Metabotropic glutamate receptor subtype-1 is essential for in vivo growth of melanoma

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Abstract

Ectopic expression of metabotropic glutamate receptor subtype 1 (mGluR1) in mouse melanocytes induces melanoma formation. Although requirement of mGluR1 for development of melanoma in the initial stage has been demonstrated, its role in melanoma growth in vivo remains unclear. In this study, we developed novel transgenic mice that conditionally express mGluR1 in melanocytes, using a tetracycline regulatory system. Pigmented lesions on the ears and tails of the transgenic mice began to appear 29 weeks after activation of the mGluR1 transgene, and the transgenic mice produced melanomas at a frequency of 100% 52 weeks after transgene activation. Subsequent inactivation of the mGluR1 transgene in melanoma-bearing mice inhibited melanoma growth with reduction of immunoreactivity to phosphorylated ERK1/2, whereas mice with persistent expression of mGluR1 developed larger melanoma burdens. mGluR1 expression is thus required not only for melanoma development but also for melanoma growth in vivo. These findings suggest that growth of melanoma can be inhibited in vivo by eliminating only one of the multiple genetic anomalies involved in tumorigenesis.

Introduction

Melanoma arises from the malignant transformation of melanocytes, the cells in the skin responsible for pigment production. In its early stages, melanoma can be surgically removed with great success. However, advanced stages of melanoma have a high mortality rate, because of the lack of responsiveness to currently available therapies. Involvement of glutamate signaling in cancer has recently been reported for various types of tumors such as breast cancer, colon cancer, astrocytoma and lung cancer (Rzeski et al., 2001; Stepulak et al., 2005). Ectopic expression of metabotropic glutamate receptor subtype 1 (mGluR1) in melanocytes has a critical function in the onset of melanoma in transgenic mice, and mGluR1 has been shown to be aberrantly expressed in human melanomas, but not in normal melanocytes (Pollock et al., 2003; Marin et al., 2005; Namkoong et al., 2007).

mGluR1 is a G-protein-coupled receptor activated by glutamate, and has a pivotal function in synaptic transmission, synaptic plasticity, neuronal development and motor learning in the central nervous system (Masu et al., 1991; Nakanishi, 1992, 1994). mGluR1 knockout mice exhibit ataxic gait, deficient long-term depression at parallel fiber-Purkinje cell synapses and impaired motor learning (Conquet et al., 1994; Aiba et al., 1994a, 1994b). mGluR1 is coupled to phospholipase Cβ, leading to triggering of inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release and activation of protein kinase C (Nakanishi, 1994; Conn and Pin, 1997). Furthermore, mGluR1 activates extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinases in neurons and model cell systems (Karim et al., 2001; Thandi et al., 2002).

Consistent with these findings in the central nervous system and cells ectopically expressing mGluR1, stimulation of mGluR1 by L-quisqualate, a group I mGluR agonist, results in IP3 accumulation and activation of ERK1/2 through a PKCɛ-dependent, but not a PKA-dependent pathway in cultured cells established from tumors that ectopically express mGluR1 (Marin et al., 2006). Furthermore, treatment of mGluR1-expressing melanoma cells with an mGluR1 antagonist or glutamate release inhibitor leads to suppression of cell proliferation as well as a decrease in levels of extracellular glutamate (Namkoong et al., 2007). Although requirement of mGluR1 during melanoma development in the initial stage and agonist stimulation of mGluR1 to obtain melanoma growth in vitro have been demonstrated, the roles of mGluR1 in melanoma growth in vivo remain unclear. Furthermore, the signaling pathway stimulated by mGluR1 activation in transformed melanocytes remains largely unknown.

To explore the requirement of continuous mGluR1 expression in growth of melanoma in vivo, we developed mGluR1 conditional transgenic mice using a tetracycline-controlled gene expression system to achieve both cell type-specific and temporal control of mGluR1 expression (Gossen and Bujard, 1992; Van Dyke and Jacks, 2002). The mGluR1 transgenic mice produced melanomas at a frequency of 100%, and subsequent inactivation of the mGluR1 transgene suppressed melanoma growth, suggesting that mGluR1 expression is required not only for melanoma development but also for melanoma growth in vivo.

Results

Generation of a conditional transgenic model for mGluR1-induced melanoma formation

To investigate the role of mGluR1 in melanoma growth in vivo, we developed the mGluR1 conditional transgenic mice using the tetracycline-regulated system (Figure 1a). In an mGluR1-conditional transgenic mouse harboring both NSE-tTA and TRE-mGluR1 transgenes, mGluR1 is expressed under the neuron-specific enolase (NSE) promoter (Chen et al., 1998) in the absence of doxycycline. To express mGluR1 only in adulthood, doxycycline was administered to the mGluR1-conditional transgenic mice during the embryonic period and postnatal 3 weeks. Pigmented tumors on the pinnae and tails of mGluR1-conditional transgenic mice began to appear 29 weeks after withdrawal of doxycycline. By 52 weeks after withdrawal of doxycycline, the mGluR1 transgenic mice (n=20) produced melanomas with a frequency of 100% (Figure 1b). In contrast, NSE-tTA transgenic mice (n=21; Figure 1b), and the mGluR1 transgenic mice treated continuously with doxycycline (n=5; data not shown) produced no lesions.

Figure 1
figure1

Generation of mGluR1 conditional transgenic mice. (a) Schematic diagram of mGluR1 expression by the tetracycline-regulated gene system. In the mGluR1 conditional transgenic mice, mGluR1 expression depends on two transgenes in the absence of doxycycline (Dox). tTA binds to the TRE sequence and activates transcription of mGluR1. In the presence of Dox, Dox binds to tTA and blocks its binding to the TRE sequence. (b) Kaplan–Meier graph of incidence of melanoma in mGluR1-conditional transgenic mice (mGluR1 Tg). NSE-tTA transgenic mice, which did not express mGluR1, served as a control (Control).

Conditional tumorigenesis by an mGluR1 transgene in melanocytes

To confirm mGluR1 expression in melanomas of the mGluR1 transgenic mice, we carried out RT–PCR and western blot analyses using total RNAs and proteins from pinnae and tail tumors of mGluR1-conditional transgenic mice 60 weeks after doxycycline depletion. We detected mGluR1 mRNA (Figure 2a) and protein (Figure 2b) in the pinnae and tail tumors of mGluR1-conditional transgenic mice, but not pinnae or tail of wild-type mice. The color of pinnae and tail became darker around 20 weeks after withdrawal of doxycycline. Histopathological examination showed that spindle-shaped dermal melanocytes were significantly increased and these melanocytes contain more melanin 30 weeks after withdrawal of doxycycline (Figure 2c) with the gradual increase of mGluR1 mRNA as well as TRP1 mRNA (Figure 2d). These results reflect enhanced melanocytic proliferation in tail of mGluR1-conditional transgenic mice. Finally, by 52 weeks of doxycycline depletion, all mGluR1 transgenic mice examined had developed melanoma (Figure 1b). These lesions as well as perianal/fungoid rectal and ocular masses began to appear gradually. There were no apparent abnormalities of the lungs, liver, kidneys, gastrointestinal tract or cardiac muscle, though lymph node involvement was observed (data not shown). Tumor formation tended to occur on the hairless skin areas rather than the trunk as reported in previous studies, probably because there are more dermal melanocytes retained in these areas (Pollock et al., 2003). Dissection of the cervical, thoracic, abdominal and inguinal lymph nodes revealed several dark masses (2–5 mm in diameter) associated with the nodes (data not shown). By 100 weeks after withdrawal of doxycycline, these mice became lean with heavy burden of skin melanoma and died.

Figure 2
figure2

Ectopic expression of mGluR1 induces melanoma formation. (a) RT–PCR analyses of mGluR1 and melanocyte marker, TRP1 mRNAs using RNAs extracted from pinnae and tail with tumors of mGluR1 transgenic mice (Tg) and normal pinnae and tail of wild-type mice (WT) and mGluR1 knockout mice (KO). Cerebellar RNA containing endogenous mGluR1 mRNA at a high level was used as a positive control. GAPDH mRNA was used as an internal control. (b) Western blot analysis of mGluR1 protein in cerebellum, pinnae and tail. Expression of α-tubulin was used as an internal control. (c) Representative histology of tails of inducible mGluR1 transgenic mice at various times in melanoma development. Spindle-shaped dermal melanocytes were significantly increased 30 weeks after withdrawal of doxycycline. The histopathology of this tumor shows the proliferation of strongly melanin-containing rounded large cells. Scale bars: 100 μm. (d) RT–PCR analysis of mGluR1 and TRP1 mRNA in tails from mGluR1 transgenic mice at various times in melanoma development.

Histopathological evaluation of tumors in the mGluR1 transgenic mice

Histologically, multifocal-pigmented tumor nests or nodules were detected in the dermis of the skin (Figures 2c and 3a and b). Large tumors exhibited invasion of melanoma cells to the skeletal muscle (Figure 3a). The tumors contained a mixture of strongly melanin-containing rounded cells grouped in irregular bundles with interspersed fibroblasts and connective tissue (Figures 2c and 3b). Infiltration of inflammatory cells into the tumors was uncommon. Examination of sections of lymph nodes stained with hematoxylin and eosin revealed rounded cells with large cytoplasm filled with melanosomes, which are similar to these cells in the dermis of multipigmented tumors (Figures 2c, 3b and c and 4). Mitoses were rare in even the most severely affected nodes (Figure 4). Although there was no significant nuclear atypia, which is commonly observed in human melanoma cells, their capacity for invasion to the muscles and metastasis to the lymph nodes was suggestive of malignancy. The similar observation that melanoma cells had invasive ability in the absence of mitoses was also reported by Chen et al. (1996). The distribution of lesions reflected expansion and metastasis, but not activation of local histiocytes. Because melanin-containing cells in the lymph nodes were stained positive with S-100 as well as TRP1 and mGluR1 (Figure 4), they were considered to be melanoma cells, but not macrophages which had phagocytosed extracellular melanin or melanosomes. Constitutive activation of ERK 1/2 has been frequently found in human melanoma tissues (Satyamoorthy et al., 2003; Huntington et al., 2004). Large, melanin-containing ballooning cells exhibited immunoreactivities for phosphorylation of ERK1/2 as well as TRP1 and mGluR1 (Figure 4). These findings indicate that ectopic expression of mGluR1 in melanocytes leads to activation of ERK1/2, and consequently the development of melanoma.

Figure 3
figure3

Hyperproliferative dermal pigment producing cells in the mGluR1 transgenic mice are metastasized in the lymph nodes. Hematoxylin and eosin-stained paraffin section of tail (a), pinnae (b) and lymph node (c). An arrow indicates invasion of melanoma cells to skeletal muscle (a). Scale bars: 1 mm (a and c), 200 μm (b).

Figure 4
figure4

Metastasized pigment producing cells express S100 and phosphorylated ERK1/2 as well as mGluR1 and TRP1. Sections of lymph node stained with antibodies to mGluR1, TRP1, S100 or phosphorylated ERK1/2. These expressions were confined to the pigmented melanoma cells in the lymph node of the mGluR1 transgenic mouse. Negative control with mouse serum showed no specific staining (Control). Scale bars: 100 μm.

Lack of mutation of B-raf or N-ras genes in mGluR1-expressing melanoma cells

S-phase promotion occurs through activated ERK signaling due for the most part to B-raf or N-ras mutation in human melanomas (Davies et al., 2002; Wong et al., 2005; Thomas, 2006). Somatic missense mutations of the B-raf gene are found in 66% of human malignant melanomas and all of the mutations of B-raf were found within the kinase domain. A single substitution of glutamate for valine (V600E) was responsible for 80% of these B-raf mutations. Ras mutations have been identified in 15% of melanomas, with most occurring at codon 61 of N-ras, whereas K-ras and H-ras mutations are relatively rare. We therefore evaluated the tumors for the presence of these activating mutations of B-raf and N-ras genes by PCR amplification and direct sequencing of PCR products. No mutation of B-raf or N-ras was observed in melanomas of the mGluR1 transgenic mice (n=10, from 9 transgenic mice), although melanomas in mGluR1-conditional transgenic mice exhibited activation of ERK. These findings suggest that mGluR1 induces melanoma without known activating mutations of B-raf or N-ras genes.

Doxycycline re-administration suppresses mGluR1 expression and melanoma growth

We next examined whether tumor growth can be suppressed by inactivation of the mGluR1 transgene by doxycycline. Mice were divided into two groups 42 weeks after withdrawal of doxycycline. The ‘−Dox’ group (mGluR1-ON) was maintained without intervention, whereas the ‘+Dox’ group (mGluR1-OFF) was treated with 50 μg/ml doxycycline in drinking water for 12 weeks. Doxycycline re-administration suppressed mGluR1 protein expression in tail tumors (Figure 5a) and reduced the level of mGluR1 mRNA in pinna and tail tumors of the mGluR1 transgenic mice (Figure 5b). The size of melanomas in ‘+Dox’ mice (n=20) was not significantly changed after 12 weeks doxycycline re-administration, whereas the size of melanomas in ‘−Dox’ mice (n=18) was significantly increased (Figure 5c). No obvious histological differences in size, attachment, or arrangement of melanoma cells were observed between melanomas in the ‘−Dox’ and ‘+Dox’ mice (Figure 5a). Melanoma cells in both groups were heavily melanized, rounded cells, which grouped in irregular bundles. Invasive and metastatic characters were still kept in ‘+Dox’ mice. None of the ‘+Dox’ mice cleared the melanoma burden and their tumor size was not significantly changed, although melanoma in ‘−Dox’ mice were continuously enlarged during the observation period of 24 weeks (data not shown). To examine the ligand dependency of mGluR1 in melanoma growth, the mGluR1 transgenic mice harboring melanoma were treated with glutamate release inhibitor, riluzole (200 μg/ml) in drinking water for 4 weeks. In contrast to untreated control mice (n=6), the growth of melanoma in mice treated with riluzole (n=8) was significantly inhibited (Figure 5d). These findings indicate that activation of mGluR1 by glutamate is required for melanoma growth. Although melanoma cells in tail melanomas of ‘−Dox’ mice exhibited phosphorylated ERK1/2, those of ‘+Dox’ mice were not stained for phosphorylated ERK1/2 (Figure 5e). Furthermore, the ratio of terminal deoxy-nucleotidyl transferase dUTP nick-end labeling positive cells were significantly increased in tail melanomas of ‘+Dox’ mice (Figure 5e; +Dox, 37.9±2.7%; −Dox,14.5±3.3%; t-test, P<0.01), suggesting that apoptosis of melanoma cells was induced by blockade of mGluR1 expression. Enhanced inflammatory cell infiltration or fibrotic change in the tumor area of ‘+Dox’ mice were not observed. These findings indicate that continuous expression of mGluR1 is required for ERK activation and melanoma growth as well as melanoma survival.

Figure 5
figure5

Doxycycline re-administration suppresses mGluR1 expression and melanoma growth. (a) Sections of tails from untreated mGluR1 transgenic mice (−Dox) and doxycycline-treated mGluR1 transgenic mice (+Dox) stained with antibodies to mGluR1 and hematoxylin–eosin. Scale bars: 200 μm (mGluR1), 20 μm (HE). (b) RT–PCR analysis of mGluR1 mRNA in pinnae, tail and cerebellum from untreated mGluR1 transgenic mice (−Dox) or doxycycline-treated mGluR1 transgenic mice (+Dox). (c) Quantification of size of tail tumors in ‘−Dox’ and ‘+Dox’ mice. The longest dimension of each tumor was measured. Values are the mean±s.e.m. (t-test, P<0.01). (d) Quantification of size of tail tumor in untreated (Control) and riluzole-treated mGluR1 transgenic mice (Riluzole). Values are the mean±s.e.m. (t-test, P<0.01). (e) Immunohistochemical analysis with antibody to phosphorylated ERK1/2, and terminal deoxynucleotide transferase-mediated nick-end labeling staining of sections of tail melanomas in ‘−Dox’ and ‘+Dox’ mice. Only the positive control endothelial cells were stained with this antibody (arrow) in the mouse re-administered doxycycline. Scale bars: 50 μm.

Discussion

We found in this study that growth of malignant melanomas induced by a conditionally expressed mGluR1 transgene can be inhibited when the transgene is inactivated. mGluR1 signaling is required for both melanocyte and melanoma growth. Additional genetic events are required to produce the malignant melanomas during the promotion stage after initiation by the mGluR1 transgene, as melanoma formation on the mice began to appear 29 weeks after ectopic expression of mGluR1. Despite these genetic events, the growth of melanomas induced by mGluR1 expression depends on the continuous expression of mGluR1. Blockade of the mGluR1 transgene leads to inhibition of melanoma growth and survival, accompanied by the reduction of immunoreactivity to phosphorylated ERK1/2. Activation of ERK1/2 has been linked to melanoma development by enhancing several key oncogenic features of the cell including increased cell proliferation, survival, invasion and tumor angiogenesis (Sharma et al., 2005). Taken together, our finding suggests that inactivation of the transgene leads to inactivation of ERK1/2, and consequent inhibition of tumor growth and survival.

In previous studies, mGluR1 transgenic mice in which mGluR1 was driven by dopachrome tautomerase (Dct) promoter produced melanomas in the pinnae, tail, snout and eyelid as well as in lymph nodes (Pollock et al., 2003) and mGluR1 was aberrantly expressed in human melanomas (Pollock et al., 2003; Namkoong et al., 2007). In the present study, an mGluR1 transgene driven by the NSE promoter produced melanomas in the same organs as in Dct-mGluR1 transgenic mice, further confirming that ectopic expression of mGluR1 in melanocytes produced melanoma, which is relevant to human disease.

Continuous mGluR1 expression on melanocytes could drive transformation as a potent initiating and gain-of-function factor, as all transgenic mice developed melanoma after a period of latency. However, abrogation of mGluR1 expression after melanoma development inhibited melanoma growth, though significant reduction of melanoma was not observed. In the case of oncogenic H-Ras (H-RasG12V) transgenic mice deficient in tumor suppressor INK4a, H-RasG12V downregulation resulted in regression of tumors, suggesting that continuous expression of H-RasG12V is essential for tumor maintenance (Chin et al., 1999). Melanomas in H-RasG12V transgenic mice and those in the mGluR1 transgenic mice exhibited different histopathological features. Melanomas in H-RasG12V transgenic mice deficient in INK4a were manifested amelanotic, invasive and highly vascular tumors with spindle morphology and pleomorphic cytology, whereas those in the mGluR1 transgenic mice were highly melanotic and invasive with round morphology and no significant nuclear atypia. These differences in the histopathological features of melanomas may be caused by the differences of the downstream effectors between H-RasG12V and mGluR1. In the present instance, the spontaneous genetic alterations that circumvented the need of mGluR1 for tumor maintenance might have been acquired de novo in the melanomas after a certain period of continuous mGluR1 expression. Indeed, development of melanoma requires the cooperative effects of multiple genetic lesions, though hot spot mutations in the B-raf and N-ras genes were not observed in our transgenic mice. It needs to be examined whether genetic lesions in our mice are similar to those previously observed in melanoma (Michaloglou et al., 2008).

Deregulated receptor tyrosine kinase signaling, leading to activation of ERK1/2 is one of the most important features of metastatic human melanoma (Satyamoorthy et al., 2003; Huntington et al., 2004; Nambiar et al., 2005). Oncogenic mutations in the B-raf and N-ras genes (Davies et al., 2002; Wong et al., 2005; Thomas, 2006) as well as autocrine growth factor signaling loops are frequently observed in human melanomas. In mGluR1 transgenic mice, melanoma cells were stained positive for phosphorylated ERK1/2 antibody, though, as noted above, hot spot mutations in B-raf and N-ras were not observed. A previous study showed that stimulation of mGluR1 by L-quisqualate, a group I mGluR agonist, resulted in IP3 accumulation and activation of ERK1/2 through a PKCɛ-dependent pathway in cultured cells established from tumors that ectopically express mGluR1 (Marin et al., 2006). Furthermore, riluzole suppresses cell proliferation and also decreases extracellular glutamate levels in culture of human melanoma cells (Namkoong et al., 2007). Similarly, riluzole suppressed melanoma growth in our mice. It thus appears possible that melanoma cells in our mice received autocrine stimulation through glutamate release and activation of mGluR1 signaling, leading to activation of ERK1/2.

Our mGluR1-expressing mice may provide an attractive model of human melanoma, as some human melanoma cells have been shown to express this receptor strongly (Pollock et al., 2003; Namkoong et al., 2007), and as cell type- and stage-specific expression of mGluR1 results in escape of the mice from diverse tumors such as soft tissue sarcomas, lymphomas and other cancers of internal organs that limit the lifespan of mice.

We found that growth of melanoma can be inhibited in vivo by eliminating only one of the multiple genetic anomalies involved in tumorigenesis. Our finding is consistent with a previous observation that an mGluR1 antagonist suppressed cell proliferation of human melanoma cells in vitro (Namkoong et al., 2007). It remains to be determined whether the targeted inactivation of mGluR1 is sufficient to inhibit tumor growth in various types of human melanoma. In addition, the further identification of successive mGuR1-induced signaling which is critically involved in conferring melanoma survival might yield additional clues to treatment of melanoma.

Materials and methods

Generation of mGluR1 conditional transgenic mice

The TRE-mGluR1 transgene was composed of rat mGluR1a cDNA (a gift from Dr Nakanishi), the TRE sequence from pTRE2 vector (Clontech, Palo Alto, CA, USA), and polyadenylation sites derived from the rabbit β-globin gene. The chicken β-globin insulator sequence from pJC5-4 (Chung et al., 1993) was introduced into the end of the construct. TRE-mGluR1 DNA fragments were microinjected into the C57BL/6N embryos. Progenies were screened for the presence of the transgenes by PCR and Southern blot analysis of tail DNAs. Because of embryonic lethality of the mGluR1 transgenic mice without doxycycline, mice were administered 50 μg/ml doxycycline (Sigma, St Louis, MO, USA) until weaning (3–4 weeks of age). Riluzole (Sanofi-Aventis, Paris, France) was dissolved in 0.01 N HCl at a concentration of 200 μg/ml and adjusted to pH 7.0 by NaOH. The riluzole solution was given as drinking water and changed two times a week. Animals were housed at 21 °C with free access to food and water. This study was approved by the Committee on Animal Experimentation of Kobe University School of Medicine and carried out according to the guidelines for the care and use of experimental animals of Kobe University School of Medicine.

Histology

A general dissection was performed, and tissues were taken for histological examination by light microscopy. Formalin-fixed, paraffin-embedded tissue sections which contain melanin were first bleached by standard procedures to decolorize melanin (0.25% potassium permanganate 60 min and 1% oxalic acid 5 min, at 20 °C) after being deparaffinized and rehydrated (Handerson and Pawelek, 2003). To retrieve antibody-binding epitopes, sections in 10 mM citric acid buffer at pH 6.0 were microwaved intermittently for a total of 10 min to maintain boiling temperature. After cooling, the slides were placed in cold methanol containing 3% H2O2 for 15 min to block endogenous peroxidase activity. Some of the samples were then incubated with monoclonal or polyclonal antibodies directed against mGluR1 (mouse monoclonal, BD Bioscience, San Jose, CA, USA), tyrosinase-related protein 1 (TRP1; goat polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA), S100 (mouse monoclonal, Abcam Ltd, Cambridge, UK), and phosphorylated ERK1/2 (rabbit monoclonal, Cell Signaling Technology, Beverly, MA, USA). Binding of these antibodies was detected using a Nichirei AEC kit (Nichirei, Tokyo, Japan). Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining was performed with an Apoptag Direct in situ apoptosis detection kit (Chemicon International, Temecula, CA, USA) according to the manufacturer's protocol.

Reverse transcription–PCR analysis

Total RNAs were isolated using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Reverse transcription (RT) was carried out using AMV reverse transcriptase (Takara, Tokyo, Japan), and RCR was carried out at an annealing temperature of 55–60 °C for each primer for 30–40 cycles. The primer pairs used were as follows: mGluR1, 5′-IndexTermTTCAAGACCCGCAACGTGCC-3′ and 5′-IndexTermCAGACTTGCCGTTAGAATTGG-3′; TRE-mGluR1, 5′-IndexTermTTCAAGACCCGCAACGTGCC-3′ and 5′-IndexTermCAGACTTGCCGTTAGAATTGG-3′; TRP1, 5′-IndexTermTGGACACACTATTATTCAGT-3′ and 5′-IndexTermCCCAGTTGCAAAATTCCAGTAG-3′; GAPDH, 5′-IndexTermATGGTGAAGGTCGGTGTGAACG-3′ and 5′-IndexTermTGGTGAAGACGCCAGTAGACTC-3′.

Western blot analysis

Tissues were homogenized in 10 volumes of buffer containing 0.32 M sucrose, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA and protease inhibitors (Complete Mini, EDTA-free; Roche, Mannheim, Germany). We determined the protein concentration of each sample with a BCA protein assay kit (Thermo scientific, Rockford, IL, USA). Twenty micrograms of proteins were separated by SDS–PAGE and transferred to Immobilon-P membrane (Millipore, Billerica, MA, USA). We probed the membrane with the antibodies to mGluR1 (BD) and α-tubulin (Sigma) followed by anti-mouse HRP-conjugated secondary antibody. Bands were visualized with ECL Plus detection reagents (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA).

Sequencing analysis of B-raf and N-ras gene mutations

Genomic DNAs isolated from melanomas of the mGluR1 transgenic mice were used as a template for PCR. Exon 15 of B-raf containing codon 637, which is equivalent to codon 600 of human B-raf, exon 1 containing codons 12 and 13 and exon 2 containing codon 61 of the N-ras gene were amplified, and the nucleotide sequences of these fragments were determined by dye terminator cycle sequencing (Applied Biosystems, Foster City, CA, USA). The primers for amplification of the exon sequences were as follows: B-raf exon 15, 5′-IndexTermTCCTTTACTTACTGCACCTCAG-3′ and 5′-IndexTermATGTGACCAACTGAGATACCTC-3′; N-ras exon 1, 5′-IndexTermTATTGTAGGTTTGGTTTGCC-3′ and 5′-IndexTermCTCTATGGTGGGATCATATT-3′; N-ras exon 2, 5′-IndexTermTCCTCACTCTTTCATATTCC-3′ and 5′-IndexTermAATATCCCCAGTACCTGTAG-3′. Amplification conditions included premelting at 94 °C for 2 min, 30 cycles of melting at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min, with extension at 72 °C for 7 min in the last cycle. The primers for sequencing were as follows: B-raf exon 15, 5′-IndexTermTCCTTTACTTACTGCACCTCAG-3′ and 5′-IndexTermATGTGACCAACTGAGATACCTC-3′; N-ras exon 1, 5′-IndexTermCGTAATTGCTGCTTTTCTAC-3′ and 5′-IndexTermCATCCACAAAGTGGTTCTGG-3′, N-ras exon 2, 5′-IndexTermTTCTTACCGAAAGCAAGTGG-3′ and 5′-IndexTermTGATGGCAAATACACAGAGG-3′.

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Acknowledgements

We thank Dr S Nakanishi for rat mGluR1a cDNA, Dr Y Ishida for β-globin insulator and the members of Aiba lab for helpful discussion. This study supported in part by Grant-in-Aid for Scientific Research on Priority Areas—Molecular Brain Science, a grant for the 21st Century COE Program ‘Center for Excellence for Signal Transduction Disease: Diabetes Mellitus as Model’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and grants from the Naito Foundation, the Takeda Science Foundation, the Uehara Memorial Foundation and the Astellas Foundation for Research on Metabolic Disorders.

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Correspondence to A Aiba.

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Keywords

  • mGluR1
  • melanoma
  • transgenic
  • tetracycline

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