Arizona Cancer Center, University of Arizona, 1515 North Campbell Avenue, PO Box 245042, Tucson, AZ 85724-5024, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; SCID, severe combined immunodeficient; TPA, 12-O-tetradecanoylphorbol-13-acetate.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The use of animal models in the study of many forms of cancer has been invaluable for dissecting the molecular events in cells as they progress towards malignancy. Animals also provide the opportunity to study the systemic effects of treatment and prevention strategies. There has been a lack of suitable melanoma models for these purposes. Reports of animal models for melanoma include the opossum (3), hamster (4), guinea pig (5) and mouse (6,7). Melanomas develop in 540% of these hosts after treatment with the phorbol ester, 12-O-decanoylphorbol-13-acetate (TPA), croton oil or 7,12-dimethylbenz[a]anthracene (DMBA). UV light exposure alone or with additional chemical tumor promoters and initiators has also been used to induce melanoma. The model with the highest incidence was guinea pigs exposed to DMBA (5). Melanoma developed in 40% of the animals after a long 18 month latency period. Hussain et al. (6) used a hairless SKHR mouse initiated with DMBA followed by repeated exposure to UV light (280320 nm) to achieve an incidence of 35%. Romerdahl et al. (7) observed that C3He mice initiated with DMBA followed by topical treatment with croton oil (or TPA) plus UV irradiation (280320 nm) resulted in a similar incidence of melanoma. The latency period of ~35 weeks was far shorter than was seen in other species.
The advent of transgenic technology has aided the development of mice that develop melanocytic lesions or tumors. Several groups targeted expression of oncogenes to pigment producing cells (810). The transgenic mice described by Mintz et al. (8) and Klein-Szanto et al. (11) express a transgene that contains the oncogenic sequences of the SV40 large T/small t complex driven by a mouse tyrosinase promoter. The mice are categorized according to susceptibility of tumor development, with transgene copy number delineating the groups. The more susceptible lines develop cutaneous tumors when exposed to UV radiation, but due to aggressive malignant ocular tumors they do not live long enough for the investigators to observe full progression of the cutaneous tumors. The tumors are instead transplanted to less susceptible mice where they progress, sometimes to metastasis.
The transgenic mouse line that we have developed expresses a transgene using a mouse tyrosinase promoter sequence to drive the expression of the activated human T-24 Ha-ras gene in pigment producing cells. The resulting mouse has an altered phenotype including mild melanocytic hyperplasia, a muted agouti coat and pigmented skin, as described previously (9). We hypothesized that the expression of an activated T24 Ha-ras gene would cause the melanocytes to be susceptible to the development of melanoma. The mice do not spontaneously develop cutaneous melanoma or other skin tumors; however, ~12% develop ocular melanoma (12).
More recently, Chin et al. (10) developed several transgenic mouse lines to study gene function in the development of melanoma. Mice that were deficient for INK4a (p16) did not develop melanoma. However, when they generated mice on the INK4a deficient background that express an activated Ha-ras in the melanocytes, the mice spontaneously developed cutaneous melanoma at a high penetrance. A fourth transgenic mouse model that has been reported expresses a transgene containing a metallothionein gene promoter regulating expression of hepatocyte growth factor/scatter factor (13). Over expression of the transgene was observed in a number of tissues and a broad array of tumors developed including melanoma.
In our model, the TPras transgenic mice, we examined the induction of melanoma. We report in this manuscript the results of a variety of carcinogenesis protocols and their efficiencies of inducing melanoma.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Treatment groups
All mice were housed in an AALAC approved University Animal Facility with 12 h light cycles. Food and water were provided ad libitum. The mice were shaved dorsally for treatments once a week with an electric razor, or as needed to remove hair. Studies were begun when the mice were 34 weeks old.
UV irradiation.
Mice were placed in open polystyrene cages with wire lids and irradiated with a light panel containing four FS40T12/UVB lamps (National Biological Corporation, Twinsburg, OH). Greater than 90% of the emitted light from the bulbs was in the 280340 nm range, as determined by a scanning spectrophotometer (model 440; Spectral Instruments, Tucson, AZ). The initial dose was 5.6 kJ/m2 per treatment, which was increased twice by 20% for a total final dose of 8.06 kJ/m2 per treatment. Doses were determined with the wire lid in place using a UVX Radiometer with a 310 nm probe (Ultraviolet Products, Upland, CA). The UV treatments were performed twice a week for 38 weeks or until tumors developed.
TPA.
Five micrograms of TPA (LC Services, Woburn, MA) were applied topically to the entire dorsal section of the mouse four times per week. The TPA was dissolved in acetone to a concentration of 100 µg/ml. An aliquot of 50 µl (5 µg TPA) was administered per treatment for 38 weeks.
UV/TPA.
A combination treatment of UV and TPA was administered as described above. The mice were treated topically with TPA (5 µg ) three times (Monday, Wednesday and Friday) per week. UV treatments were performed twice (Tuesday and Thursday) per week.
DMBA.
Fifty or 100 µg DMBA (Sigma, St Louis, MO) were applied once per week. The duration of the treatments was either 5 or 16 weeks. The DMBA was dissolved in acetone to a concentration of 0.5 mg/ml or 1.0 mg/ml. Aliquots of 100 µl of the appropriate DMBA solution were applied to the shaved backs of the mice. Due to the photosensitivity of DMBA, the solutions were prepared and applied in minimal, low-level lighting. Additionally, the cages were covered with dark cloth sheets for 24 h after treatment.
DMBATPA.
An aliquot of 100 µg DMBA was applied topically to the dorsal section of the mouse once a week, as described above, for 2 weeks. Additionally, 5 µg TPA was applied twice per week, on days when DMBA was not administered. The TPA treatments were performed for 38 weeks.
Cell lines
Cell lines from DMBA induced melanoma from the TPras mice were established and cultured as described previously (16). Briefly, cell lines from tumors were derived by placing the tumors in trypsin/EDTA and then mincing the tumor into a cell suspension. During early passage the cells were maintained either in melanocyte media with 5 ng/ml TPA or in M15 with the addition of 3.5% fetal bovine serum and 3.5% newborn calf serum (16). Later passage cells were maintained in M15 media supplemented with fetal bovine and newborn calf serum.
Severe combined immunodeficient (SCID) mouse injections
Cell lines (passage 10 to 20) were grown in tissue culture flasks to ~80% confluency, removed by trypsinization, and counted using a hemocytometer with Trypan blue staining (Sigma) to determine the number of viable cells. Cells were centrifuged and resuspended in sterile saline. Five million cells were then injected subcutaneously into SCID mice (obtained from the Arizona Cancer Center SCID mouse facility). Mice were observed for tumor development and killed when tumors progressed to 1.5 cm in diameter or were present for 45 days. Mice were killed and examined for the presence of metastatic lesions.
Histology
Histological sections were prepared from skin samples and tumors from each group of mice and for each treatment. Tissues were fixed in 10% buffered formalin, parafin embedded and cut into 45 µm sections. The histologically prepared samples were stained with hematoxylin and eosin (H&E) or Mason Fontana stain. Selected tumor samples were fixed in glutaraldehyde for examination by electron microscopy.
TPras transgene and mouse Ha-ras expression
Tumors from the DMBA-treated TPras mice and tumors that developed on SCID mice injected with cell lines established from TPras mouse melanomas were screened for expression of the TPras transgene and expression of the endogenous mouse Ha-ras gene using RTPCR. RNA was extracted by using a RNeasy kit (Qiagen, Chatsworth, CA) with a modified protocol (17). Briefly, fresh or frozen pieces of tumors were homogenized in RNeasy lysis buffer and subjected to a phenolchloroform extraction. RNA was precipitated in isopropanol, resuspended in RNeasy lysis buffer, and purified using RNeasy spin columns. DNA contaminates were removed by DNase I digestion prior to RT reaction. Two micrograms of RNA were reverse transcribed with the addition of Superscript Reverse Transcriptase (Gibco BRL, Grand Island, NY), 10 mM dNTPs, 25 mM MgCl2, 10x PCR Buffer II, 0.5 mg/ml oligo(dT)16, and Rnasin (40 U/µl). The cDNA PCR was carried out in a 25 µl reaction mixture containing 2 µl of a 1:20 dilution of the RT reaction product, 10x NH4 reaction buffer, 2.0 mM MgCl2 (TPras) or 3.0 mM MgCl2 (endogenous Ha-ras), 10 mM dNTP (2.5 mM of each) and 1.25 U Biolase DNA polymerase (Intermountain Science, Kaysville, UT). Reaction mixtures were heated to 95°C for 5 min and amplified for 30 cycles at 95°C for 35 s, 60°C for 35 s, and 72°C for 45 s. Samples were then held at 72°C for 10 min. The primer sequences for the TPras transgene and mouse Ha-ras were as follows: TPras 30, 5'-GTTGCTGGAAAAGAAGTCTGTG-3'; Ras 433, 5'-CCGTTTGATCTGCTCCCTGTAC-3'; and MRas 226, 5'-CCACTTTGTGGACGAGTATGAT-3'; and Ras 433 as described above.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The UV light (280340 nm) exposures induced papillomas in the negative littermates (22%). Melanocytic nevi and melanoma developed on 20% of the TPras mice. When the UV light exposure was combined with TPA treatment, papillomas were induced on the TPras mice (33%) and negative littermates (11%). Carcinoma developed on 11% of the negative littermates. Nevi (67%) and melanoma (11%) were induced on the TPras mice by this combined protocol with 15% of the negative littermates also developing melanocytic nevi. Of particular interest was the development of melanomas in the UV-treated TPras group and the UVTPA treated TPras group. The TPras mice that developed melanomas all had an albino phenotype.
Our observations with DMBA treatments demonstrated a high incidence of melanoma in the TPras mice. A total of 8688% of the mice developed both nevi and melanoma with 50 or 100 µg DMBA administered once per week for 5 weeks (Figure 1B and C). With 16 weekly doses of 100 µg DMBA, 100% of the TPras mice developed melanoma; however, we also observed some ulcerated lesions due to toxicity of DMBA. Papillomas and carcinomas were observed with the higher doses of DMBA (100 µg). With 16 weeks of the high dose DMBA treatment, papilloma (100%) and carcinoma (75%) developed on the negative littermates. Nine percent of the TPras mice developed carcinomas and 55% developed papillomas on this protocol. When the number of DMBA treatments was reduced to 5 weeks with 50 or 100 µg, the incidence of papillomas on the TPras mice dropped to 13% and no carcinoma were observed. No carcinomas were observed with the 5 week DMBA treatment in the negative littermates; however, papillomas were observed on 82% of the negative littermates treated with 100 µg DMBA (Figure 1D
). When the mice were given an even lower dose of DMBA (20 µg for 16 weeks) none of the mice developed any tumors (data not shown).
|
Histopathology of the skin lesions and tumors
Histological sections were prepared from skin samples or cutaneous tumors after 27 weeks of treatment. The control, TPras mice treated with acetone alone, showed no stimulation of melanin (Figure 2A). In the TPras mice treated with TPA, we observed a marked acanthosis of the epidermis along with increased melanin depositions in the reticular and papillary dermal layers (Figure 2B
). UV irradiation caused similar epidermal thickening as was observed in TPA treated mice, but UV also induced some cellular disorganization (Figure 2C
). The combination of UV and TPA treatments resulted in significant melanin deposits in the reticular dermis and to a lesser extent, in the papillary dermis (Figure 2D
).
|
|
|
Evaluation of tumorigenicity in SCID mice
Four cell lines established from the DMBA-induced melanoma in TPras mice, including cutaneous tumors and lung metastases, were evaluated for tumorigenicity in SCID mice. Cells harvested from tissue culture were injected into SCID mice and the animals were observed for tumor formation. Tumors formed in mice injected with melanoma cells within 1 to 2 weeks. The tumors were invasive to adjacent muscle and metastasized in some of the mice. The lesions had histologically identifiable melanoma characteristics. The non-malignant melanocyte line derived from the untreated transgenic mouse did not form tumors when injected into SCID mice.
Cell lines designated 1998 and 1984-1, both derived from DMBA-induced primary cutaneous melanoma, formed tumors within 1 week of injection. The tumors invaded muscle and the body cavity, though no attachment to internal organs was observed. Additionally, 1984-1 was very vascularized and formed metastatic lesions in both lungs. Cell lines designated 1996 and 1984-2, from a DMBA-induced cutaneous lesion and a lung metastasis (from mouse with 1984-1 cutaneous lesion), respectively, formed tumors by 2 weeks after injection. The tumors demonstrated slower progression than the 1998 and 1984-1 cell lines.
Expression of the TPras transgene in the melanoma
Expression of the transgene and endogenous Ha-ras in tumors was analyzed by RTPCR (Fig. 5). cDNAs were made from pieces of tumors from the DMBA-treated TPras mice and tumors that developed when SCID mice were injected with cells lines derived from the TPras mice tumors. Skin samples taken from regions that had not been treated were also analyzed. As shown in Figure 5
, tumors that developed on the DMBA-treated TPras mice (1984-1, 2391a, 2391b, 2395a, 2421a, 2452a) and the tumors on the SCID mice that developed from TPras melanoma cells (SC1984-1a, SC1984-1b, SC1998a) all expressed the TPras transgene. In most samples of untreated TPras skin (Sk2391, Sk2395, Sk2421 and Sk2425) we did not detect the transgene using the conditions described. However, if the number of cycles that the samples were amplified was increased then low levels of the TPras transgene were detected in some samples (data not presented). Endogenous mouse Ha-ras was detected in all of the tissues and tumors analyzed.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
These studies demonstrate that the TPras mice are highly susceptible to developing melanoma and represent a model in which melanoma is induced in multiple stages of progression, from nevi to metastatic tumors. This model provides an ideal system for the evaluation of chemopreventive and therapeutic treatments due to the presence of multiple stages, a high incidence (>80%) and a moderate latency period (1225 weeks).
We chose a mutated ras gene for the transgenic construct because of its integral role in the mitogenic response pathway and a high rate of mutation in many forms of cancer. In melanomas, ras mutation rates of 525% have been reported by several groups (2022), and Platz et al. (21) observed increased ras expression in 70% of the melanomas they studied. The Ha-ras gene has been shown to be involved in non-melanoma skin cancers (2325) initiated by DMBA and promoted by TPA. DMBA, unlike many other carcinogens, preferentially causes AT point mutations through its epoxide metabolites. As shown by Cheng et al. (26), a point mutation in codon 61 of ras causes oncogenic conversion of the protein, which is integral in the transformation of the cells. We have screened the tumors for additional mutations in endogenous ras and the Ha-ras transgene (M.B.Powell, J.Morreale and L.Martin, manuscript in preparation). No mutations were observed in codons 12, 13 or 61. The evaluation of the DMBA-induced tumors in SCID mice demonstrated that the melanoma cell lines are malignant, invasive and have a propensity for metastasis.
We also have observed the induction of melanoma with UV alone and UVTPA treatments. The melanoma developed in a small number of TPras mice that had an albino phenotype. These tumors developed after 2535 weeks of treatments. Several other models of UV-induced melanoma have been reported. Mintz et al. (8) observed in their SV40 transgenic mice that UV exposure increased the incidence of melanoma in transplanted skin grafts from 25 to 39%. This model represented a major step forward in mouse melanoma models, as the incidence reached nearly 40% and the latency period was between 24 and 37 weeks in UV-irradiated subjects. Ley et al. (3) observed in an opossum model, Monodelphis domestica, that UVB induced melanoma. Ley reported recently that UVA is nearly as efficient as UVB in inducing melanocytic lesions in these animals (27). A third group, Setlow et al. (28), have observed the induction of melanoma at UV wavelengths of 365, 405 and 436 nm in platyfish. We are presently developing several lines of TPras mice with different pigmentation phenotypes to allow a more careful evaluation of the role of UV and pigments in melanoma development.
We have characterized the melanoma cell lines for cytogenetic abnormalities (16). A key feature discovered during the characterization was a conserved alteration in chromosome 4. Accompanying this alteration was a loss of gene material for two tumor suppressor genes, p15 and p16, which have been identified by several studies as being involved in melanoma development (29,30). A transgenic system developed by Chin et al. (10) examines the functional role of ras and INK4a (p16) in melanoma development. They generated p16 deficient mice that alone did not develop melanoma. However, when they crossed the p16-deficient mice with a transgenic mouse with an activated Ha-ras controlled by a tyrosinase promoter (similar to our TPras mice), they observed multiple spontaneous, locally invasive melanomas. Chin's observations support our report (16) that melanoma development in the DMBA treated TPras mice was accompanied by either complete or partial loss of the p16 gene.
These studies provide evidence that malignant melanoma is inducible in the TPras transgenic mice that we have developed. The most effective carcinogenesis protocol, five weekly doses of 100 µg of DMBA, that we have identified has yielded a >80% incidence of malignant melanoma, including some invasive metastatic disease. These studies suggest that expression of an activated ras in the melanocytes contributes to their susceptibility to develop melanoma.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|