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Tumor Biology |
Department of Pathology, Nagoya University School of Medicine, Showa-ku, Nagoya 466-8550 [K. K., T. I., H. M., K-i. I., A. N., M. T.]; Division of Experimental Animal Research, Life Science Tsukuba Research Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba, 305-0074 [N. H., A. Y., M. K.]; and Department of Pathology, Tosei General Hospital, Seto 489-8642 [K. O.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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arginine) mutation, we generated transgenic mice by introducing
the RET-MEN2A gene fused to Moloney murine leukemia
virus long terminal repeat. Expression of the transgene and its product
was detected at variable levels in a variety of tissues including
thyroid, heart, liver, colon, parotid gland, and brain. All of 29 mice
analyzed developed thyroid C-cell hyperplasia or medullary carcinoma,
accompanying high levels of serum calcitonin. In addition, development
of mammary or parotid gland adenocarcinoma was observed in one-half of
the transgenic mice. RET dimerization and its complex formation with
Shc and Grb2 adaptor proteins were detected in tumor tissues.
Unexpectedly, no tumor formation was found in other tissues despite
RET-MEN2A expression where RET dimerization was
undetectable. Because these tissues but not tumors expressed glial cell
line-derived neurotrophic factor family receptor 
(GFR) at high
levels, this suggested that GFR expression may interfere in the
dimerization of the RET-MEN2A mutant proteins, leading to
tissue-specific tumor development in vivo. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(3, 4, 5, 6, 7, 8, 9, 10, 11)
. On the basis of
analysis of Ret-/-,
Gdnf-/-, and
Gfr1-/- mice, it
turned out that the GDNF/GFR1/RET signaling complexes play a crucial
role in the development of the enteric nervous system and kidney
(12, 13, 14, 15, 16, 17)
.
Germ line mutations in RET result in human hereditary
diseases including MEN 2A and MEN 2B, FMTC and Hirschsprung’s disease
(18, 19, 20, 21, 22, 23)
. MEN 2A and MEN 2B are autosomal dominant cancer
syndromes characterized by medullary thyroid carcinoma and
pheochromocytoma. In addition to these tumors, 
20–30% of MEN 2A
patients develop parathyroid hyperplasia, whereas MEN 2B includes
mucosal neuroma, intestinal ganglioneuromatosis, and skeletal
abnormalities. Most MEN2A mutations were identified in one
of six cysteine residues in the extracellular domain of the
RET gene (24)
. Biochemical and biological
analyses revealed that these cysteine mutations induced
disulfide-linked dimerization of the RET protein, leading to
constitutive activation of its intrinsic tyrosine kinase (25
, 26)
. On the other hand, mutations in the kinase domain of
RET that result in substitution of threonine for methionine
at codon 918 or of phenylalanine for alanine at codon 883 are
responsible for MEN 2B (20
, 22
, 27
, 28)
. The
MEN2B mutations appear to activate RET without dimerization
(26
, 29 , 30)
. Despite different molecular mechanisms of
RET activation by MEN2A and MEN2B mutations,
however, the binding of Shc adaptor proteins to tyrosine 1062 of RET
was important for the transforming activity of both RET-MEN2A and
RET-MEN2B mutant proteins in vivo (31
, 32)
.
Recently, transgenic mice expressing RET-MEN2A or RET-MEN2B mutant proteins were reported by two groups (33 , 34) . In these cases, the RET-MEN2A and RET-MEN2B genes were linked to the calcitonin gene-related peptide/calcitonin promoter and dopamine ß-hydroxylase promoter to induce their expression specifically in calcitonin-secreting parafollicular C cells and in developing sympathetic and enteric nervous systems, respectively. As expected, transgenic mice carrying the RET-MEN2A gene developed thyroid CCH and medullary carcinoma (33) . Transgenic mice that carried the RET-MEN2B gene developed ganglioneuromas in the sympathetic nervous system and adrenal glands as well as renal malformation, although the enteric nervous system was not affected (34) .
To investigate further the action of the RET-MEN2A gene in
various tissues, we used MoMuLV LTR. All of the transgenic mice from
the high copy line developed CCH or medullary thyroid carcinoma, and
one-half of them developed mammary or parotid gland adenocarcinoma.
Interestingly, although mutant RET proteins were expressed in liver,
heart, and brain at high levels, no tumor formation was observed in
them. Analysis of GFR expression in various tissues suggested that
high levels of its expression may interfere with RET dimerization,
which results in suppression of tumor development. In addition, the
present study may partly explain the differences of clinical phenotypes
between MEN 2A and MEN 2B.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Arg) was introduced by PCR. The mutation and absence of
polymerase errors were verified by sequencing.
Generation of Transgenic Mice.
The MoMuLV/RET-MEN2A transgene was excised from the vector
by DraI digestion and purified from agarose gels using Gene
Clean II (BIO101 Inc.). The linearized DNA (7.5 kb) was injected into
the pronuclei of fertilized oocytes from (C57BL/6 x BALB/cA) mice at a concentration of 2 µg/ml in TE buffer [5
mM Tris-HCl (pH 7.4)/0.2 mM
EDTA]. Eggs surviving microinjection were transferred into the
oviducts of pseudopregnant ICR females. The offspring were
weaned at 4 weeks of age, and genomic DNA was extracted from their
tails. Integration of the transgene was screened by PCR analysis and
Southern blotting. The forward primer used for PCR
(5'-TCCCTTTTTGATCATATCTACACCA-3') was derived from exon 16 of the human
RET gene, and the reverse primer
(5'-AATCCATGTGGAAGGGAGGGCTCGA-3') from exon 19. Two transgenic mouse
lines (lines 121 and 180) were established by crossing founder mice
with BALB/cA or C57BL/6 mice.
Analysis of Transgene Expression.
Mouse tissues were removed under general anesthesia and total cellular
RNAs were isolated using RNeasy Mini Kit (QIAGEN). RNAs were
reverse-transcribed with AMV reverse transcriptase XL (TaKaRa)
for 30 min at 55°C, and the resulting cDNA was subjected to 30 cycles
of PCR with a thermal cycler. (94°C for 30 s, 56°C for 30 s, and 72°C for 30 s). The forward primer
(5'-CCTCCGCGAGAGCCGCAAAG-3') annealed to exon 10 of the human
RET gene, and the reverse primer
(5'-TGGAATCCGACCCTGGCTCC-3') hybridized to exons 15 and 16. This primer
set was designed not to anneal to the mouse Ret gene at the
3' end of the primers. Primer sets for other genes are as follows:
mouse Ret (forward primer, 5'-CCTCCGTGACAGCCGCAAGA-3';
reverse primer, 5'-GGGAATCCGGCCCTTGCTTT-3'); mouse GFR1 (forward
primer, 5'-TCATTGGCAGAAACATCGTAG-3'; reverse primer,
5'-GCTCAGCTTGCTTTACAGTCC-3'); mouse GFR2 (forward
primer, 5'-GCTGCCCTGCGGACAACTAC-3'; reverse
primer, 5'-GAGCTCTGTGAAACACATGC-3'); mouse GFR3 (forward primer,
5'-CTTGTGCAACTGAGCAGTCC-3'; reverse primer,
5'-CCACAGGCTGCAAATCAGTC-3'); and mouse ß-actin (forward primer,
5'-AGCTGCCTGACGGCCAGGTC-3'; reverse primer,
5'-GCTCAGGAGGAGCAATGATC-3').
Antibodies.
Anti-RET antibody was raised against the COOH-terminal 19 amino acids
of the long isoform as described previously (10)
. Anti-Shc
polyclonal antibody and anti-Grb2 monoclonal antibody were purchased
from Transduction Laboratories. Antiphosphotyrosine monoclonal antibody
was purchased from Upstate Biotechnology. Anti-phosphoErk polyclonal
antibody and anti-Erk2 monoclonal antibody were purchased from New
England Biolabs and Santa Cruz Biotechnology, respectively.
Histology and Immunohistochemistry.
Fresh tissues sampled from transgenic mice or normal littermates were
fixed in 10% neutral buffered formalin and embedded in paraffin.
Five-µm sections were used for H&E staining or immunohistochemistry.
Slides were deparaffinized in xylene and rehydrated through graded
alcohols. They were subjected to microwave pretreatment for 12 min in
10 mM citrate buffer (pH 6.0) and cooled at room
temperature. Nonspecific binding sites were blocked with 10% goat
serum for 30 min. The sections were incubated with primary antibodies
[rabbit anticalcitonin (Dako) or rabbit anti-RET antibody] overnight
at 4°C. Endogenous peroxidase was blocked in 0.3% hydrogen peroxide
in methanol for 15 min. The slides were incubated with secondary
antibody conjugated to peroxidase-labeled polymer (EnVision+, Dako) and
the reaction products were visualized using diaminobenzidine and
H2O2. Counterstaining was
performed with hematoxylin.
Ultrastructural Analysis.
Thyroid tumors were cut into 1-mm3
cubes and
fixed in 2% glutaraldehyde/2% paraformaldehyde for 1 h prior to
routine processing for transmission electron microscopic analysis.
Protein Analysis.
Harvested mouse tissues were homogenized in SDS sample buffer [50
mM Tris-HCl (pH 6.8), 5 mM EDTA, 2% SDS, 10%
glycerol, 20 mg/ml bromphenol blue, and 10 mM
iodoacetamide] with or without 80 mM DTT. Equal protein
amounts of the lysates were subjected to SDS-PAGE and transferred to
polyvinylidene difluoride membranes (Nihon Millipore Kogyo). Membranes
were blocked for 30 min at room temperature in 3% albumin in TPBS (PBS
containing 0.5% Tween 20) with gentle shaking and incubated with
primary antibody overnight at 4°C. After washing the membranes with
TPBS three times, they were incubated with the secondary antibody
conjugated to horseradish peroxidase (swine antirabbit IgG-HRP, rabbit
antimouse IgG-HRP, Dako) for 1 h at room temperature. The reaction
was examined by an enhanced chemiluminescence detection kit (ECL,
Amersham) according to the directions of the supplier. For
immunoprecipitation, tissues and cells were lysed in
radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH
7.5), 150 mM NaCl, 5 mM EDTA, 1% Triton
X-100] containing 1 mM phenylmethylsulfonyl fluoride
(PMSF), complete protease inhibitor cocktail set (Roche), and 0.5
mM sodium orthovanadate. The lysates were clarified by
centrifugation (15,000 x g) for 1 h,
incubated with Sepharose beads conjugated with antibodies at 4°C for
1 h, and washed with radioimmunoprecipitation assay buffer three
times. The resulting immunocomplex was eluted by boiling in SDS sample
buffer in the presence of 80 mM DTT and was
subjected to Western blotting.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Arg) mutation was fused to MoMuLV LTR, followed by an intron
and SV40 poly(A) signal. By injecting a 7.5-kb linearized fragment into
fertilized eggs from C57BL/6 x BALB/cA mice, we
obtained two founder mice carrying the MoMuLV/RET-MEN2A
gene. Transgenic mouse lines 121 and 180 were established by crossing
these founder mice with C57BL/6 or BALB/cA mice. Southern blot analyses
revealed that the number of transgene copies in line 121 (22 copies)
was approximately seven times higher than that in line 180 (three
copies; data not shown). Although the founder mouse of the high copy
line was mosaic for the transgene (data not shown), F1 mice of the high
copy line, and the founder of the low copy line, transmitted the
transgene in a Mendelian fashion.
Expression of the Transgene.
Mice from the F1 to F3 generations were sacrificed at 3 months of age
and transgene expression was analyzed by RT-PCR. Total RNAs were
prepared from various tissues of transgenic mice as well as
nontransgenic littermates and were amplified using primers specific for
the human RET proto-oncogene. In the case of transgenic mice
from the high copy line, the transgene expression was detected in
brain, parotid gland, heart, liver, and testis at high levels and in
lung, kidney, colon, and thyroid at low levels (Fig. 1A)
, whereas no expression was observed in any organs of
control nontransgenic littermates (data not shown). Four transgenic
mice examined showed the same tissue distributions of the transgene
expression. Transcription of endogenous mouse Ret gene was
detected in submandibular gland at high levels and in brain, heart,
colon, and testis at low levels, although it was undetectable in
thyroid gland under our experimental conditions (Fig. 1A)
.
When amplification of actin mRNA was carried out as a control,
approximately equal efficiency was observed in each tissue sample (Fig. 1A)
. The transgene expression in transgenic mice from the
low copy line was relatively high in heart but very low or undetectable
in other tissues (Fig. 1B)
. The tissues used for RT-PCR were
histologically normal, except the thyroid gland of transgenic mice from
the high copy line in which CCH or medullary carcinoma already
developed at 3 months of age as described below.
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. Gross appearance of thyroids was normal at 4 weeks of age but
microscopic analysis revealed nodular CCH (Fig. 2A)
or MTC in all of the four mice examined. Thyroids from
older transgenic mice (
3 months old) were bigger in size than normal
thyroids and often developed bilateral MTC (9 of 25 mice examined).
Although 17% of transgenic mice at 3–5 months of age (2 of 12 mice)
still showed the development of only CCH, all of the mice at 6–9
months of age (13 mice) developed MTC (Table 1)
. MTC cells grew in a
solid or papillary pattern (Fig. 2D)
, and mitotic figures
were infrequently seen. Some MTCs were associated with amyloid-like
material (data not shown). No evidence of metastasis of MTC to other
organs and lymph-nodes was found.
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. In addition, it was
shown by transmission electron microscopy that tumor cells contain many
electron-dense neurosecretory-type granules (Fig. 2H)
.
Consistent with these findings, serum calcitonin levels were elevated
in all of the transgenic mice from line 121 and appeared to correlate
with tumor sizes. For example, serum calcitonin levels in transgenic
mice with only CCH and with bilateral MTC were approximately, 2-fold
and 8-fold, respectively, higher than those in normal mice (Table 2)
.
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. Nearly one-half of the mice developed one of the two
types of carcinoma at 3–9 months of age (Table 1)
. Four transgenic
mice exhibited lung metastasis of mammary carcinoma. On the other hand,
no histological change was observed in adrenal glands in which the
RET-MEN2A transgene was not expressed.
Detection of Disulfide-linked RET Homodimer and RET-Shc-Grb2
Complex Formation.
We and others previously demonstrated that the MEN2A
mutations that involve cysteine residues present in the RET
extracellular domain activate RET by inducing its ligand-independent
dimerization (25
, 26)
. To examine the state of the
RET-MEN2A mutant proteins in tumor tissues, lysates that were prepared
from MTC and mammary and parotid gland adenocarcinomas that developed
in transgenic mice were analyzed by Western blotting with anti-RET
antibody under nonreducing conditions. A lysate from NIH 3T3 cells
stably expressing the RET-MEN2A protein [designated NIH(C634R)] was
used as a positive control. As shown in Fig. 3A
, 350-kDa RET dimers, in addition to 155-kDa and 175-kDa
monomers, were detected in lysates of these tumors from transgenic mice
under nonreducing conditions.
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, Shc
was highly phosphorylated on tyrosine, and the mutant RET-MEN2A and
Grb2 proteins were coprecipitated with Shc, which showed the complex
formation of these signaling molecules.
When phosphorylation levels of Erk in tumors were analyzed by Western
blotting with anti-phosphoErk (pErk) antibody, they were variable,
depending on tumors (Fig. 3C)
. Among six tumors examined
(one MTC, four parotid gland carcinomas, and one mammary carcinoma),
the phosphorylation was almost undetectable in one parotid gland
adenocarcinoma (Fig. 3C)
, although increased tyrosine
phosphorylation of Shc was observed in the same tumor (Fig. 3B)
. In addition, the phosphorylation levels of Erk in two
parotid gland carcinomas were similar to those in normal parotid glands
(Fig. 3C)
.
Inverse Correlation between GFR Expression and Dimerization of
RET-MEN2A Mutant Proteins.
Because no tumor development was observed in several tissues despite
high levels of expression of the RET-MEN2A transgene, we
further investigated the expression of the RET-MEN2A mutant protein in
them. The expression of the mutant protein was detected in liver,
heart, and brain at high levels (Fig. 4A)
. The expression level was also high in colon from a few
transgenic mice (Fig. 4A)
. RET-MEN2A was expressed as
155-kDa and 175-kDa proteins in liver and heart as observed in MTC and
parotid gland carcinoma, but the molecular mass was a bit different in
colon and brain (Fig. 4A)
, probably because of the
difference in glycosylation form of the mutant proteins. Although a
155-kDa band was present in liver from a normal littermate, it could be
a nonspecific band because the expression of mouse Ret gene
in liver was not detected by RT-PCR (Fig. 1)
.
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, and brain (data not shown).
Consistent with this result, the level of tyrosine-phosphorylation of
the mutant protein was not increased (Fig. 4C)
, which
confirms that dimerization was required for its activation.
Recently, Trupp et al. (11)
reported that the
spontaneous dimer formation of RET expressed in COS cells could
be inhibited in a dose-dependent manner by coexpression with GFR
receptors (GFR1, GFR2 or GFR3). It was also shown that GFRs
were expressed in heart, liver, and brain at high levels
(35)
. Thus, we analyzed the expression of GFR in
tumors developed in transgenic mice to investigate whether it
influences the RET dimerization. RT-PCR analysis revealed that the
expression of GFR1, GFR2, or GFR3 mRNA was undetectable or was
very low in tumors (Fig. 5)
. Heart, liver, brain, and testis in which no tumor formation was
observed expressed at least one of the GFRs at high levels, whereas
their expression in mammary and parotid glands was undetectable or was
very low (Fig. 5)
. These results suggested the possibility that high
levels of expression of GFR receptors at the cell surface may
interfere with dimerization of the RET-MEN2A mutant proteins.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20),
using MoMuLV LTR as a regulatory unit. In this line, the
RET-MEN2A mutant gene was expressed at high levels in brain,
parotid gland, heart, liver, and testis and at low levels in lung,
kidney, colon, and thyroid. Although MoMuLV LTR contains promoter and
enhancer elements known to be active in hematopoietic cells in
vitro, expression of the transgene was undetectable in the spleen
and thymus of our transgenic mice. It is possible that the unique
combination of the sequences present in the MoMuLV/RET-MEN2A
transgene and/or its integration site may affect the transcription
pattern of the transgene.
Despite the widespread transgene expression, however, transgenic mice
displayed a very peculiar tissue-restricted phenotype. That is,
transgenic mice developed calcitonin-secreting CCH and/or medullary
thyroid carcinoma at as early as 4 weeks of age with a complete
penetrance, and about one-half of them developed mammary and/or parotid
gland adenocarcinoma at 5–9 months of age. In these tumors,
disulfide-linked dimerization of the mutant RET protein was clearly
detected as expected. In contrast, no tumor development was observed in
liver, heart, and brain in which the RET-MEN2A mutant proteins were
highly expressed. Unexpectedly, the levels of RET dimerization and
tyrosine phosphorylation were very low in these tissues. Trupp et
al. (11)
reported recently that constitutive tyrosine
autophosphorylation of RET overexpressed in COS cells was inhibited in
a dose-dependent manner by coexpression with GFR receptors (GFR1,
GFR2, or GFR3), which suggests that GFRs could inhibit the
spontaneous formation of RET homodimers in COS cells. In addition, the
same group showed that RET and GFR1 preassemble at the cell surface
in the absence of GDNF (40)
. Thus, to investigate the
relation between GFRs expression and tumor development, we analyzed
the expression of the receptors in tumors. RT-PCR analysis demonstrated
that the expression of GFR1, GFR2, or GFR3 mRNA was
undetectable or very low in the tumors. In contrast, consistent with
the previous report (35)
, GFR1, GFR2 ,and/or GFR3
mRNAs were expressed in heart, liver, and brain at high levels.
Moreover, it has recently been demonstrated that GFR1 expression was
undetectable in thyroid C cells (41)
. Taken together,
these findings supported the view that high levels of GFR expression
may interfere in the dimer formation of the RET-MEN2A mutant proteins
expressed in liver, heart, and brain, which results in suppression of
tumor development in those organs, although it is possible that other
cell surface proteins could also influence the RET-MEN2A dimerization.
In this respect, it is interesting to note that MEN 2A does not develop
the mucosal neuroma and ganglioneuromatosis of the enteric nervous
system that are the characteristic features of MEN 2B. These neuronal
cells affected in MEN 2B are known to express GFR1 at high levels
(4)
. Because the MEN2B mutations activate RET
in a monomeric form (26
, 29
, 30)
, this fact implied that
expression of GFR1 may not impair activation of the RET-MEN2B mutant
protein in neuronal cells. Our results, thus, suggested the possibility
that the expression pattern of GFRs could be one of factors that
determine the clinical features of MEN 2A and MEN 2B, in addition to
possible differences of intracellular signaling activated by RET-MEN2A
and RET-MEN2B proteins (26
, 42
, 43)
.
As a result of RET-MEN2A dimerization, its complex formation with Shc and Grb2 adaptor proteins was induced in tumors. In vitro experiments demonstrated that Shc binds to tyrosine 1062 of RET and is phosphorylated on tyrosine (31 , 36 , 38 , 39) . Then phosphorylated Shc binds to Grb2, leading to activation of Ras-MAPK signaling pathway. When tyrosine 1062 in RET was replaced with phenylalanine, the transforming activity of both RET-MEN2A and RET-MEN2B mutant proteins markedly decreased (30, 31, 32) , which confirmed that Shc binding to RET is crucial for their activity. Despite tyrosine phosphorylation of Shc, however, the levels of Erk phosphorylation were variable, depending on tumors developed in transgenic mice. Because the levels were low in some tumors examined, this suggested that Erk activation may be unnecessary at least for maintenance of malignant phenotype. Interestingly, Sweetser et al. (34) reported similar results using RET-MEN2B transgenic mice. Their study also failed to show a consistent correlation between RET-MEN2B overexpression and activation of Erk. Thus, Shc binding to RET-MEN2A mutant proteins in tumor cells may activate different signaling pathways through other members of MAPK or phosphatidylinositol-3 kinase (44) . Further investigation searching alternative signaling pathways will be necessary to elucidate the mechanisms of tumor development in RET-MEN2A transgenic mice.
All of the 29 transgenic mice analyzed thus far developed CCH and/or MTC. Both types of tumors have already been detected in mice as early as 4 weeks of age. In addition, because one mouse each, at 3 and 5 months of age, developed only CCH, this suggested that secondary genetic alterations could be required for the development of MTC. Consistent with this view, it is well known that loss of heterozygosity was frequently found on chromosomes 1p, 3q, and 22q in human MTC, which suggests the presence of tumor suppressor genes or modifier genes (45) . However, it is still possible that the expression of the RET-MEN2A transgene is sufficient for the development of MTC, depending on its expression levels in thyroid C cells. On the other hand, mammary or parotid gland adenocarcinoma developed at an incidence of 17–46% at 3–9 months of age in a stochastic fashion. This implied that additional genetic events could be more important to acquire tumorigenic properties in these tissues.
In conclusion, the fact that CCH and/or MTC developed in our transgenic
mice with a complete penetrance indicates that they constitute a useful
animal model for developing therapeutic strategies for hereditary MTC
caused by RET mutations. In addition, our results suggest
that the tissue distributions of GFRs may partly explain the
differences in clinical phenotypes between MEN 2A and MEN 2B.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by grants-in-aid
for COE research, Scientific research, and Cancer
research from the Ministry of Education, Science, Sports and Culture of
Japan. 
2 To whom requests for reprints should be
addressed, at Department of Pathology, Nagoya University School of
Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Fax:
81-52-744-2098; Email: mtakaha{at}med.nagoya-u.ac.jp
3 The abbreviations used are: GDNF, glial
cell line-derived neurotrophic factor; GFR, GDNF family receptor
; MEN, multiple endocrine neoplasia; FMTC, familial MTC; MTC,
medullary thyroid carcinoma; MoMuLV LTR, Moloney murine leukemia virus
long terminal repeat; CCH, C-cell hyperplasia; RT, reverse
transcription; MAPK, mitogen-activated protein kinase.
Received 2/ 7/00. Accepted 7/20/00.
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REFERENCES |
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, a novel receptor for GDNF. Cell, 85: 1113–1124, 1996.
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-4 and the tyrosine kinase Ret form a functional receptor complex for persephin. Curr. Biol., 8: 1019-1022, 1998.[Medline]
1 is an essential receptor component for GDNF in the developing nervous system and kidney. Neuron, 21: 53-62, 1998.[Medline]
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| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
