Original Paper

Oncogene (2004) 23, 2347–2356. doi:10.1038/sj.onc.1207405 Published online 26 January 2004

Genomic alterations in spontaneous and carcinogen-induced murine melanoma cell lines

Vladislava O Melnikova1, Svetlana V Bolshakov1, Christopher Walker1 and Honnavara N Ananthaswamy1

1Department of Immunology, The University of Texas MD Anderson Cancer Center, PO Box 301402, Unit 902, Houston, TX 77030, USA

Correspondence: HN Ananthaswamy, E-mail: hanantha@mdanderson.org

Received 5 September 2003; Revised 6 November 2003; Accepted 26 November 2003; Published online 26 January 2004.

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Abstract

We have conducted an analysis of genetic alterations in spontaneous murine melanoma cell line B16F0 and its two metastatic clones, B16F1 and B16F10 and the carcinogen-induced murine melanoma cell lines CM519, CM3205, and K1735. We found that unlike human melanomas, the murine melanoma cell lines did not have activating mutations in the Braf oncogene at exon 11 or 15. However, there were distinct patterns of alterations in the ras, Ink4a/Arf, and p53 genes in the two melanoma groups. In the spontaneous B16 melanoma cell lines, expression of p16Ink4a and p19Arf tumor suppressor proteins was lost as a consequence of a large deletion spanning Ink4a/Arf exons 1alpha, 1beta, and 2. In contrast, the carcinogen-induced melanoma cell lines expressed p16Ink4a but had inactivating mutations in either p19Arf (K1735) or p53 (CM519 and CM3205). Inactivation of p19Arf or p53 in carcinogen-induced melanomas was accompanied by constitutive activation of mitogen-activated protein kinases (MAPKs) and/or mutation-associated activation of N-ras. These results indicate that genetic alterations in p16Ink4a/p19Arf, p53 and ras-MAPK pathways can cooperate in the development of murine melanoma.

Keywords:

melanoma, Braf, ras, ERK1/2, p53, INK4a/ARF

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Introduction

Melanoma is the second most rapidly growing type of cancer in humans (Rigel and Carucci, 2000). It is characterized by a high metastatic propensity, resistance to any known cancer therapies, and extremely poor patient survival rate (Herlyn, 1993; see references in Jhappan et al., 2003). Development of melanoma is associated with epigenetic factors such as excessive exposure of skin to intense ultraviolet (UV) irradiation (Elwood and Jopson, 1997), and a growing number of genetic alterations. Inactivation of the INK4a/ARF melanoma susceptibility locus has been identified in approximately 20–30% of multiple case melanoma families and 15–30% of sporadic melanomas (Fountain et al., 1990; Flores et al., 1996; Piccinin et al., 1997; Halushka and Hodi, 1998; Monzon et al., 1998; Gruis et al., 1999; Bishop et al., 2002). The INK4a/ARF locus encodes two independent growth inhibitors and effectors of cellular senescence: the cyclin-dependent kinase (CDK) inhibitor p16INK4a and the p53 activator p14ARF (mouse p19Arf). These two protein products arise from transcription of overlapping sequences using alternative reading frames. The transcripts are initiated from separate promoters in 5' exons 1alpha (p16INK4a) and 1beta (p14ARF) and are spliced to include the common exons 2 and 3 (Mao et al., 1995; Quelle et al., 1995; Stone et al., 1995). p16INK4a inhibits cell-cycle progression from G1 to S phase by binding and inactivating CDK4/6, thus, promoting sequestration of the transcription factor E2F1 by nonphosphorylated retinoblastoma-susceptibility (pRb) tumor suppressor protein (Serrano et al., 1993; Koh et al., 1995; Lukas et al., 1995). p14ARF mediates G1 and G2 arrests by binding to human double minute protein 2 (HDM2, mouse Mdm2) and blocking HDM2-induced translational silencing and degradation of p53 (Pomerantz et al., 1998; Tao and Levine, 1999), which itself has been found to be mutated in 10–30% of cultured human melanoma cell lines (Volkenandt et al., 1991; Weiss et al., 1993; Albino et al., 1994), and at 0% (Albino et al., 1994; Lubbe et al., 1994; Papp et al., 1996) or 20–25% frequency (Sparrow et al., 1995; Hartmann et al., 1996) in melanoma tumor tissues. Both p16INK4a and p14ARF are bona fide tumor suppressor proteins (Hussussian et al., 1994; Serrano et al., 1996; Kamijo et al., 1997; Sharpless et al., 2001). This is explained at least in part because of the unique ability of p16INK4a to constrain cell proliferation and induce density-dependent growth arrest (Sharpless et al., 2002) and the ability of p14ARF to upregulate p53 in response to abnormal proliferative signals such as activation of the ras oncogene (Bates et al., 1998; Palmero et al., 1998; Lin and Lowe, 2001).

A search for genetic alterations in melanomas recently revealed oncogenic somatic mutations in the BRAF gene in 66% of primary sporadic human melanomas, and at lower rate in other cancers (Brose et al., 2002; Davies et al., 2002; Rajagopalan et al., 2002; Cohen et al., 2003; Dong et al., 2003; Gorden et al., 2003; Pollock et al., 2003; Weber et al., 2003; Xu et al., 2003). All mutations were within the kinase domain and 80% of them were a V599E (exon 15) substitution. Additional mutations were in codon 465 (exon 11). There is an inverse correlation between the occurrence of mutations in the BRAF gene and in the ras oncogene, which is activated in approximately 10–30% of human melanomas (Albino et al., 1989; Van't Veer et al., 1989; Jafari et al., 1995; Omholt et al., 2002). This suggests that the two proteins are in the same pathway of melanomagenesis. Activation of BRAF protein by ras binding triggers transduction of proliferative signaling to the mitogen-activated protein kinase ERK1/2 kinase (MEK1) and extracellular signal regulated (ERK1/2) MAP kinases. The human BRAF gene is located on chromosome 7q34 and is 97.8% similar to the mouse Braf gene, which is on chromosome 6. It is not known whether similar mutations in the Braf gene occur in mouse melanomas. Finally, the loss of the PTEN (phosphatase and tensin homologue deleted on chromosome 10) gene product occurs in 30–60% of sporadic human melanomas (Bastian et al., 1998).

Rodents are known to be resistant to spontaneous and induced melanoma development, but some transgenic mice are important models for investigating the interaction between environment and genetics in melanoma development. Transgenic H-ras(G12V)/Ink4a-/-/Arf-/- and H-ras(G12V)/p53-/- mice frequently develop spontaneous melanomas. Melanoma genesis and maintenance in these mice depends strictly on overexpression of inducible mutant H-ras(G12V) (Chin et al., 1999; Wong and Chin, 2000; Bardeesy et al., 2001). In addition, a single erythemal dose of UV light to neonatal, but not adult, transgenic mice that overexpress hepatocyte growth factor/scatter factor results in the development of cutaneous melanoma (Noonan et al., 2001; Jhappan et al., 2003). However, very few spontaneous or carcinogen-induced melanomas have developed in wild-type mice, and many of the tumors have not been fully characterized genetically.

In this study, we analysed the spontaneous mouse melanoma cell line B16F0 and its metastatic clones B16F1 and B16F10 (Fidler, 1973), and the carcinogen-induced melanoma cell lines CM519, CM3205, and K1735 (Kripke, 1979; Romerdahl and Kripke, 1986) for genetic alterations in p16Ink4a, p19Arf, ras, Braf, and p53. We found that in the spontaneous melanoma cell lines, expression of p19Arf and p16Ink4a was lost as a result of simultaneous deletion of INK4a/ARF exons 1alpha, 1beta, and 2. In the three carcinogen-induced melanoma cell lines, mutations in p19Arf or p53 cooperated with constitutive activation of MAPKs or/and mutation-induced activation of N-ras. We also found that unlike human melanomas, murine melanomas did not have mutations in the Braf gene exon 11 or 15.

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Results

p16ink4a and p19arf alterations

We analysed six murine melanoma cell lines for the expression of the tumor suppressor proteins p16Ink4a and p19Arf (Figure 1). Immunoblotting of whole-cell lysates revealed high levels of p16Ink4a expression in CM519 and CM3205 cells. In contrast, relatively low levels of p16Ink4a expression were observed in K1735 cells. In addition, p16Ink4a protein was not expressed in B16F0, B16F1, or B16F10 cells. Similarly, p19Arf protein was expressed in CM519 and CM3205 cells but was not detected in K1735 cells or the B16 cell lines. Expression of p16Ink4a protein was further confirmed by immunocytochemical detection (Figure 2). CM519, CM3205, and K1735 cells showed positive reactivity with an anti-p16Ink4a antibody. In these cells, p16Ink4a was expressed mostly in the nucleus. As expected p16Ink4a staining was not observed in the B16 cell lines. Immunocytochemical analyses with an anti-p19Arf antibody revealed strong expression and nucleolar localization of p19Arf in CM519 and CM3205 cells. This pattern of localization is characteristic of overexpressed functional p19Arf (Zhang and Xiong, 2001). However, no p19Arf staining was observed in the B16 cell lines (Figure 2).

Figure 1.
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Expression of p16Ink4a and p19Arf proteins. Whole-cell lysates were subjected to Western blot analysis. Equal loading of protein was verified by using anti-beta-actin antibody. The results are representative of three independent experiments

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Figure 2.
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Immunocytochemical staining showing intracellular localization of p16Ink4a (left panels) and p19Arf (right panels) proteins. Note the diffuse nuclear and cytoplasmic expression of p16Ink4a in CM519, CM3205, and K1735 cells and nucleolar expression of p19Arf in CM519 and CM3205 cells. B16F0 (and B16F1, and B16F10 cells, data not shown) did not express p16Ink4a and p19Arf

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To determine whether the loss of p16Ink4a and p19Arf was due to deletions or mutations, we amplified and sequenced exons 1alpha, 1beta, and 2 of the Ink4a/Arf locus. Figure 3 shows that polymerase chain reaction (PCR) amplification of p16Ink4a exon 1alpha yielded a band of the expected size (246 bp) in CM519, CM3205, and K1735 cells and normal control samples (DNA from C3H and C57/BL mice). Direct sequencing of the PCR products revealed a wild-type p16Ink4a exon 1alpha sequence in these three-cell lines. In contrast, PCR amplification of B16F0, B16F1, and B16F10 DNA produced faint bands of unexpected sizes (283 and 224 bp). These aberrant bands were not PCR artifacts because reextraction of DNA from the B16 cell lines (two times) and repeated PCR analysis (four times, including PCR with different set of primers) produced the same results (data not shown). Nucleotide sequencing of the aberrant bands and a BLAST homology search (Altschul et al., 1997) revealed that these two mutant sequences did not correspond to any known mouse genes (data not shown). Most probably, the two bands were the result of a rearrangement or translocation of unknown DNA segments. Nonetheless, these results indicate that p16Ink4a exon 1alpha was deleted in the B16 melanoma cell lines.

Figure 3.
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PCR amplification of the p16Ink4a and p19Arfgenes (Ink4a/Arf exons 1alpha, 1beta, and 2). The large arrows indicate the positions and sizes of the expected wild-type transcripts. Marker (M) size is indicated by small arrows

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Analogous to exon 1alpha, PCR amplification of exon 1beta also gave rise to a band of the expected size (218 bp) in both CM cell lines but not in K1735 and B16 cells (Figure 3). In addition, exon 2 was deleted in the B16 cell lines but not in the other melanoma cell lines (Figure 3). Sequencing analysis revealed only wild-type sequences in exon 1beta in CM519 and CM3205 cell lines, and in exon 2 in both CM and K1735 cell lines. Therefore, loss of p19Arf expression in K1735 cells was a result of a deletion of only exon 1beta, whereas loss of p19Arf and p16Ink4a expression in B16 cells were due to simultaneous deletion of exons 1alpha, 1beta, and 2 (Table 1).


ras mutations

Genomic DNA from murine melanoma cell lines was analysed for oncogenic mutations in codons 12 and 13 (exon 2) and 61 (exon 3) of the H-, K-, and N-ras genes by PCR amplification followed by nucleotide sequencing. The results indicated that CM519 had mutations in both alleles of N-ras codon 61 (CAA right arrow CAT, Table 1), and K1735 in both alleles of N-ras codon 13 (GGT right arrow GAT, ACC right arrow ATC on the opposite strand, Table 1). In contrast, CM3205 and the B16 cell lines did not have mutations in any of the ras genes.

Braf status

Activating mutations in the BRAF gene were recently shown to occur in 66% of human melanomas (Davies et al., 2002). We therefore examined mouse melanoma cell lines for the presence of activating mutations in codon 465 (exon 11) and codon 599 (exon 15) of the Braf gene. Both exons were successfully amplified by PCR in all six-cell lines (Figure 4a). However, sequencing of the PCR products did not reveal any mutations in these two exons (data not shown). In addition, Western blot analysis revealed that all the melanoma cell lines expressed a 95-kDa variant of the Braf kinase protein (Figure 4b).

Figure 4.
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PCR amplification of the Braf gene and expression of Braf and PTEN proteins. (a) PCR amplification of Braf exons 11 and 15 showing correct-size bands in normal skin and melanoma samples. (b) Immunoblotting of whole-cell lysates with anti-Braf, anti-PTEN, and anti-beta-actin antibodies. The results are representative of three independent experiments

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PTEN expression

The loss of the PTEN protein has been implicated in melanomagenesis in humans (Bastian et al., 1998). Immunoblotting experiments showed that all the murine melanoma cell lines expressed similar amounts of PTEN, indicating that the loss of PTEN is not involved in murine melanoma development (Figure 4b).

Activation of ras-MEK1-ERK1/2 MAPK signaling pathway

Mutations in the ras gene in tumor cells should lead to constitutive activation of the downstream MAPK pathway. As two of the six murine melanoma cell lines (CM519 and K1735 cell lines) had mutations in N-ras, we determined whether these mutations led to hyperphosphorylation of the MEK1 and ERK1/2 MAPKs, which is indicative of high kinase activity. We found that the amounts of MEK1 and ERK1/2 proteins were approximately equal in all the cell lines (Figure 5). However, the amounts of phosphorylated MAPKs varied. The highest levels of MEK1 and ERK1/2 phosphorylation were detected in CM519 and CM3205 cells, and low levels were observed in K1735 cells and the B16 cell lines. This experiment indicated that the level of constitutive phosphorylation of MAP kinase proteins is much higher in cells carrying N-ras mutation in codon 61 (CM519 cells) than in codon 13 (K1735 cells). Furthermore, hyperphosphorylation of MAPKs was not restricted to cell lines with mutant ras (CM519) but also occurred in cells with wild-type ras genes (CM3205). Expression of activated MAPKs in CM519 and CM3205 cells did not depend on the presence of serum in the medium (data not shown), suggesting that MAPK activation may be caused by an increase in autocrine growth factor stimulation (Satyamoorthy et al., 2003).

Figure 5.
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Expression of total and phosphorylated MEK1 and ERK1/2 proteins. The blots were first probed with antibodies against total MEK1 or ERK1/2, then stripped and reprobed with anti-p-MEK1 or anti-p-ERK1/2 antibodies, and then stripped and reprobed again with an antibody against beta-actin. The results are representative of three independent experiments

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p53 alterations

Analysis of p53 protein expression revealed large amounts of stabilized p53 protein in CM519 and CM3205 cells (Figure 6). In addition, p53 protein in these cell lines was phosphorylated at serine 15 (Figure 6) and localized in the nuclei (Figure 7), which is typical of mutant p53 (Melnikova et al., 2003). In contrast, K1735 and the B16 cell lines expressed only residual levels of total and phosphorylated p53 (Figure 6). Immunocytochemical staining with anti-p53 antibody showed cytoplasmic localization in K1735 cells and nuclear localization in all other cell lines (Figure 7, left panels). Both K1735 and the B16 cell lines reacted only very weakly with anti-p-Ser15-p53 antibody (Figure 7, right panels and data not shown).

Figure 6.
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Expression of the p53, p-Ser15-p53, and Mdm2 proteins. Whole-cell lysates were subjected to immunoblotting with specific antibodies. Note the high levels of p53 expression and phosphorylation at serine 15 residue in CM519 and CM3205 cells and high levels of Mdm2 expression in K1735 cells. These blots are representative of three independent experiments

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Figure 7.
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(a) Immunocytochemical staining showing intracellular localization of total p53 protein (left panels) and p53 phosphorylated at serine 15 (right panels). B16F0 cells staining is representative of staining of all B16 cells. In case of K1735 and B16F cells, which express small amounts of wild-type p53, the image acquisition time was increased for better visualization. (b) UV irradiation (75 J/m2) induced p53 nuclear translocation (left panels) and phosphorylation at serine 15 (right panels) in K1735 cells

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To determine whether the p53 gene was mutated in the melanoma cell lines, we analysed p53 exons 2–10 by PCR amplification followed by direct sequencing of the PCR products. The results indicated that CM519 and CM3205 cells had C right arrow T mutation at dipyrimidine sequences in codons 270 and 246, respectively. In contrast, K1735, B16F0, B16F1, and B16F10 cells did not have mutations in p53 exons 2–10.

That wild-type p53 protein was localized in the cytoplasm in K1735 cells suggested that p53 may have undergone Mdm2-mediated nuclear export. K1735 cells did express very high levels of Mdm2 (Figure 6). In contrast, B16 cells expressed p53 in the nucleus and low levels of Mdm2 (Figures 6 and 7). This suggests that in the absence of p19Arf, p53 localization may depend on the level of Mdm2 expression.

UV-induced phosphorylation and nuclear translocation of wild-type p53 in K1735 cells

Overexpression of Mdm2 protein in K1735 cells can render wild-type p53 inactive (Oliner et al., 1993). Furthermore, absence of p19Arf protein could also hinder p53's capacity to be activated. Therefore, we determined whether the wild-type p53 in these cell lines could respond to UV irradiation, which induces phosphorylation and stabilization of p53. In K1735 cells irradiated with UVB (75 J/m2), p53 protein underwent nuclear translocation (Figure 7b), stabilization (Figure 8), and concomitant phosphorylation at serine 15 residue (Figures 7b and 8). Furthermore, UV stimulated expression of a downstream target of p53, p21Waf1Cip1 protein (Figure 8). These results suggest that neither loss of p19Arf nor overexpression of Mdm2 prevented functional activation of wild-type p53 in response to UV irradiation in K1735 cells.

Figure 8.
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UV irradiation induced accumulation of wild-type p53 protein, its phosphorylation at serine 15, and expression of its downstream target p21Waf1/Cip1 protein, in K1735 cells. Cells were irradiated (75 J/m2) with an FS40 sunlamp (Westinghouse Electric Corp., Bloomfield, NJ, USA), and cell lysates were prepared at the indicated time points and subjected to immunoblotting. The blots are representative of three independent experiments

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Discussion

Our analysis of spontaneous and carcinogen-induced murine melanoma cell lines for genetic alterations in critical tumor suppressor genes (p16Ink4a, p19Arf, p53) and oncogenes (ras and Braf) revealed several major trends. First, unlike sporadic human melanomas, none of the murine melanoma cell lines tested had activating mutations in the Braf gene at exon 11 or 15. All of the cell lines also expressed PTEN protein, indicating that loss of PTEN is not involved in the development of murine melanomas. Second, all the murine melanoma cell lines lost expression of either p19Arf (K1735 and the B16 cell lines) or functional p53 (CM519 and CM3205 cells), emphasizing the role of the p19Arf/p53 axes in melanomagenesis in mice. Third, concomitant with the loss of p19Arf, the B16 cell lines lost the p16Ink4a gene as a result of a large deletion spanning exons 1alpha, 1beta, and 2, suggesting that inactivation of the Ink4a/Arf locus plays an important role in the development of spontaneous B16 murine melanoma. Finally, in contrast to the spontaneous B16 melanoma cell lines, all three carcinogen-induced melanoma cell lines either overexpressed phosphorylated MEK1 and ERK1/2 MAPKs (CM519 and CM3205) and/or had activating mutations in the N-ras oncogene (CM519 and K1735). This observation suggests that inactivation of p19Arf or p53 cooperates with activated ras-MAPK signaling in the development of carcinogen-induced mouse melanomas.

The cooperation between the loss of p19Arf and constitutively activated ras in tumorigenesis has been previously observed in transgenic mice overexpressing oncogenic H-ras. In these animals, the deficiency in p19Arf (and/or p16Ink4a) facilitated melanoma formation (Chin et al., 1997). Furthermore, H-ras transgenic mice crossed with p53-/- mice developed melanomas with a higher incidence, compared to either strain alone (Bardeesy et al., 2001). A functional link between the two pathways is provided by studies indicating that p19Arf and p53 are induced by oncogenic ras to suppress transformation. This occurs by initiating apoptosis or premature senescence in murine keratinocytes and fibroblasts in vitro and in vivo (Kamijo et al., 1997; Palmero et al., 1998; Groth et al., 2000; Lin and Lowe, 2001). Therefore, inactivation of p19Arf or p53 tumor suppressors may provide survival and proliferative advantages to cells with activated ras-MAPKs.

Our data showed that inactivation of p19Arf or p53 occurred in all the melanoma cell lines, albeit in a mutually exclusive manner, contributing to the hypothesis that the two proteins function in the same pathway to suppress transformation of melanocytes (Chin et al., 1997). As mentioned earlier, human melanomas also carry mutations in these two genes. Most of the mutations detected in the p53 gene in human tumors were UV signature mutations characterized by C right arrow T and CC right arrow TT transitions at dipyrimidine sites (Hartmann et al., 1996). Such UV-type mutations in p53 largely dominate in nonmelanoma skin cancers, which have been linked to a cumulative lifetime exposure to UV (Brash et al., 1996; Ananthaswamy et al., 1998b). The presence of C right arrow T transitions in the p53 gene in CM519 and CM3205 melanomas, initiated by DMBA, and promoted by UV plus croton oil or TPA, indicates that those mutations were most likely induced by chronic UV exposure. Unlike squamous cell carcinomas, a history of severe sunburns together with intermittent UV exposure is implicated in the etiology of human melanoma (Elwood and Jopson, 1997). Perhaps more relevant to human melanomas, cells from K1735 murine melanoma initiated by a single UV exposure and promoted by croton oil have lost p19Arf but retained the wild-type p53 genotype. In accordance with these findings, Ink4a/Arf deficiency has been recently shown to cooperate in the promotion of melanoma in HGF/SF transgenic mice, after a single UV irradiation of neonates, thus placing Ink4a/Arf among the list of UV-responsive tumor suppressor genes (Recio et al., 2002). Interestingly, K1735 cells also carried a UV-type ACC right arrow ATC mutation in N-ras codon 13 (reverse strand). In fact, previous studies have revealed mechanistic and topological association between UV and activating N-ras mutations, which often occurred at or near dipyrimidine sequences, and were detected in tumors localized on sun-exposed body sites (Van't Veer et al., 1989; Van Elsas et al., 1996). Still, the mechanism by which UV radiation participates in melanoma formation is widely debated due to the lack of direct evidence for the UV-induced genetic alterations in a large proportion of primary human melanomas. Nevertheless, our study shows that UV-signature mutations in p53 or ras genes are present in carcinogen-induced murine melanomas. The presence of p53 or ras mutations may be specific to carcinogen-induced transformation in general. For example, in chemical-induced lymphomas, mutations in p53 or/and ras genes were more common than mutations in the Ink4a/Arf or Ink4b loci (Zhuang et al., 1997, 1998). Further emphasizing the role of carcinogens in acquisition of genetic alterations in murine melanomas, a CAA(Gln) right arrow CAT(His) mutation in N-ras codon 61 was found in CM519 cells. This mutation was most probably caused by exposure to DMBA, which was shown to induce A right arrow T transversions, specifically, in the third position of N-ras codon 61 in rat bone-marrow cells (Osaka et al., 1996). However, a nucleotide change at this position is rarely observed in human melanomas, where codon 61 CAA(Gln) right arrow AAA(Lys) or CAA(Gln) right arrow CGA(Arg) mutations are typical (Albino et al., 1989; Van't Veer et al., 1989; Jafari et al., 1995; Omholt et al., 2002).

Interestingly, a comparison of MAPK activity in CM519, CM3205, and K1735 cells revealed that the levels of activation of the MEK1 and ERK1/2 kinase proteins were similarly high in CM519 cells, which had a mutation in N-ras codon 61, and CM3205 cells, which had a wild-type N-ras gene. In contrast, MAPK activation was low in both K1735 cells, which have a mutation in N-ras codon 13 and in the B16 cell lines, which have a wild-type N-ras (Figure 5). Further, despite the absence of ras and Braf mutations, the level of constitutive MAPK activation in CM3205 cells was equal to that of CM519 cells, which have mutant N-ras. This suggests that in both CM519 and CM3205 cells, MAPKs are activated through a mechanism that overrides the activity of mutant ras. Constitutive phosphorylation of MAPKs in CM519 and CM3205 cells did not depend on the presence of serum in the medium (data not shown), suggesting that MAPK activation may be related to an increase in autocrine growth factor stimulation (Satyamoorthy et al., 2003).

As in the B16 cell lines, in many human cancers, including melanomas, the INK4a/ARF locus undergoes homozygous deletion of exons 1alpha, 1beta, and 2 or alterations in the common exon 2 that inactivate both the p16INK4a and p14ARF proteins (Pollock et al., 1996). Alterations in only exon 1alpha or 1beta have also been described in familial as well as sporadic human melanomas (Fitzgerald et al., 1996; Flores et al., 1996; Kumar et al., 1998; Randerson-Moor et al., 2001; Rizos et al., 2001; Hewitt et al., 2002). Although such targeting of one of the INK4a/ARF genes is somewhat rare, it indicates the importance of both of the two gene products in melanomagenesis in humans. We found that K1735 cells lacked p19Arf expression because of deletion of exon 1beta alone.

Lastly, we have observed a cytoplasmic sequestration of wild-type p53 in K1735 melanoma cells. Similar p53 localization is common to several tumor types including colorectal carcinoma, retinoblastoma, inflammatory breast carcinoma, and undifferentiated neuroblastoma, where it has been linked to a poor response to chemotherapy, greater tumor metastasis and a low expectation of long-term patient survival (Moll et al., 1992, 1996; Sun et al., 1992; Bosari et al., 1995; Schlamp et al., 1997). Cytoplasmic accumulation of p53 in K1735 cells could be associated with the loss of p19Arf and simultaneous overexpression of Mdm2 protein, which itself would have been targeted for degradation if p19Arf was expressed. On the other hand, p53 was detected mostly in the nuclei of p19Arf-deficient B16 cells, which could be attributed to the low level of Mdm2 expression. Alternatively, cytoplasmic sequestration of p53 in K1735 cells could be explained by other mechanisms, for example, a defective nuclear import. Nonetheless, no mutations in the C-terminal regulatory domain encoding nuclear localization signals of p53 were found in K1735 cells. Furthermore, UV irradiation of K1735 cells resulted in p53 phosphorylation and nuclear translocation (albeit with the kinetics slower than that in B16 cells (data not shown), indicating that p53 is still functional, and that p19Arf is dispensable for its activation by UV. A number of proteins have been proposed to serve as cytoplasmic anchors for p53, including heat shock protein70, ribosomal proteins, vimentin, tubulin, F-actin, and, most recently, PARC protein (Gannon and Lane, 1991; Fontoura et al., 1992; Klotzsche et al., 1998; Metcalfe et al., 1999; Giannakakou et al., 2000; Nikolaev et al., 2003). Further experiments are needed to elucidate whether p53 is sequestered in the cytoplasm of K1735 cells by one of these molecular chaperons, and to determine whether this phenomenon can be also detected in human melanoma cell lines.

In summary, our studies demonstrate that inactivation of the p19Arf and p16Ink4a genes cooperate in the development of spontaneous mouse melanomas, whereas loss of p19Arf or p53 tumor suppressors cooperate with ras and/or MAPK cascade to induce malignant transformation in carcinogen-induced melanomas.

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Materials and methods

Cell lines

The K1735 cell line was derived from a melanoma induced in a C3H/HeN mouse by initiation with UV radiation and promotion with croton oil (Kripke, 1979). The parental B16F0 cell line was derived from a spontaneous melanoma in a C57/BL6 mouse (Fidler, 1973). Cell lines B16F1 and B16F10 were obtained by serial passage of B16F0 cells in C57/BL mice, isolation of a lung metastasis, and establishment of cells in culture (Fidler, 1973). The CM519 and CM3205 cell lines were derived from tumors induced in a C3H mouse by DMBA initiation and subsequent promotion with UV and croton oil (CM519) or with UV and TPA (CM3205) (Romerdahl and Kripke, 1986). All cell lines were maintained in Dulbecco's modified Eagle's medium (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT, USA) in 5% CO2.

Reagents

Mouse monoclonal immunoglobulin G (IgG) against p53 (Ab-1, clone PAb421) and against p21Waf1/cip1 (Waf-4) and rabbit polyclonal IgG against p19Arf (Ab-4) were obtained from Oncogene Research Products (Boston, MA, USA) and CM5 rabbit polyclonal IgG against p53 from Novocastra (Newcastle, UK). Immunocytochemical analysis of p19Arf protein was performed with an antibody from Novus Biologicals (Littleton, CO, USA). Phosphospecific rabbit polyclonal IgG against p53 phosphorylated at serine 15, rabbit polyclonal anitibodies against phospho(Thr202/Tyr204)-ERK1/2 and phospho(Ser217/221)-MEK1/2, and antibodies against total ERK1/2 and MEK1/2 were obtained from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal IgG against Mdm2 (SMP14) and p16Ink4a (M-156) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against PTEN was from Cascade Bioscience (Winchester, MA, USA).

PCR and nucleotide sequencing

Genomic DNA was analysed by using intronic upstream and downstream primers specific for each exon of each gene (Table 2). All the oligonucleotide primers were synthesized by Genosys Technologies, Inc. (The Woodlands, TX, USA). The PCR products were directly sequenced.


Western blot analysis

Whole-cell lysates were analysed for protein expression by Western blotting as described previously (Melnikova et al., 2003).

Immunocytochemical analysis

Immunocytochemical staining was performed in fixed cells as described previously (Melnikova et al., 2003). The antibody dilution factors used were: 1 : 100 for M-156 anti-p16Ink4a, anti-p19Arf, and Ab-1 anti-p53, and 1 : 1000 for antiphospho(Ser15)-p53. Biotinylated secondary antibody was added at a 1 : 500 dilution. Fluorescein-labeled avidin D was diluted 1 : 2000. Propidium iodide (1 mug/ml in phosphate-buffered saline) was added to visualize chromatin.

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Acknowledgements

We thank Dr Maureen Goode for editing the manuscript. This work was supported by National Cancer Institute grant CA 46523 (to HNA), National Institute of Environmental Health Sciences Center Grant ES07784, and The University of Texas MD Anderson Cancer Center institutional core Grant CA 16672.

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