Grm5 expression is not required for the oncogenic role of Grm1 in melanocytes

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Abstract

Melanoma is the aberrant proliferation of melanocytes, the cells in the skin responsible for pigment (melanin) production. In its early stages, melanoma can be surgically removed with great success, however, advanced stages of melanoma have a high mortality rate due to the lack of responsiveness to currently available therapies. We have previously characterized a mouse melanoma model, TG-3, which has implicated the ectopic expression of metabotropic glutamate receptor 1 (Grm1, formerly mGluR1), in melanomagenesis and metastasis [Pollock et al., 2003. Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia. Nat Genet. 34, 108–112.]. Here we report the characterization of several in vitro cell lines derived from independent mouse melanoma tumors. These cell lines show characteristic phenotypes of transformed melanocytes, and express Grm1, and Grm5 (another metabotropic glutamate receptor), as well as melanocyte-specific protein markers. To investigate the possible role of Grm5 in vivo during melanoma development in our mice, we have crossed Grm5 null mice with TG-3, generating a new line of transgenic mice, TGM. TGMs, which are homozygote knockouts for Grm5 and carry the TG transgene, develop tumors with onset, progression, and metastasis very similar to that described for TG-3. Taken together, these results indicate that Grm1 can act as an oncogene in melanocytes independently of Grm5 expression.

Introduction

In our lab, we have characterized the histological and molecular alterations in TG-3, a transgenic mouse line that spontaneously develops melanoma (Chen et al., 1996, Zhu et al., 1998, Zhu et al., 2000, Pollock et al., 2003, Marín and Chen, 2004). Primary tumors arose in ears, tail, and perianal region of the animals, and subsequent pigmented lesions detected in additional tissues were likely due to metastasis (Zhu et al., 1998, Zhu et al., 2000). Molecular characterization of TG-3 showed that integration of the transgene (TG) disrupted part of intron 3 of the gene that encodes for the metabotropic glutamate receptor 1 (Grm1, formerly known as mGluR1 or Gprc1a). Expression of Grm1 was only detected in the tumor tissues, but not in normal tissues (Pollock et al., 2003). No mutations in the coding region of tumor derived Grm1 were found. These results suggest that ectopic expression of Grm1 is sufficient to transform melanocytes and gives rise to malignant melanoma in vivo.

Albino versions of TG-3 have been genetically engineered and characterized previously by our laboratory. Spontaneous cutaneous amelanotic melanomas developed in these albino transgenic mice (Cohen-Solal et al., 2001). The onset and progression of these amelanotic melanoma tumors are very similar to that reported for TG-3. Here we show that tumors in these mice also bear aberrant expression of Grm1 protein as compared to normal melanocytes. These tumors provide an excellent source for the derivation of melanoma cell cultures without melanin. Their characterization with respect to their growth phenotype is described here.

Grm1 is normally expressed and functional in the brain, and is stimulated by the neurotransmitter glutamate. Glutamate receptors belong either to the ionotropic or to the metabotropic families of receptors. Ionotropic glutamate receptors are ligand-gated cation channels, whereas, metabotropic glutamate receptors (mGluRs) are seven-transmembrane domain G-protein-coupled receptors (GPCRs). mGluRs are divided into three groups based on sequence homology, agonist selectivity, and effector coupling. Grm5 (also known as mGluR5 or Gpcr1e) together with Grm1 comprise Group I mGluRs and are positively coupled to adenylate cyclase and inositol triphosphate (IP3) formation (Pin and Duvoisin, 1995, Conn and Pin, 1997).

Grm5, but not Grm1, is normally expressed in melanocytes (Frati et al., 2000). Grm1 and Grm5 null mouse lines have been described. These mice showed reduced long-term potentiation, impaired context specific associative learning, and mild ataxia, but no melanocyte-related abnormalities have been reported (Aiba et al., 1994, Conquet et al., 1994, Lu et al., 1997). To clarify any possible role that Grm5 may play in vivo in melanomagenesis we crossed TG-3 with a Grm5 knockout line (Jia et al., 1998), to generate a TG positive-Grm5 null mouse line. This line of transgenic animals develops melanoma in a way indistinct from TG-3. These results provide compelling evidence that Grm1 serves as an oncogene when ectopically expressed in melanocytes independently of Grm5 expression.

Section snippets

Establishment of melanoma cultures

Ear tumors were excised from several independent mice and divided into two parts. Western blots of Grm1 expression were performed with one part of the tumor and the other part was minced and allowed to establish in culture in RPMI 1640 containing 10% fetal bovine serum (FBS) at 37 °C in a 10% CO2 atmosphere. Melanocytes were gradually selected by incubation of cells with (75–150 μM) geneticin for 3–4 days (Halaban and Alfano, 1984, Sviderskaya et al., 1995). Expression of melanocyte markers and

Derivation and characterization of cells

Several independent cultured cell lines were derived from ear tumors of albino TG-3 lines characterized previously (Cohen-Solal et al., 2001). Tumors removed from these mice were divided into two parts. Protein extraction was performed on one part of the tumors and used in Western immunoblots to verify the ectopic expression of Grm1 (Fig. 1). The other half was used to generate homogeneous in vitro melanoma cell lines, as described previously, by selection of melanocytic cells over

Discussion

Cell lines derived from mouse melanomas are scarce, with B16 cells and its derivatives being by far the most commonly used. The TG-3 mouse melanoma model gave us the opportunity to develop new in vitro melanoma lines derived from a system with well characterized molecular alterations, where ectopic expression of Grm1 in melanocytes promotes melanomagenesis (Pollock et al., 2003). These cell lines are free of other cell types and express melanocyte markers as well as Grm1. We also show that our

Acknowledgments

We thank Drs. D. Bennett and R. Halaben for the normal mouse melanocyte cell line Melan-a and B10Br; Brian Wall for technical assistance, and Jeffrey J. Martino for critical review of this manuscript. This work has been supported by grants: NCI RO1CA108720, NIEHS ES05022, MRF GM55145, NJCCR Summer Fellowship 041261CCRE0, and NCI F31CA103364.

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