c-RET Molecule in Malignant Melanoma from Oncogenic
RET-Carrying Transgenic Mice and Human Cell Lines
Yuichiro Ohshima1,2., Ichiro Yajima1., Kozue Takeda1, Machiko Iida1, Mayuko Kumasaka1, Yoshinari
Matsumoto2, Masashi Kato1*
1Units of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai-shi, Aichi, Japan,
2Department of Dermatology, Aichi Medical University School of Medicine, Nagakute-cho, Aichi-gun, Aichi, Japan
Malignant melanoma is one of the most aggressive cancers and its incidence worldwide has been increasing at a
greater rate than that of any other cancer. We previously reported that constitutively activated RFP-RET-carrying
transgenic mice (RET-mice) spontaneously develop malignant melanoma. In this study, we showed that expression
levels of intrinsic c-Ret, glial cell line-derived neurotrophic factor (Gdnf) and Gdnf receptor alpha 1 (Gfra1) transcripts in
malignant melanomas from RET-transgenic mice were significantly upregulated compared with those in benign
melanocytic tumors. These results suggest that not only introduced oncogenic RET but also intrinsic c-Ret/Gdnf are
involved in murine melanomagenesis in RET-mice. We then showed that c-RET and GDNF transcript expression levels in
human malignant melanoma cell lines (HM3KO and MNT-1) were higher than those in primary cultured normal human
epithelial melanocytes (NHEM), while GFRa1 transcript expression levels were comparable among NHEM, HM3KO and
MNT-1. We next showed c-RET and GFRa1 protein expression in HM3KO cells and GDNF-mediated increased levels of
their phosphorylated c-RET tyrosine kinase and signal transduction molecules (ERK and AKT) sited potentially
downstream of c-RET. Taken together with the finding of augmented proliferation of HM3KO cells after GDNF
stimulation, our results suggest that GDNF-mediated c-RET kinase activation is associated with the pathogenesis of
Citation: Ohshima Y, Yajima I, Takeda K, Iida M, Kumasaka M, et al. (2010) c-RET Molecule in Malignant Melanoma from Oncogenic RET-Carrying Transgenic Mice
and Human Cell Lines. PLoS ONE 5(4): e10279. doi:10.1371/journal.pone.0010279
Editor: Benjamin Edward Rich, Dana-Farber Cancer Institute, United States of America
Received August 6, 2009; Accepted March 19, 2010; Published April 21, 2010
Copyright: ? 2010 Ohshima et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported in part by Grants-in-Aid for Scientific Research (B) (No. 19390168 and 20406003) from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT), COE Project for Private Universities (No. S0801055) from MEXT, Grant-in-Aid for Young Scientists (B) (No. 20790775,
20790821, 20700370) from the Japan Society for the Promotion of Science (JSPS), the ROHTO Award, the Uehara Memorial Foundation and Chubu University
grants A and C. MEXT: http://www.mext.go.jp. JSPS: http://www.jsps.go.jp. ROHTO Award: no web sites. Uehara Memorial Foundation: http://www.ueharazaidan.
com/. Chubu University grants:http://www.chubu.ac.jp/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
It has recently been reported that the incidence of cutaneous
malignant melanoma is increasing at a greater rate than that of
any other cancer . Since malignant melanoma is known as one
of the most aggressive human cancers, malignant melanoma is one
of threats for public health.
The c-RET proto-oncogene (Figure 1A) encodes a receptor-
tyrosine kinase . Previously, glial cell line-derived neurotrophic
factor (GDNF) has been reported to be one of ligands for c-RET
. GDNF, which is structurally related to members of the
transforming growth factor- ß (TGF-ß) superfamily, exerts its effect
on target cells by binding to a glycosyl phosphatidylinositol (GPI)-
anchored cell surface protein (GFRa1), which, in turn, recruits the
receptor tyrosine kinase c-RET to form a multi-subunit signaling
complex. Formation of this complex results in c-RET autopho-
sphorylation and a cascade of intracellular signaling including the
extracellular signal-regulated kinase (ERK) and Akt kinase to
regulate cell survival [2–6]. Results of our previous study suggest
that autophosphorylation of tyrosine 905 in c-RET is crucially
important for its kinase and transforming activity . On the other
hand, it has been shown that mutationally enhanced activity of c-
RET kinase caused the development of human carcinomas,
including multiple endocrine neoplasia (MEN) and papillary
thyroid carcinoma (PTC) [4,8].
RFP-RET (Figure 1B) is a hybrid oncogene between c-RET
and RFP that was isolated by NIH3T3 transfection assays .
RFP-RET is constitutively activated without GDNF and GFRa1
. Previously, we developed metallothionein-I (MT)/RFP-RET-
transgenic mice of line 304/B6 [10,11]. Systemic skin melanosis,
skin benign melanocytic tumor(s) and skin malignant melanoma(s)
developed stepwise in the RET-mice accompanying metastasis to
lymph nodes and lungs [10,11]. These observations suggest that
activated RET signaling is correlated with the development of
malignant melanoma in mice. However, it remains unknown
whether intrinsic c-RET/GDNF signaling is associated with
melanomagenesis in RET-mice.
The correlation between c-RET and human malignant
melanoma has been denied by a report of no c-RET transcript
expression in human cultured-normal melanocytes and malignant
PLoS ONE | www.plosone.org1April 2010 | Volume 5 | Issue 4 | e10279
melanoma cell lines in Northern blotting analysis . However, a
recent study showed that c-RET protein was expressed in human
melanomas . These contradictory results indicate that the
biological significance of c-RET in malignant melanoma remains
unclear. In fact, not only GDNF-mediated c-RET kinase
activation but also GFRa1 expression in human malignant
melanoma cells has still not been elucidated. In this study, we
analyzed c-RET/GDNF signaling in malignant melanoma cells
from RET-mice and human cell lines to address the above
Tumor stage-dependent RFP-RET transcript expression
levels in tumors from RET-mice
We first examined dynamics of the introduced oncogenic RET
(RFP-RET: Figure 1B) transcript expression levels in benign and
malignant tumors from RET-mice. RFP-RET transcript expres-
sion levels were significantly upregulated with increase in tumor
size (Figure 2A). In fact, expression levels of RFP-RET transcript
in malignant melanoma were about 2-fold higher than those in
Figure 1. Scheme of c-RET and RFP-RET cDNA constructs. (A) c-RET cDNA. (B) RFP-RET cDNA. The sites of c-RET (A) and RFP-RET (B) primers for
real-time PCR analysis are shown by arrows. SS, signal sequence; CAD, cadherinlike domain; CYS, cysteine-rich region; TM, transmembrane domain;
TK1, tyrosine kinase domain 1; TK2, tyrosine kinase domain 2; aa, amino acids; RFM, RING finger motif; BB, B box; CC, coiled coil.
Figure 2. Stage-dependent RFP-RET transcript expression levels in tumors from RET-mice. (A) Levels of RFP-RET transcript expression in
tumors of various sizes from RET-mice. Histopathologically benign and malignant tumors are shown by open and closed squares, respectively. (B)
Levels of RFP-RET transcript expression (mean 6 SD) in benign melanocytic tumors (open bar) and malignant melanoma (closed bar) from RET-mice.
RFP-RET transcript levels measured by real-time PCR were adjusted by hypoxanthine guanine phosphoribosyl transferase (Hprt) transcript levels.
Difference between expression levels of RFP-RET in benign melanocytic tumors and malignant melanoma from RET-mice was statistically analyzed by
the Mann-Whitney U test. *, Significantly different (p,0.05) from the control.
c-RET Signaling in Melanoma
PLoS ONE | www.plosone.org2April 2010 | Volume 5 | Issue 4 | e10279
benign melanocytic tumors (Figure 2B). Results of our previous
study using RET-mice also showed that levels of protein
expression and activity of RFP-RET in malignant melanoma
were increased compared with those in benign melanocytic tumors
. These results suggest that constitutively activated RET kinase
plays a role in the development of malignant melanoma in mice.
Tumor stage-dependent expression levels of c-Ret, Gfra1,
Gdnf transcripts in tumors from RET-mice
We next examined expression levels of intrinsic c-Ret, Gfra1
and Gdnf transcripts in benign and malignant tumors from RET-
mice (Figure 3). Levels of c-Ret transcript expression in malignant
melanomas were 4-fold upregulated compared with those in
Figure 3. Stage-dependent c-Ret, Gfra1 and Gdnf transcripts expression levels in tumors from RET-mice. (A, C, E) Levels of c-Ret (A),
Gfra1 (C) and Gdnf (E) transcripts expression in tumors of various sizes from RET-mice. Histopathologically benign and malignant tumors are shown
by open and closed squares, respectively. (B, D, F) Levels of c-Ret (B), Gfra1 (D) and Gdnf (F) transcripts expression (mean 6 SD) in benign melanocytic
tumors (open bar) and malignant melanoma (closed bar) from RET-mice. c-Ret (A, B), Gfra1 (C, D) and Gdnf (E, F) transcript levels measured by real-
time PCR were adjusted by hypoxanthine guanine phosphoribosyl transferase (Hprt) transcript levels. Differences in expression levels of c-Ret (B),
Gfra1 (D) and Gdnf (F) between benign melanocytic tumors and malignant melanoma from RET-mice were statistically analyzed by the Mann-
Whitney U test. *, Significantly different (P,0.05) from the control.
c-RET Signaling in Melanoma
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benign melanocytic tumors (Figure 3A, B). The difference between
c-Ret transcript expression levels in benign tumors and malignant
melanomas from RET-mice was statistically significant (p,0.05;
Figure 3B). Gfra1 and Gdnf transcript expression levels in
malignant melanomas were also 13-fold and 5-fold upregulated,
respectively, compared with those in benign melanocytic tumors
(Figure 3C–F). The difference in Gfra1 and Gdnf transcript
expression levels between benign tumors and malignant melano-
mas from RET-mice was statistically significant (p,0.05;
Figure 3D, F).
Expression levels of c-RET, GFRa1, GDNF transcripts in
human malignant melanoma cell lines
We next examined the expression levels of c-RET, GFRa1 and
GDNF in primary-cultured normal human epithelial melanocytes
and human malignant melanoma cell lines (G361, SK-Mel28,
MNT-1 and HM3KO) [14–16]. Transcript expression levels of c-
RET and GDNF in MNT-1 cells were around 5-fold and 12-fold
upregulated, respectively, compared with those in NHEM cells
(Figure 4A, C). Transcript expression levels of c-RET and GDNF
in HM3KO cells were around 10-fold and 35-fold increased,
respectively, compared with those in NHEM cells (Figure 4A, C).
There were no differences in GFRa1 transcript expression levels
among NHEM, MNT-1 and HM3KO cells (Figure 4B). The
expression levels of c-RET, GFRa1 and GDNF in G361 and
SK-Mel28 human malignant melanoma cells were definitely lower
or undetectably low compared with those in NHEM cells
Levels of c-RET and GFRa1 protein expression in human
We next examined levels of c-RET protein expression in
NHEM and human malignant melanoma cells (G361, HM3KO
and MNT-1) (lanes 2–5 in Figure 5). Expression of 155-kD c-RET
protein was detected in HM3KO and MNT-1 cells (lanes 4 and 5
in Figure 5) but not in NHEM and G361 cells (lanes 2 and 3 in
Figure 5), though the level of c-RET protein expression in MNT-1
cells was weak. After selecting HM3KO as a highly expressing c-
RET cell line and G361 as a barely expressing c-RET cell line, we
confirmed GFRa1 protein expression in the cells. NHEM
(Figure 6A, B) and HM3KO cells (Figure 6E, F), but not G361
cells (Figure 6C, D), expressed GFRa1 protein. These results for c-
RET (Figure 5) and GFRa1 (Figure 6) protein expression in
NHEM, G361 and HM3KO cells correspond to c-RET and
GFRa1 transcript expression (Figure 4A, B).
c-RET/GDNF signaling in human malignant melanoma
Since both c-RET and GFRa1 were found to be expressed in
HM3KO cells (Figures 4–6), we next examined whether c-RET
tyrosine kinase in HM3KO cells is activated by its ligand (GDNF).
Not only biochemical analysis (lane 4 in Figure 5) but also
immunocytochemical analysis (Figure 7A, B) revealed that c-RET
protein was expressed in HM3KO cells. Phosphorylation of
tyrosine 905 in HM3KO cells was increased by GDNF (Figure 7C,
D). On the other hand, immunocytochemical analysis also
revealed that there was no c-RET protein expression and no
augmentation of c-RET kinase activity by GDNF in G361 cells
without c-RET protein expression (data not shown), partially in
accordance with the previous results (lane 3 in Figure 5). These
results suggest that c-RET/GDNF signaling worked in HM3KO
cells but not in G361 cells. We next examined whether signal
transduction molecules potentially sited downstream of c-RET
[2,3,6] in HM3KO cells are activated by GDNF. Phosphorylation
levels of ERK and AKT in HM3KO cells (Figure 8), but not in
G361 cells (data not shown), were increased 15 min after
stimulation with GDNF, while expression levels of ERK, AKT
and ß-actin proteins were comparable.
Augmented proliferation of human malignant melanoma
cells by GDNF stimulation
We finally examined the physiological effect of GDNF
stimulation on proliferation of HM3KO and G361 human
malignant melanoma cells, which have no polymorphism at the
G691S juxtamembrane region in c-RET as shown in a
previous study . GDNF increased the number of HM3KO
cells (Figure 9A) but not the number of G361 cells (data not
shown). MTT assay also showed significant proliferation of
GDNF-stimulated HM3KO cells (Figure 9B) but not G361
cells (data not shown). GDNF-stimulated proliferation of
HM3KO cells, but not that of G361 cells (data not shown),
was suppressed by the RET kinase inhibitor SU5416 
(Figure 9A and B).
We previously demonstrated that cutaneous malignant mela-
nomas develop in oncogenic RET (RFP-RET)-carrying transgenic
mice [10,11]. Our previous and present results showed that
constitutively activated RFP-RET increased RFP-RET protein
expression [10,18] and transcript (Figure 2) expression levels in the
process of melanomagensis in the RET-mice. On the other hand,
the present results showed that not only RFP-RET but also c-Ret,
Gfra1 and Gdnf expression levels in malignant melanomas were
definitely increased compared with those in benign melanocytic
tumors (Figures 2, 3) in RET-mice. Since continuously activated
RET kinase increased the levels of RET transcript and protein
expression , these results suggest that constitutively activated
RFP-RET enhances intrinsic c-RET protein expression in the
process of melanomagensis in the RET-mice. These dynamics of
intrinsic c-Ret/Gfra1/Gdnf and introduced RFP-RET signaling
in melanocytic tumors from RET-mice encourage us to examine
the correlation between RET and human malignant melanoma.
Previous studies showed that overexpression of c-RET, GFRa1
and/or GDNF indicated poor prognosis in human pancreatic 
and bile duct  carcinomas. Overexpression of GDNF was also
reported to be involved in tumorigenesis of human lung cancer
. A recent study has biochemically provided evidence that c-
RET/GDNF signaling promotes proliferation of human breast
carcinoma cells . These results suggest that c-RET/GDNF
signaling is correlated with the pathogenesis of human cancers. In
this study, we examined whether c-RET/GNDF signaling worked
in human malignant melanoma cells. Our results showed that
transcript expression levels of c-RET and GDNF in MNT-1 and
HM3KO human malignant melanoma were definitely higher than
those in NHEM cells, while GFRa1 expression levels were
comparable in these cells (Figure 4). Further analysis biochemically
(Figure 5) and immunohistochemically (Figure 7) revealed that c-
RET protein was expressed in HM3KO cells but not in NHEM
and G361 cells. As shown in a previous study , a 155-kDa c-
RET protein, but not a 175-kDa c-RET protein, was detected in
our study (Figure 5). Then we showed that transcript and protein
of GFRa1, which is essential for c-RET/GDNF signaling, was
expressed in malignant melanoma cells by real-time PCR (Figure 4)
and immunohistochemistry (Figure 6). We also detected for the
first time increased phosphorylated levels of tyrosine 905 in c-RET
in HM3KO cells by immunohistochemical analysis (Figure 7),
c-RET Signaling in Melanoma
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Figure 4. Levels of c-RET, GFRa1 and GDNF transcripts expression in primary-cultured normal human epithelial melanocytes
(NHEM) and human malignant melanoma cell lines. (A, B, C) Levels of c-RET (A), GFRa1 (B) and GDNF (C) transcripts expression in NHEM (lane 1)
and 4 kinds of malignant melanoma cell lines (lanes 2–5; SK-Mel28, G361, MNT-1 and HM3KO). The transcript levels measured by real-time PCR were
adjusted by TATA-box-binding protein (TBP) transcript levels. Differences in expression levels of c-Ret (A), Gfra1 (B) and Gdnf (C) between NHEM (lane
1; open bar) and malignant melanoma cell lines (lanes 2–5; closed bars) were statistically analyzed by the Kruskal-Wallis test. **, Significantly different
(P,0.01) from the control.
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although we failed to detect increased phosphorylated levels by
immunoblot analysis. Thus, we further showed GFRa1 expression
and GDNF-mediated phosphorylation of c-RET kinase in human
melanoma cells (Figures 4–7) in addition to the recent report
showing a correlation between RET and human melanoma .
We next showed that signal transduction molecules (ERK and
AKT) potentially sited downstream of c-RET were activated by
GDNF in HM3KO cells (Figures 8). These results suggest that
GDNF stimulates both RET-RAS-RAF-ERK and RET-phospha-
tidylinositol 3-kinase (PI3K)-Akt pathways in HM3KO cells. We
finally showed that a c-RET agonist (GDNF) promoted cell
proliferation and that a c-RET antagonist (SU5416)  inhibited
proliferation of HM3KO cells (Figure 9). These results suggest that
c-RET/GFRa1/GDNF signaling plays a role in proliferation of
HM3KO human malignant melanoma cells. Furthermore, a
previous report revealed that GDNF stimulation significantly
amplified proliferation of human melanoma cells with polymor-
phism at G691S in c-RET . Our results presented in Figure 9
newly showed that GDNF stimulation also amplifies the
proliferation of HM3KO human malignant melanoma cells
without the polymorphism.
In summary, we newly showed c-RET protein expression in
HM3KO and MNT-1 melanoma cells in addition to its
expression in the previously reported five human melanoma cell
lines . Moreover, we for the first time demonstrated not only
GFRa1 protein expression but also GDNF-mediated c-RET
kinase activation via phosphorylated tyrosine 905 in human
malignant melanoma cells. Our results for activated RET and
(Figures 4–9) partially correspond to our previous reports of
increased activation and protein expression levels of RET and
signal transduction molecules sited downstream in the process of
melanomagenesis in RET-mice [10,18]. Thus, we might have
partially addressed the correlation between RET and malignant
melanoma by integrating the previous and present results for
mice and humans.
Materials and Methods
We previously established RET-mice (line 304/B6) with
C57BL/6 background by introducing the RET oncogene (RFP-
RET) fused to the mouse MT promoter-enhancer . All mice
were kept on a 12-h light-dark cycle in a temperature- and
humidity-controlled environment in the Animal Research Center
of Chubu University. This study was formally approved by Chubu
University (approval no.: 2010001)
Total RNA was prepared from frozen tumor and human cell
line samples using a High Pure RNA Kit (Roche Diagnostics)
according to the method previously described . cDNA was
then synthesized by reverse transcription of total RNA using
SupercriptTMIII reverse transcriptase included in the RT enzyme
Figure 5. Levels of c-RET protein expression in human
malignant melanoma cell lines. The levels of c-RET protein
expression were examined in c-RET-transfected NIH3T3 cells as a
positive control (c-RET transfectant; lane 1), primary-cultured normal
human epithelial melanocytes (NHEM; lane 2), G361 (lane 3), HM3KO
(lane 4) and MNT-1 (lane 5) by immunoblotting analysis with anti-RET
antibody after immunoprecipitation with anti-RET antibody.
Figure 6. GFRa1 protein expression in human melanocytic cells. GFRa1 protein expression was examined in primary-cultured normal human
epithelial melanocytes (NHEM; A, B), G361 cells (C, D) and HM3KO cells (E, F) by immunocytochemistry with anti-GFRa1 antibody (A, C, E) using DAPI
counterstaining (B, D, F).
c-RET Signaling in Melanoma
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mix and RT reaction mix according to the protocol of the
manufacturer (Invitrogen). Real-time quantitative RT-PCR with
SYBR green was performed using power SYBRH Green PCR
master mix (Applied Biosystems) in an ABI Prism7500 sequence
detection system (Applied Biosystems). The expression levels of c-
RET, GFRa1 and GDNF transcripts measured by quantitative
RT-PCR (real-time PCR) were adjusted by the transcript
expression level of TATA-box-binding protein (TBP) for human
samples or hypoxanthine guanine phosphoribosyl transferase
(Hprt) for mice samples. The following pairs of forward and
reverse primers were prepared for amplification: mice c-Ret, 59-
GCTGCATGAGAATGACTGGA-39 and 59-TGGCATfTCTC-
CCTCTCTCTG-39 (PCR product size, 177 bp); mice Gfra1, 59-
GACCTGGAAGATTGCCTGAA-39 and 59-CAGTGGTAGT-
CGTGGCAGTG-39 (PCR product size, 148 bp); mice Gdnf, 59-
GTCCAACTGGGGGTCTACG-39 and 59-AGCAACACCA-
GGCAGACAG-39 (PCR product size,101 bp); mice Hprt, 59-
CTTTGCTGACCTGCTGGATT-39 and 59-TATGTCCCCCG-
TTGACTGAT-39 (PCR product size, 121 bp); human RFP-
RET, 59-TGACGGAGAGTCTAAAGCAG-39 and 59-GCT-
TTAATCCCCCGGGGC-39 (PCR product size, 139 bp); human
c-RET, 59-GCTCCACTTCAACGTGTC -39 and 59-GCAG-
CTTGTACTGGACGTT-39 (PCR product size, 158 bp); human
GFRa1, 59-CACTGCCACTACCACCACTG-39 and 59-GTGT-
human GDNF, 59-CTGGGCTATGAAACCAAGGA-39 and 59-
(PCR productsize, 146 bp);
CAACATGCCTGCCCTACTTT-39 (PCR product size, 143 bp);
human TBP, 59-CACGAACCACGGCACTGATT-39 and 59-
TTTTCTTGCTGCCAGTCTGGAC-39 (PCR product size,
89 bp). PCR was carried out using 10 ml of power SYBRH
Green PCR master mix (Applied Biosystems) containing 5 mM
forward primer and 5 mM reverse primer in a final volume of
20 ml. The PCR conditions were as follows: 50uC for 2 min,
95uC for 10 min and 40 cycles of 95uC for 15 s and 60uC for
Analysis for polymorphism at the G691S juxtamembrane
region in c-RET
Polymorphism at the G691S juxtamembrane region in c-RET
in melanoma cells was examined according to the method
previously reported .
Immunoprecipitation and Immunoblotting
Immunoprecipitation with anti-c-RET rabbit polyclonal anti-
body was performed by the method previously described .
Immunoblotting with anti-c-RET (#18128, IBL, Fujioka, Japan),
anti-ERK (#9102 Cell Signaling, MA), anti-phosphorylated
ERK (#E7028 SIGMA), anti-AKT (#9272 Cell Signaling,
MA) and anti-phosphorylated AKT (#9271 Cell Signaling,
MA) antibodies was performed according to the method
described previously .
Figure 7. Augmentation of c-RET tyrosine kinase activity in HM3KO cells by c-RET ligand (GDNF). Expression of c-RET protein (A, B) and
phosphorylated tyrosine 905 in c-RET (C, D) in HM3KO cells in the absence (A, C) or presence (B, D) of GDNF were examined by immunocytochemistry
with anti-c-RET and anti-phosphorylated tyrosine 905 in c-RET antibodies using hematoxylin counterstaining (A–D).
c-RET Signaling in Melanoma
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G361 and HM3KO cells were treated with 100 ng/ml GDNF
for 15 minutes at 37uC, and then the cells were fixed in freshly
prepared phosphate-buffered 2% paraformaldehyde for 15 min at
4uC. For DAB staining, the fixed cells were incubated in 3%
hydrogen peroxide for 10 min at RT. All specimens were treated
with a blocking reagent (phosphate-buffered 1% BSA (fraction V)
solution containing 0.2% gelatin and 0.05% tween20) and then
incubated with the following primary antibodies for 60 min at
room temperature: anti-GFRa1 (H-70, Santa Cruz Biotechnology,
CA), anti-c-RET (#18128, IBL, Fujioka, Japan) and anti-
phosphorylated tyrosine 905 in c-RET (#3221, Cell Signaling,
MA) antibodies. Then the cells were stained using a DAKO
Envision-HRP/DAB system (K1390) (DAKO, CA) or labeled with
Alexa fluore 488-conjugated donkey anti-rabbit IgG (A21206)
(Invitrogen, OR). Counterstaining was performed using hematox-
ylin or DAPI.
Cell proliferation in the presence or absence of GDNF was
evaluated by counting cells with trypan blue staining  and by 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay following previous methods .
Normal human normal melanocytes (NHEM) were purchased
from Cell Applications Inc, and were maintained with melanocyte
growth medium containing hydrocortisone and growth supple-
ments (Cell Applications Inc.). MNT-1 and HM3KO human
malignant melanoma cell lines were kindly provided by Dr. Tamio
Suzuki (Department of Dermatology, Yamagata University School
of Medicine, Yamagata, Japan). G361 cells were kindly provided
by Cell Resource Center for Biomedical Research, Tohoku
University. SK-Mel28 cells were purchased from Riken Bior-
esource Center Cell Bank. SK-Mel28, MNT-1 and HM3KO cells
were maintained in Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% fetal bovine serum (FBS).
Other cell lines were maintained in RPMI 1640 supplemented
with 10% FBS.
We thank Tomoko Kunogi, Kazumi Shigeta and Yuriko Hara for
Conceived and designed the experiments: YO IY YM MK. Performed the
experiments: YO IY KT MI MK. Analyzed the data: YO IY KT MI MK
MK. Contributed reagents/materials/analysis tools: YO MK YM. Wrote
the paper: YO IY MK.
Figure 9. Proliferation of HM3KO cells. Proliferation of HM3KO
cells treated with a solvent (0.1% of DMSO) (open circle in A, lane 1 in
B), GDNF (100 ng/ml) (closed circle in A, lane 2 in B), SU5416 (5mM)
(open square in A, lane 3 in B) and GDNF plus SU5416 (closed square in
A, lane 4 in B) for 24 (A) and 48 hours (A, B) was examined by cell
counting with trypan blue staining (A) and MTT assay (B). Difference
between proliferation levels of DMSO-treated control cells and other
cells was statistically analyzed by the Kruskal-Wallis test. *, Significantly
different (P,0.05) from the control.
Figure 8. Signal transduction molecules potentially sited
downstream of c-RET in HM3KO cells. Expression and phosphor-
ylation levels of ERK and AKT in HM3KO cells before (0 min) and at 15
and 60 min after stimulation with GDNF (100 ng/ml) were examined by
immunoblotting. Equality of protein amounts in each lane was
confirmed by immunoblotting with anti-b-actin antibody.
c-RET Signaling in Melanoma
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c-RET Signaling in Melanoma
PLoS ONE | www.plosone.org9 April 2010 | Volume 5 | Issue 4 | e10279