1. ^*Queensland Institute of Medical Research, Brisbane, Queensland, Australia
2. ^†School of Medicine, University of Tasmania, Hobart, Tasmania, Australia
3. ^‡Division of Radiation and Cancer Biology, Stanford University, Stanford, California, USA

Correspondence: Graeme Walker, Queensland Institute of Medical Research, Brisbane, Queensland, Australia. Email: graew@qimr.edu.au

Received 7 February 2005; Revised 16 June 2005; Accepted 28 June 2005; Published online 25 October 2005.

To the Editor:Malignant melanoma (MM) in humans develops within a complex etiologic framework of genetic, host, and environmental factors (^{Goldstein and Tucker, 2001}). The strongest environmental risk factor is sun exposure (^{Sulaimon et al, 2003}). In the mouse, wild-type animals are resistant to MM development even when exposed to repeated treatments with ultraviolet radiation (UVR) (^{Gallagher et al, 1984}). Chronic UVR treatment regimens, however, have increased MM penetrance by up to 26% in mice carrying various transgenes capable of inducing spontaneous MM development, or melanocytic hyperplasia, e.g., Tyr-SV40Tag (^{Klein-Szanto et al, 1994;Kelsall and Mintz, 1998}), TPras (^{Broome Powell et al, 1999}) and Mt-Hgf/Sf (^{Noonan et al, 2000}) mice. More recently,^{Noonan et al (2001)} showed that a single neonatal dose of 9 kJ per m² was far more effective than chronic treatments at inducing MM in the Mt-Hgf/Sf transgenics.^{Kannan et al (2003)} used the neonatal UVR regimen on mice with melanocyte-specific activation of Hras on a background of either Ink4a or Arf nullizygosity. At 22 wk, Ink4a^-/-:Tyr-Hras and Arf^-/-:Tyr-Hras animals developed spontaneous MM, with an incidence of 35% and 53%, respectively (^{Chin et al, 1997}). Importantly, neonatal UVR exposure resulted in a marked increase in MM development only in the Arf^-/-:Tyr-Hras animals (penetrance rose to 88%) (^{Kannan et al, 2003}), implying that a defect in the p53 pathway may be necessary for UVR-induced MM. Arf^-/-:Tyr-Hras tumors were characterized by Cdk6 amplification and Ink4a mutation, genetic lesions that were never observed in non-UVR-induced MM. Notably, these secondary mutations indicate that these UVR-induced MM may only arise on an activated Hras background when both the p53 and pRb pathways are compromised.

It has previously been demonstrated that pigmented Tyr-SV40Tag mice treated with repeated neonatal UVR doses from days 3–10, show increased rates of MM compared to untreated animals (^{Klein-Szanto et al, 1994;Kelsall and Mintz, 1998}). These mice did not, however, develop melanomas over their limited lifespan when treated with a single neonatal dose. All subsequent studies have used albino mouse strains for neonatal UVR experiments.

Evidence suggests that the Ras pathway is pivotal for MM development. The RAS family is comprised of NRAS, HRAS, KRAS, which are all involved in the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signalling cascades (^{Busca et al, 2000}). There is a high frequency of BRAF and NRAS mutation in melanocytic nevi and MM (e.g.,^{Davies et al, 2002;Pollock et al, 2003} and reviewed in^{Rodolfo et al, 2004;de Snoo and Hayward, 2005}). Human cutaneous MM from chronically sun-exposed body sites commonly harbor NRAS mutations, thus supporting a possible role for solar UVR in their genesis (^{van Elsas et al, 1996;Jiveskog et al, 1998}). As discussed above, activated Hras has been shown to be a potent amplifier of tumorigenesis in genetically modified mice carrying deletions of the Ink4a or Arf genes, however, to date there have been no reports assessing the capability of Hras alone to induce MM in mice treated with a neonatal dose of UVR. We thus treated brown mice (mixed C3H/Sv129 strain background) carrying a melanocyte-specific mutant Hras (G12V) transgene (TPras) (^{Powell et al, 1995}), with a similar neonatal UVR regimen to that used by^{Noonan et al (2001)}. We used an apparatus that holds three Phillips FS40 UVB lamps mounted 34 cm above the mice (Phillips, Andover, MA). Pups (2–3 d old) were exposed for 16 min, to give a total dose of 8.15 kJ m² (UVA 320–400 nm, 2.36 kJ m² UVB 280–320 nm, 5.77 kJ m², UVC 250–280, 0.02 kJ m²), a slightly lower dose than previously used (^{Noonan et al, 2001}). All experiments were undertaken after receiving institutional approval. The UVR cohort (n=14) developed in situ cutaneous MM with a penetrance of 57% by 12 mo, whereas none of the untreated cohort (n=42) developed tumors (Figure 1). This is markedly higher penetrance and earlier average age of onset than that of the similarly treated Mt-Hgf/Sf transgenics (^{Noonan et al, 2001;Recio et al, 2002}). Previous experiments in which our pigmented TPras animals were exposed to a chronic UVR regimen (5.6–8.06 kJ m² biweekly for 38 wks) did not induce MM (^{Broome Powell et al, 1999}). To assess the reasons for the differences in UVR-induced MM susceptibility observed, we examined skin sections from adult TPras and neonatal TPras and wild type littermates. Adult TPras skin was highly pigmented, with scattered melanin in the upper dermis and large aggregates in the deep dermis (Figure 2a). In contrast, 2-d-old TPras skin lacked visible melanin (Figure 2b). The majority of melanocytes in neonatal skin of wild-type mice were located within hair follicles (Figure 2c), whereas there were noticeably more melanocytes in the extra-follicular dermis of TPras mice (Figure 2d).

Figure 1.

Melanoma penetrance in ultaviolet radiation (UVR)-treated TPras mice. A single UVR dose of 8.15 kJ m² to neonatal skin cooperates with melanocyte-specific activated Hras (n=14, dashed line) to facilitate malignant melanoma (MM) formation. Untreated animals (n=42) are represented by a solid line. Animals that died without developing MM are represented by a cross (UVR treated) or a dash (untreated). There is a significant difference (p<0.001, log rank test) in MM incidence between the treated and untreated groups.

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Figure 2.

Morphology and histopathology of melanomas from ultravioler radiation (UVR)-treated TPras mice. (a) Hematoxylin and eosin (H&E) skin section from an untreated adult TPras mouse. Note the scattered melanin deposits throughout the dermis and large, dense melanin deposits in the deep dermis above the muscle layer, see arrow (b) H&E skin section from an untreated neonatal TPras mouse (2 d old). Note the lack of visible melanin. Brightfield images of untreated (2-d-old) mouse skin sections (c) wild-type and (d) Tpras, stained for Tyrp1 (red) and counter-stained with Mayers' haematoxylin (blue). The majority of wild-type melanocytes are located within the hair follicles, see arrow. In contrast, TPras melanocytes are more frequency observed in the extra-follicular dermis, see arrow. (e) Cutaneous melanoma arising on the dorsal skin surface. Scale bar increments are of 1 mm. (f–h) H&E sections of a melanoma from an UVR-treated TPras mouse. Note the dermal origin of the lesion (f), hyperplasia of the epidermis and lack of epidermal junctional involvement, see arrow (g), atypical highly pigmented melanocytes within the tumor, see arrow (h). In all cases except (e), scale bar=50 m.

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UVR-induced MM ranged in size from 0.4–1.2 cm² and were heavily pigmented (Figure 2e). These lesions appear to originate in the dermis (Figure 2f), lack the classical junctional changes observed in human MM (Figure 2g), and contain large atypical melanocytes (Figure 2h). The dermal origin of these lesions reflects the location of the hyperplastic melanocytes in the adult animals (i.e., not along the basal layer as in humans) (^{Powell et al, 1995}). Hyperplasia of the epidermis was frequently observed in UVR-treated mouse skin (Figure 2g).

The mutation status of the Cdkn2a locus in these tumors was determined by immunohistochemistry (IHC) and analysis of tumor DNA and RNA (Figure 3 and Table I). No genomic deletion was observed and both transcripts were expressed in two of three tumors examined. Ink4a was detected in five of seven MM by IHC. The melanoma cell-line derived from one of the tumors lost Ink4a as it was passaged.

Figure 3.

Molecular status of Cdkn2a locus in melanomas from ultraviolet radiation (UVR)-treated TPras mice. Products from semi-quantitative PCR analysis electrophoresed in ethidium bromide-stained agarose gels. Panels (a) and (b) show results of genomic PCR to assess relative copy number of the Ink4a and Arf genes, respectively. The three lanes on the left contain DNA isolated from a melanoma cell line (derived from tumor malignant melanoma (MM)-1), at passage 4, 7, and 19, respectively. Ink4a is initially present but subsequently lost as the culture is passaged. Exon 1 (Arf) was already lost at passage 4, indicating that is was deleted prior to exon 1 (Ink4a). At each of these passages, all cells were of melanocytic origin as assessed by morphology and pigmentation. Lanes MM-1 and MM-2 represent two melanomas. Liver and water were used as controls. All TPras MM DNAs showed similar copy number of both exon1 (Ink4a, 300 bp) and exon1 (Arf, 283 bp) as judged by their ratios to Gapdh (220 bp). (c) Brightfield image of MM-3 showing strong nuclear staining for Ink4a (red) in the majority of tumor cells, using SC-1207 rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, California) diluted 1:300, counterstained with Mayers' hematoxylin (blue) (scale bar=50 m).

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Table I - Molecular status of Cdkn2a locus in melanomas from UVR-treated Tpras mice.

Full table

This study confirms the importance of a single neonatal UVR dose, compared with chronic UVR regimens in adult mice, for MM induction. The reason neonatal melanocytes are more susceptible to transformation than adult melanocytes is unclear, but it may be that they are immature and not fully differentiated, and/or, some are still located in the epidermis in newborn skin whereas in adult dorsal skin they are invariably located in the dermis (reviewed in^{Hirobe, 1995}).

In conclusion, our work has demonstrated that neonatal UVR treatments are probably as effective at inducing MM in pigmented mice as albino strains. Furthermore, we have shown that RAS activation alone is sufficient to predispose melanocytes to UVR-induced transformation, and, although the precise mechanism is yet to be determined, it does not always involve loss of Ink4a or Arf. It may be that activated Ras simply promotes melanocyte proliferation, or alternatively, that it may interfere with the DNA damage response and apoptotic pathways. This mouse model further consolidates the mounting evidence that NRAS or BRAF mutations co-operate with solar UVR in the development of melanoma.