Ultraviolet Radiation-Induced Malignant Skin Melanoma in Melanoma-Susceptible Transgenic Mice1

[CANCER RESEARCH 54, 4569-4572. September 1, 1994] Advances in Brief

Ultraviolet Radiation-induced Malignant Skin Melanoma in Melanoma-susceptible Transgenic Mice1

Andres J. P. Klein-Szanto, Willys K. Silvers,2 and Beatrice Mintz3

Institute for Cancer Research, Fox Chase Cancer Center. Philadelphia. Pennsylvania 19111

Abstract several other mammalian species, especially in mice. Protocols are based largely on the application of 7,12-dimethylbenz[a]anthracene as A new mouse model of IV radiation-induced melanoma is described. an initiator, followed by repeated use of croton oil as a promoter (5, Unlike previous models, melanoma induction requires only short-term 6); UVR has been either added or substituted in place of the initiator irradiation, does not require the application of chemical carcinogens, and or promoter (7-10). These experiments have demonstrated a mela- does not cause any other tumors. The model takes advantage of the fact that Tyr-SV40E (C57BL/6 strain) transgenic mice are all melanoma- nomagenic contribution of UVR, they have shown that UVR has an susceptible, and that different inbred lines are susceptible to different immunosuppressive effect enhancing tumor outgrowth (11), and they extents. Four-day-old mice of a moderately susceptible line (line 9 trans have yielded several new melanoma cell lines (5, 8). The difficulties genic homozygotes) were exposed for 20 min/day to 328 mj/cm2 to UVB are the prolonged repeated treatments required in all the protocols (280-320 nm wavelength, comprising 70% of the total irradiance) for up (over 1 year in some), and that other skin tumors (papillomas, squa- to 4 consecutive days. Melanocytic lesions resembling macules, nevi, or mous cell carcinomas, and fibrosarcomas), and even lymphomas, arise early melanomas gradually appeared in the irradiated mice that were not along with melanomas. seen in unirradiated transgenic controls of similar age. To afford ongoing We were interested in the possibility that both problems might be observation beyond the short life span of line 9 homozygous mice, skin overcome by using a transgenic mouse strain, Tyr-SV40E, that is samples containing a total of 26 selected lesions were grafted at 20 weeks specifically melanoma susceptible but ordinarily has a very low after UV radiation to longer-lived unirradiated hosts of transgenic line 12, spontaneous incidence of skin melanoma (12, 13). The transgene is in which melanoma susceptibility is low. Ten lesions in the grafts became melanomas; all melanomas had ulcerated and two had metastasized. At comprised of SV40 oncogenic sequences, including large T, under the transcriptional control of the mouse tyrosinase-gene promoter, and is the stages examined, all the tumors were deeply melanotic. The remaining 16 lesions were still indolent when the experiment was terminated at 57 therefore expressed in pigment cells. Different inbred lines of these weeks post-1 'V radiation. The present protocol lends itself to variations in mice, each derived from one founder, have the same standard strain the choice of transgenic line, the age of the treated mice, and the intensity background (C57BL/6) but different levels of transgene expression and duration of ultraviolet light; appropriate combinations of these vari and, correspondingly, of melanoma susceptibility. Eye melanomas ables would be expected to yield melanomas in relatively long-lived trans develop before any skin melanomas and are fatal in young mice of the genic mice without skin grafting. The new model provides an opportunity more susceptible lines; less susceptible mice have much later onset to determine the melanoma action spectrum, to characterize at the mo eye tumors and longer lives. The eye melanomas have never been lecular level the melanomas induced by ultraviolet light in comparison found to metastasize to the skin. Skin melanomas were obtained in the with those of other origins, and to investigate in vivo the photoprotective role of melanin. absence of advanced eye melanomas by grafting skin from high susceptibility (unirradiated) donors to low susceptibility hosts, thereby Introduction greatly prolonging the life of the donor skin (14, 15). More direct evidence of the tumorigenic potential of skin melanocytes was ob Based on epidemiological evidence, UVR4 in sunlight, especially served after explanting them from newborn transgenic mice. Progres UVB, is thought to be a major cause of skin melanoma, and increased sion continued in culture and the cells formed melanomas after they sun exposure is thought to be responsible for the rapidly rising were injected into appropriate hosts (16). A single exposure to very incidence of this malignancy (1). Genetic susceptibility may play a low intensity (1.75 mj/crrr) UVB at an early culture passage resulted role, for example, in familial melanoma (2) and xeroderma pigmen- in accelerated progress toward tumorigenesis, whereas the same UVB tosum (3), possibly through different mechanisms that may ultimately intensity had no apparent effect on wild-type C57BL/6 melanocytes share some common pathway. The mechanisms linking UVR to (17). Transgene expression, therefore, has lowered the threshold for melanoma are little understood; hence, experimental animal models response to UVB. have long been sought. Among laboratory mammals, only in the By modifying the foregoing transgenic experiments, we have pro South American opossum does treatment with UVR alone result in duced a mouse model of UVR-induced melanoma in which no other malignant melanomas, albeit after many exposures over a very long skin tumors occur. Short-term exposure to UVR within a few days period (4). Combinations of carcinogens and UVR are effective in after birth causes melanocytic skin lesions from which malignant melanomas arise. Received 6/2/94; accepted 7/11/94. The costs of publication of this article were defrayed in part by the payment of page Materials and Methods charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by USPHS grams CA-42560 (B. M.), CA-06927, and Transgenic Mice. Homozygous Tyr-SV40E transgenic mice of line 9(12) RR-05539 to the Fox Chase Cancer Center, and by an appropriation to the Center from the have a moderate level of transgene expression relative to our other lines with Commonweallh of Pennsylvania. the same transgene. By 6 months of age, line 9 mice develop fatal eye 2 Visiting Scientist from the University of Pennsylvania. melanomas but rarely have macroscopically visible melanocytic lesions in the *To whom requests for reprints should be addressed, at Institute for Cancer Research, skin. Mice were UV irradiated at 4 days of age for 20 min; some were Fox Chase Cancer Center, 77(11 Burholme Avenue. Philadelphia, PA 19111. 4 The abbreviations used are: UVR. ultraviolet radiation: UVB. ultraviolet B (280-320 reexposed for 20 min on consecutive days for a total of 2 or 4 days. The eyes nm wavelength). were still closed and therefore were not exposed to the UVR. In order to 4569

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1994 American Association for Cancer Research. UVR-INDUCED MELANOMA [N TRANSGENIC MICE observe for the longest possible time the melanocytic lesions that appeared in Table 1 UVB-induced skin melanomas in Tyr-SV40E (C57BU6) transgenic mice the UVR-treated skin, samples of skin containing such lesions were grafted 20 Line 9 transgenic homozygotes were irradiated at 4 days of age with 328 mJ/cm2 of weeks after UVR to unirradiated line 12 hemizygous hosts, which have a low UVB for 20 min; some were reexposed for 20 min on 1 or 3 more consecutive days. After 20 weeks discs of full-lhickness skin with selected melanocytic lesions were grafted level of transgene expression, can live well over a year without advanced eye to longer-lived unirradiated transgenic recipients (line 12 hemizygotes) for further melanomas, and do not develop skin melanomas. observation. Exposure to UVR. The same UVM-57 midrange lamp (UVP, San Gabriel, melanomas'" grafts lesions in CA) as used previously to irradiate mouse pigment cells in culture was used No.UVB"exposures124No.fromNo.mice6310Body024Ear516No.graftsfromBody028Ear718No.inBody025Ear003from lesions here and has been described in detail (17). Calculations based on records of the spectral energy distribution of the lamp, transmission characteristics of the filter, and response of the sensor revealed that 70.0% of the total irradiance reaching the mice was in the UVB range (280-320 nm); 29.7% was in the lower end (320-380 nm) of the UVA range (320-400 nm), and 0.3% was in " 70.0% of the total irradiance was UVB (280-320 nm wavelength); 29.7% was in the the upper end (275-280 nm) of the UVC range. The distance from the lamp to low end (320-380 nm) of the UVA range; 0.3% was in the upper end (275-280 nm) of the mice and the exposure time were calibrated for each set of mice. Unlike the UVC range. some sunlamps, this lamp caused no measurable increase in temperature inside Metastasis occurred from two of the melanomas. The remaining 1ft lesions under the chamber during irradiation. Erythema was not observed in the mice went little change. following UVR. Skin Grafting. Skin with recorded lesions was grafted according to stan ing along with other cells in the growing skin. To determine the dard procedures (18). In the case of ear skin grafts, the ear was cut off at the long-term fate of examples of the lesions beyond the expected lifetime base, the two halves were separated, and cartilage and fat were removed; if both halves were grafted on the same host, they were placed base-to-base in the of line 9 mice, donor skin samples containing a total of 26 lesions same graft bed. With body skin grafts, a 1-cm disc of full thickness skin was were grafted. Of these, 16 lesions were present in 12 grafts of ear skin used and scraped free of underlying fat. Grafts were placed on the lateral trunk and 10 lesions were included in 6 grafts of body skin. The subsequent of an anesthetized host after removal of a slightly larger piece of host skin. development of each lesion could easily be determined from its Grafts were transferred from females to either sex and from males to males. original position in the graft area. Bandages were removed after 9 days. The graft area was occasionally shaved As summarized in Table 1, 10 of the 26 selected lesions in 7 of the with an electric clipper for observation. grafts gave rise to discrete melanomas. No other skin tumors resulted. Results and Discission At the time the grafts were prepared under a dissecting microscope at 20 weeks of age, 4 of the 10 lesions (in body skin) were recorded as The aims of the study were to determine whether melanocytic possible early melanomas on the basis of their thickness, elevation lesions appear after exposure to UVB that are absent in unexposed above the surface, and apparent presence deep in the dermis; 3 lesions transgenic mice of the same line and age and to learn whether any of (also in body skin) were scored as thick blue nevi and 3 lesions (in ear those lesions become malignant. It should be emphasized that the skin) were regarded as large fÃatmacules or as slightly darker and experiments were not designed to yield quantitative information on thicker early nevi. Thus, while 38% (10 of 26) of this nonrandom the overall frequency of UVB-induced lesions or melanomas. sample of lesions developed into histologically diagnosed melanomas, Experimental and control transgenic mice were shaved and the skin 70% (7 of 10) of the more advanced ones (judged at the time of was examined with a hand lens at 20 weeks of age. Among unirra grafting as blue nevi and candidate melanomas) either were already diated line 9 homozygotes (6 females, 5 males), no obvious macules early melanomas 20 weeks after UVR or later became melanomas. or nevi were seen under these conditions and no melanomas were Three lesions scored previously as incipient melanomas did not be present. Unirradiated line 12 mice (7 females, 4 males) were also come melanomas. It therefore appears that an early effect of UVR is negative. In contrast, flat greyish or black macules (some of the latter melanocytic hyperplasia, which is evident in the lesions that failed to were up to 3 millimeters in diameter), thicker black or blue nevi, and progress as well as in those lesions that did progress; in addition, it possible early melanomas were found in some of the 19 irradiated appears that further changes are not inevitable. This is comparable to mice (8 females and 11 males) of line 9 that had been exposed to 328 the situation in sun-exposed human skin, in which lesions arise that mJ/cm2 of UVB for 20 min a day on 2 or 4 consecutive days. The may remain indolent (19). number of these lesions was greatest after four exposures. The sharp The melanomas grew at different rates and became grossly identi outline and somewhat ameboid shape of the larger flat, dark lesions fiable in the grafts over a wide time range. At 57 weeks after UVB, suggests initial clonal expansion from a single affected cell multiply- when the experiment was terminated, three melanomas were still in an

Fig. 1. Melanomas from UVB-induced lesions in skin grafted from irradiated Tyr-SV40E transgenic mice to unirradiated transgenic hosts, (a) Graft containing an unaffected and an affected one-half ear from case 226 was excised at autopsy 56 weeks post-UVB and photographed after fixation in formalin. Both the large and the small lesion in the affected half (right) are nodular melanomas, (b) In the ear skin graft from case 231, shown on the host 37 weeks after UVB, the melanoma forms an ulcerated mound, (c) Melanoma in a body skin graft from case 235, 36 weeks after UVB, has caused a large ulcÃ©rationand scattered small ones over the tumor mass. 4570

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Fig. 2. Histological sections of UVB-induced melanomas, (a) The larger tumor in case 226 (see Fig. la) is a melanotic nodular melanoma localized in the dermis and is pressing toward the s.c. tissue and ulcerating the epidermis. Many large pigment-laden cells are in the periphery, probably representing remnants of the precursor blue nevus, (b) In case 231 (see Fig. \b), there is an ulcerated advanced melanotic melanoma with a lobulated pattern, (c) A melanotic nodular melanoma is invading muscle tissue (righi) in case 228. (d) Two patterns of melanoma growth are present in case 235 (see Fig. le). A nodular melanotic melanoma is seen in the dermis (bottom) while a more superficial radial growth occupying the dermis is found just under the epidermis. The intensely pigmented melanophages and spindle cells (Â¡eft)resemble cells in a blue nevus, (e) In the nodular melanoma of the same case, at a higher magnification, there are many small homogeneous tumor cells with clear cytoplasm containing powder-like melanin granules and also large heavily pigmented melanophages. Mitotic figures are present. (/) A lymph node from the same case contains a micrometastasis comprising small tumor cells and large melanin-containing macrophages in the interfollicular space. H & E Staining, a, b, and d X30; c, X80; e,f, X160. early stage and were partly associated with a blue nevus component. unirradiated grafts (14, 15) have usually developed near the periphery By the same cutoff time, 16 nontumorous lesions had undergone only of the grafted skin, whereas in the irradiated skin they developed from very slight enlargement, chiefly by radial growth. the lesions already present at grafting, regardless of location. The The following lines of evidence indicate that the melanomas re latency between grafting of line 9 skin and the histologically con ported here must have resulted largely from exposure of the skin to firmed presence of melanoma was 44-60 weeks in unirradiated skin UVR before grafting, rather than from grafting alone. Melanomas in and only 24-37 weeks in irradiated skin grafted at a similar age. 4571

Moreover, 25% of the unirradiated grafts yielded melanomas, with nient modifications of this protocol are readily foreseeable, based on one tumor distinguishable per graft, while 39% of the irradiated grafts the choice of transgenic mouse line, the age of the mice at the time of developed melanomas, with two separate tumors in three of the grafts. exposure, and the intensity and duration of UVR. An appropriate Melanomas at different stages of advancement are shown in Figs. 1 combination of these changes is likely to favor the occurrence of and 2. Virtually all the tumors were nodular melanomas, including the melanomas after brief treatment in relatively long-lived transgenic smaller ones (Figs, la and 2a). They were localized at first in the mice of minimal susceptibility, without skin grafting. dermis where they grew expansively (Fig. 2b), spreading into the s.c. tissue, and then invading neighboring tissues such as muscle (Fig. 2c). References Destruction of the overlying basement membrane and epidermis was 1. Rigel, D. S., Kopf, A. W.. and Friedman. R. J. The rate of malignant melanoma in the characteristic (Fig. 2 a and b) and did not occur in the remaining United States: are we making an impact? J. Am. Acad. Dermatol.. 17: 1050-1053, 1987. lesions which did not become melanomas. The presence of some 2. Cannon-Albright, L. A., Goldgar. D. E.. Meyer, L. J., Lewis, C. M, Anderson, D. E., melanomas contiguous with a blue nevus was evidence for their Fountain. J. W.. Hegi. M. E., Wiseman, R. W., Petty, E. M.. Bale, A. E., Olopade, derivation from the nevus (Figs. 2 a and d). In one case, there was a O. I., Diaz. M. O., Kwiatkowski, D. J., Piepkom, M. W.. Zone, J. J., and Skolnick, M. H. Assignment of a locus for familial melanoma. MLM. to chromosome 9pl3- more superficial radial growth pattern that seemed to be independent p22. Science (Washington DC), 258: 1148-1152, 1992. of the subjacent nodular melanoma; the former included islands or 3. Cleaver, J. E., and Kraemer. K. H. Xeroderma pigmentosum. In: C. R. Scriver. A-L. Beaudet, W. S. Sly, and D. Valle (eds.). The Metabolic Basis of Inherited Disease, pp. cords of melanoma cells spreading closer to the epidermis (Fig. 2d). 2949-2971. New York: McGraw-Hill, Inc., 1989. A striking feature of the UVB-induced tumors was their dense 4. Ley, R. D., Applegate, L. A., Padilla. R. S., and Stuart. T. Ultraviolet radiation blackness throughout. Whether the smaller melanomas, which were induced malignant melanoma in Monadelphus domestica. Photochem. Photobiol., 50: 1-5, 1989. also black, would have remained melanotic if they were allowed to 5. Berkelhammer, J.. Oxenhandler, R. W., Hook, R. R., Jr., and Hennessy, J. M. grow further in place is, of course, unknown. Small pieces of two of Development of a new melanoma model in C57BL/6 mice. Cancer Res., 42: 3157- the largest melanomas (case 231, Figs. \b and 2b; case 235, Figs, le 3163, 1982. 6. Berkelhammer. J., and Oxenhandler. R. W. Evaluation of premalignant and malignant and 2d) were transplanted s.c. by trocar to line 12 hosts and then lesions during the induction of mouse melanomas. Cancer Res., 47: 1251-1254. 1987. 7. Epstein. J. H., Epstein, W. L., and Nakai, T. Production of melanomas from DMBA- passaged a second time. The intense pigmentation was maintained in induced "blue nevi" in hairless mice with ultraviolet light. J. Nati. Cancer Inst., 38: the grafts in all 12 hosts. This was especially noteworthy because 19-30, 1967. melanomas arising in our grafts of transgenic skin not subjected to 8. Kripke, M. L. Speculations on the role of ultraviolet radiation in the development of UVR were all largely hypomelanotic or amelanotic with one or two malignant melanoma. J. Nati. Cancer Inst., 63: 541-545, 1979. very melanotic zones (14)5. The melanomas obtained in mice by other 9. Romerdahl, C. A., Stephens, L. C., Bucana, C., and Kripke. M. L. The role of ultraviolet radiation in the induction of melanocytic skin tumors in inbred mice. investigators have varied from highly melanotic (5) to melanomas Cancer Commun.. /: 209-216, 1989. with a mixture of melanotic and amelanotic areas (9); all those tumors 10. Husain, Z., Pathak, M. A., Flotte, T., and Wick, M. M. Role of ultraviolet radiation in the induction of melanocytic tumors in hairless mice following 7,12-dimethylben- were from chemical carcinogen-treated animals and thus may differ in z(n)anthracene application and ultraviolet irradiation. Cancer Res., 5/.- 4964-4970, some respects from the melanomas in the present study. The mela 1991. noma cells in our UVB-induced tumors contained clear cytoplasm 11. Donawho, C. K., and Kripke. M. L. Evidence that the local effect of ultraviolet radiation on the growth of murine melanomas is immunologically mediated. Cancer with fine dust-like melanized granules and were accompanied by Res., 51: 4176-4181, 1991. numerous large melanin-filled macrophages or melanophages (Fig. 12. Bradi, M., Klein-Szanto. A.. Porter. S., and Mintz, B. Malignant melanoma in transgenic mice. Proc. Nati. Acad. Sci. USA, 88: 164-168, 1991. 2e). Tumor cells were usually relatively small epithelioid cells with 13. Klein-Szanto, A., Bradi, M.. Porter. S.. and Mintz. B. Melanosis and associated round nuclei, a loose chromatin meshwork, and small nucleoli. tumors in transgenic mice. Proc. Nati. Acad. Sci. USA, 88: 169-173, 1991. 14. Mintz. B., and Silvers, W. K. Transgenic mouse model of malignant skin melanoma. Two instances of metastasis from the original tumors were ob Proc. Nail. Acad. Sci. USA, 90: 8817-8821, 1993. served, both from melanomas arising after four exposures to UVB. 15. Mintz, B.. Silvers, W. K., and Klein-Szanto, A. J. P. Histopathogenesis of malignant One melanoma had spread to the lymph nodes (Fig. 2f) and another skin melanoma induced in genetically susceptible transgenic mice. Proc. Nati. Acad. Sci. USA, 90: 8822-8826, 1993. had spread to the lungs. In both cases, the tumor cells were melanotic, 16. Larue, L., Dougherty, N., Bradi. M., and Mintz, B. Melanocyte culture lines from present as micrometastases, and accompanied by melanophages. Tyr-SV40E transgenic mice: models for the molecular genetic evolution of malignant The present protocol yields a mouse model of UVR-induced mel melanoma. Oncogene, 8: 523-531, 1993. 17. Larue, L., Dougherty, N., and Mintz, B. Genetic predisposition of transgenic mouse anoma in which many questions may be examined. These include the melanocytes to melanoma results in malignant melanoma after exposure to a low UV action spectrum leading to melanoma and to no other skin tumors, ultraviolet B intensity nontumorigenic for normal melanocytes. Proc. Nati. Acad. Sci. the role of DNA damage, and the photoprotective function of melanin. USA, 89: 9534-9538, 1992. 18. Billingham, R. E. Free skin grafting in mammals. In: R. E. Billingham and W. K. The latter will be facilitated by the availability of numerous pigment- Silvers (eds.). Transplantation of Tissues and Cells, pp. 1-26. Philadelphia: Wistar gene mutations in mice of the C57BL/6 inbred strain. More conve- Institute, 1961. 19. Holman, C. D. J., and Armstrong, B. K. Cutaneous malignant melanoma and indi cators of total accumulated exposure to the sun: an analysis separating hislogenetic 1B. Minlz and W. K. Silvers, unpublished data. types. J. Nail. Cancer Inst., 73: 75-82, 1984.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1994 American Association for Cancer Research. Ultraviolet Radiation-induced Malignant Skin Melanoma in Melanoma-susceptible Transgenic Mice

Andres J. P. Klein-Szanto, Willys K. Silvers and Beatrice Mintz

Cancer Res 1994;54:4569-4572.

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