11].
Adjuvant interferon (IFN) alfa therapy combined with
surgical excision is approved method of treatment for
patients with high-risk melanomas. Patients with advance
disease with distant metastasis may be treated with com-
bination of immuno and chemotherapy. Raf inhibitor,
vemurafenib has been approved therapy in patients with
advanced disease with mutant B-rafV600E tumors [12].
*This research is supported in part by the National Institutes of Health
through MD Anderson’s Cancer Center Support Grant CA016672.
#Corresponding author.
Melanocytic nevi, a transient in vivo proliferation of
melanocytes, originate from a transdifferentiation proc-

Distinct Subcellular Localization of GSK-3β in Melanocytic Nevi: Implications in Melanocyte Senescence
114
ess that allows melanocytes to escape from the early
triggers of melanoma development and enter a cellular
mechanism of senescence or restricted growth [13]. In
the skin, melanocytic nevi are stable neoplasms that be-
come clinically detectable after an initial period of mel-
anocyte proliferation followed by induction of mecha-
nisms of cellular senescence [14]. B-Raf mutations are
the most common activating mutations in melanocytic
nevi, and overexpression of mutant B-RafV600E induces
cell cycle arrest and senescence [15,16]. The mecha-
nisms of growth arrest in melanocytic nevi are attributed
to several cell cyclin-dependent kinase inhibittors in-
cluding p16INK4a, p27KIP1, and p57KIP2 [17,18].
Glycogen synthase kinase-3 (GSK-3) is a serine/
threonine protein kinase involved in phosphorylation of
the glycogen synthase (GS) [19]. A proportion of GSK-3
is present in an active, multiprotein complex composed
of axin, adenomatous polyposis coli (APC) protein, and
β-catenin [20]. GSK-3α and GSK-3β are the two iso-
forms of GSK-3 which are structurally similar, but func-
tionally diverse [21]. GSK-3β null mice die in utero since
activity of GSK-3α cannot rescue impaired functions of
GSK-3β [22]. GSK-3β has been associated with senes-
cence and melanocyte differentiation and melanogenesis
[23,24]. GSK-3β is a critical downstream effector of the
AKT signaling pathway, which accounts for 10% - 30%
of melanomas [25]. Primary and metastatic melanomas
demonstrate increased expression of phosphorylated
AKT, and the identification of AKT mutations in me-
lanoma cell lines supports the role of this signaling
pathway in melanomagenesis [26]. Furthermore, animal
studies have demonstrated that AKT has the ability to
convert melanoma cells from a radial to a vertical
growth phase phenotype [27,28]. Thus an AKT/GSK-3β
pathway may be a crucial component in the sequence of
biologic interactions that initiates melanocytes to form
nevi or melanoma. To further examine AKT/GSK-3β
pathway in melanocytic neoplasm, we sought to exam-
ine the immunoreactivity of GSK-3β in melanocytic
nevi and melanoma. We sought to examine the im-
muno-reactivity of GSK-3β in melanocytic nevi and
melanoma.
2. Materials and Methods
2.1. Nevi and Melanoma Tissue Samples
After this study was approved by the University of Texas
MD Anderson Cancer Center Institutional Review Board
for human subject research, we obtained the following
formalin-fixed paraffin-embedded tissues from the ar-
chives of the Department of Pathology, Section of Der-
matopathology at MD Anderson Cancer Center: 144 me-
lanocytic lesions including benign nevi (10 cases), pri-
mary cutaneous melanoma without metastasis (41 cases),
primary cutaneous melanoma with metastasis (78 cases),
and metastatic melanoma (15 cases). All diagnoses had
been rendered by dermatopathologists.
2.2. Tissue Microarray
Tissue microarray construction was performed as previ-
ously described [29]. Briefly, hematoxylin and eosin (H
& E)-stained sections were reviewed from tissue blocks,
and 0.6 mm or 1.0 mm cylindrical cores from the se-
lected areas were punched out from donor blocks and
inserted into a standard 4.5 × 2.0 × 1.0 cm recipient
block with use of a manual tissue arrayer (Beecher In-
struments, Silver Spring, MD) with an edge-to-edge dis-
tance of 0.10 or 0.15 mm. Care was taken to preserve the
original tissue block. At least two tissue cores were taken
for each case for a total of 144 cores in four tissue mi-
croarrays. Control cases were included in each microbar-
ray. Serial 5-μm-thick sections of each microarray were
cut and stained with H & E to verify presence of lesional
cells in the tissue cores.
2.3. Immunohistochemical Studies
Immunohistochemical studies were performed as previ-
ously described [18]. Briefly, 5-µm-thick tissue sections
were deparaffinized, rehydrated in a graded series of
ethanol, and subjected to heat-induced antigen micro-
wave retrieval (using 10 mM citrate buffer [pH 6.0] and
microwaving for 15 minutes). Sections were incubated
with primary antibody in a buffer containing 0.1% bo-
vine serum albumin for 1 hour at room temperature. The
following antibodies were used: polyclonal anti-GSK-3β
(1:100 dilution, Santa Cruz Biotechnology, Santa Cruz,
CA). Subsequent immunostaining was performed by us-
ing avidin-biotin immunoperoxidase and/or a micro-po-
lymer technique according to the manufacturer’s in-
structions (Vectastain, Vector Laboratories, Burlingame,
CA). Endogenous peroxidase activity was blocked with
hydrogen peroxide, and the color was developed with
either 3-amino-9-ethyl carbazole or diaminobenzidine
(Vectastain, Vector Laboratories) as the chromogen, pro-
ducing a positive red or brown reaction product, respec-
tively. Sections were counterstained with hematoxylin.
2.4. Immunofluorescence Studies
Deparaffinized embedded tissue samples of melanoma
and nevi were subjected to a graded series of ethanol
washes and antigen microwave retrieval in citrate buffer
as described earlier. Tissue sections were blocked in
PBS-T in 5% milk for 30 minutes and incubated with
primary antibody in a buffer containing 0.1% bovine
serum albumin for 1 hour at 37˚C. Incubation with ap-
propriate anti-rabbit secondary antibody conjugated with
tetramethylrhodamine isothiocyanate or FITC (1:500;

Distinct Subcellular Localization of GSK-3β in Melanocytic Nevi: Implications in Melanocyte Senescence 115
Santa Cruz Biotechnology, Santa Cruz, CA) in 5% milk
with PBS-T was performed for 1 hour at 37˚C after three
5-minute washes in FA buffer. After incubation with
secondary antibody, three 5-minute washes in 5% milk
with PBS-T were performed. One drop of DAPI (Vector
Laboratories) was added to each slide, and after a final
wash, the slides were mounted with antifade aqueous
mounting media (Thermo Fisher Scientific Inc., Waltham,
MA) and examined with a fluorescence microscope.
2.5. Blocking Studies with GSK-3β Substrate
Tissue sections were deparaffinized as described earlier
and subjected to heat-induced antigen in DAKO Target
Retrieval Solution at a high pH for 20 minutes. Primary
antibody (1:200) and substrate (1:100) were mixed and
incubated for 15 minutes at 37˚C. Sections were incu-
bated with the antibody and substrate mix for 1 hour at
37˚C, and subsequent immunostaining was performed as
described earlier.
2.6. Statistical Analysis
The pattern and intensity of immunolabeling (diffuse
cytoplasmic vs perinuclear) in the tissue samples and
tissue microarray were scored by dermatopathologists
(JLC and VGP) as 0, 1, 2, or 3, corresponding respec-
tively to no labeling, weak, moderate, and strong labeling.
The percentage of cells that were positive for GSK-3β
was scored from 0% to 100%. Associations between
GSK-3β and categorical variables were examined with
use of the Fisher’s exact test, Kruskal-Wallis test, or
Spearman’s correlation coefficient as appropriate. A
time-to-event analysis was conducted by using Cox pro-
portional hazards regression models. Survival end points
included overall survival duration (measured from the
time of diagnosis for the particular specimen assayed
until death or last follow-up) and disease-free survival
duration (measured from the time of diagnosis until the
next event: metastasis, new primary, death, or last fol-
low-up). Results were considered significant if P values
were less than 0.05. Individual significance levels were
not adjusted for multiple comparisons. All analyses were
performed with use of SAS 9.1 software (SAS Institute,
Cary, North Carolina).
3. Results
Distinct Perinuclear Subcellular Localization of
GSK-3β in Melanocytic Nevi
Benign nevi are composed of growth-arrested melano-
cytes that have entered cellular senescence in response to
activating B-raf mutations. Oncogene induction of cy-
clin-dependent kinase inhibitors such as p16INK4a,
p27KIP1, and p57KIP2 in nevi creates a barrier to further
melanocytic proliferation. GSK-3β is a crucial enzyme in
many cellular processes, including senescence [30].
Striking perinuclear dot-like reactivity of GSK-3β was
detected in all cases of benign nevi by immunohisto-
chemical studies (Figure 1(a)). To better localize GSK-3β
immunoreactivity in benign nevi, immunofluorescence
studies were performed on paraffin-embedded tissue. The
subcellular localization of GSK-3β was found to be in the
cytoplasm at a perinuclear location (Figure 1(b)).
Invasive melanoma may be associated with melano-
cytic nevi; therefore, we next examined GSK-3β in le-
sions that demonstrated both melanomas and nevi in the
same tissue section (Figure 2(a)). The nevus component
(a) (b)
Figure 1. Distinct perinuclear dot-like cytoplasmic immu-
noreactivity of GSK-3β in benign nevi. (a) GSK-3β immu-
nohistochemical image at 400× magnification; (b) GSK-3β
immunofluorescence image at 400× magnification. Arrows
highlight GSK-3β reactivity in nevus cells. Insert: Subcellu-
lar cytoplasmic and perinuclear localization of GSK-3β.
(a)
(b)
(c)
(d)
Figure 2. Cytoplasmic reactivity for GSK-3β in cutaneous
melanoma. (a) H & E-stained image of primary cutaneous
melanoma (*) with coexisting benign nevus (short arrow) at
20× magnification; (b) GSK-3β immunohistochemical im-
age of invasive melanoma at 200× (c) and at 400× magnifi-
cation. Insert: Absence of cytoplasmic dot-like reactivity for
GSK-3β in melanoma cells; (d) Perinuclear GSK-3β reac-
tivity in coexisting nevus cells (long arrow). Insert: Subcel-
lular cytoplasmic and perinuclear localization of GSK-3β.

Distinct Subcellular Localization of GSK-3β in Melanocytic Nevi: Implications in Melanocyte Senescence
116
intensity of GSK-3β immunoreactivity did not appear to
be significantly associated with patient age at diagnosis
or between histologic factors that included ulceration,
Clark level, vertical growth phase, and regression. There
was no association between GSK-3β and overall sur-
vival.
was composed of banal nevomelanocytes with minimal
cytologic atypia and absence of dermal mitoses com-
pared with the invasive melanoma component with en-
larged, atypical epithelioid melanoma cells and an in-
creased mitotic rate. Immunohistochemical studies for
GSK-3β detected perinuclear labeling in benign nevus
cells but not in melanoma cells (Figures 2(b)-(d)).
4. Discussion
To ensure that the immunoreactivity was specific to
GSK-3β, blocking studies with GSK-3β peptide demon-
strated decreased perinuclear immunoreactivity for GSK-
3β in benign melanocytes after the antibody was ab-
sorbed with GSK-3β peptide (Figure 3).
We have described the presence of GSK-3β in human
nevi and melanoma in vivo. Subcellular localization of
GSK-3β was detected at significantly higher levels in
nevi than in melanoma, although some melanomas did
demonstrate perinuclear subcellular localization of GSK-
3β (data not shown). The expression of GSK-3β (perinu-
clear or cytoplasmic labeling) in melanoma showed no
We next examined the melanoma tissue microarray for
GSK-3β for differences in labeling in nevi and the sub-
groups of invasive melanoma (melanoma with metastasis,
melanoma without metastasis, and metastatic melanoma).
The clinical characteristics of the 144 patients in the tis-
sue microarray included median age of 57 years (range
11 to 86 years). The male to female ratio was 1 to 1.1.
Among the patients with melanoma, 69% of the patients
died during follow-up. The estimated median disease-
free survival was 6.2 years (955 CI 5.2 to 7.9 years) and
the estimated median overall survival was 6.1 years (90%
CI 5.1 to 7.8 years). Perinuclear labeling of GSK-3β was
greater in melanocytic nevi than it was in melanomas of
any subgroup (Figure 4; P < 0.0019). The cytoplasmic
percentage of GSK-3β did not vary significantly between
nevi and the subgroups of melanoma. The percentage and
(a)(b)
Figure 3. GSK-3β immunohistochemical image in benign
nevi at 600× magnification. ((a) and insert) Dot-like immu-
noreactivity for GSK-3β; (b) GSK-3β immunohistochemical
image with blocking peptide at 600× magnification. Note the
absence of immunoreactivity.
Melanoma with Mets Melanoma without Mets Metastatic Melanoma Nevus
100
80
60
40
20
0
CS
K
-3β Perinuclear Percen
t
Figure 4. Perinuclear immunoreactivity of GSK-3β in melanoma and nevi. Melanocytic nevi demonstrated higher levels of
perinuclear GSK-3β positivity compared with all groups of melanomas. These differences were statistically significant (P <
0.0019).

Distinct Subcellular Localization of GSK-3β in Melanocytic Nevi: Implications in Melanocyte Senescence 117
correlation with prognostic parameters (e.g., ulceration,
tumor thickness, regression, or tumor-infiltrating lym-
phocytes) in melanoma, metastatic potential, or overall
patient survival. However, perinuclear subcellular local-
ization of GSK-3β was more commonly seen in benign
nevi, which may have a role in growth arrest of melano-
cytes.
Regulation of GSK-3β activity is achieved by a com-
bination of inhibitory phosphorylation at the Ser9, pro-
tein complex formation, and subcellular localization
[31,32]. GSK-3β is seen in several subcellular fractions
including the cytoplasm, nucleus, and mitochondria [32].
The effects of GSK inhibition promotes GSK-3β medi-
ated phosphorylation and repression of the Wnt signal
pathway involved in cell proliferation and carcinogenesis
through stabilization of β-catenin. Activation of AKT
inhibits GSK3 through phosphorylation of S21 and S9
residues of GSK-3α and GSK-3β isoforms, respectively
[33]. This inactivation of GSK-3β affects cellular me-
tabolism and allows cellular conversion of glucose into
glycogen by glycogen synthase [34,35]. Induction of the
senescence phenotype and augmentation of glycogenesis
by GSK inhibitors (e.g., SB415286 and LiCl) were seen
in multiple cell lines including human liver cells, HeLa
cells, and primary human diploid fibroblasts. In addition,
overexpression of the active GSK-3β-S9A mutant could
weakly drive senescence with glycogen accumulation
[36]. Collectively, these tissue culture studies suggest a
potential role for GSK-3β as a regulator of melanocyte
senescence.
The subcellular localization of GSK-3β in melanocytic
nevi maybe related to protein sequestration (and its inhi-
bition) or compartmentalization of GSK-3β in distinct
inactive cellular pools, quality control of the protein, or
protein complex formation. GSK-3β is central to many
cellular pathways including signaling by insulin, growth
factors, cell division, apoptosis, and microtubule forma-
tion [20,37]. Insight into the cellular mechanisms of
GSK-3β in postnatal expansion of melanocytes in nevo-
genesis and melanocyte senescence may allow us to
combine therapies targeted at induction of senescence
along with inhibition of proliferating signals.
5. Conclusion
Melanocytic nevi demonstrate distinct peri-nuclear dot
like labeling of GSK-3β that was detected by immuno-
histochemistry and immunofluorescence. Further inves-
tigation is necessary to examine the biologic significance
of GSK-3β accumulation in melanocytic nevi. In vivo
studies with proliferating neonatal human melanocytes
and evaluation of phosphorylated GSK-3β in melano-
cytic neoplasms may provide additional insight into the
role of GSK-3β in melanocyte senescence.
6. Acknowledgements
We like to thank Dr. Nikolaj Timchenko for kindly pro-
viding the use of GSK-3β antibody and Tamara K. Locke
for editorial review of the manuscript.
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