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[Cancer Research 63, 3503-3510, July 1, 2003]
© 2003 American Association for Cancer Research

Carcinogenesis

Molecular Characterization of New Melanoma Cell Lines from C3H Mice Induced by Ethanol plus Ultraviolet Radiation

Faith M. Strickland1, Sen Pathak, Asha S. Multani, Ronald P. Pelley and Cherrie K. Donawho2

Departments of Immunology-178 [F. M. S.] and Cancer Biology [S. P., A. S. M., C. K. D.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and Pangea Phytoceuticals, Harlingen, Texas 78550 [R. P. P.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A major obstacle in understanding the etiology of malignant melanoma is the lack of mouse models and transplantable cell lines. We have recently developed a model of primary melanoma in C3H mice induced by ethanol and UV light. The present study characterizes three cell lines, SM190.2, SM190.626, and SD0302, derived from two melanomas produced in the dorsal skin of two C3H mice treated thrice weekly for 28–33 weeks with UV radiation and ethanol. In both tumors, the N-ras oncogene was mutated. Tumor SM190 lacked exon 2 of the p16INK4a tumor suppressor gene. Cell line SM190.2, which was derived from tumor SM190, produced pigmented tumors when transplanted into syngeneic severe combined immunodeficient mice and normal mice. None of the cell lines produced metastases. All three cell lines were highly aneuploid, even at low passage numbers. SM190.2 and SD0302 cells contained an interstitial deletion in the long arm of chromosome 4, where the p16INK4a gene resides, and SM190.2 had an additional segment in chromosome 6. The third cell line, SM190.626, had three consistent Robertsonian translocation markers involving chromosomes 7, 14, and 17. The translocation involving mouse chromosome 14 may prove especially valuable because translocations in this chromosome are associated with metastatic behavior. These reagents will provide opportunities to search for new tumor suppressor genes that may contribute to the growth and metastasis of primary melanoma.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The incidence of melanoma skin cancer has been sharply rising for the past 50 years (1, 2, 3) . One of the melanoma risk factors is periodic, high-dose UV exposure of young adults (4 , 5) . But whereas solar UV exposure is clearly the dominant risk factor for nonmelanoma skin cancer, the development of melanoma in humans appears to be much more complex. In addition to chemical exposure and the immune response, other factors such as genetic predisposition, DNA repair enzymes, skin pigmentation, and cell cycle regulators also appear to be involved (2 , 4 , 6, 7, 8, 9, 10) . Unlike basal cell and squamous cell skin carcinomas, which feature high incidence but low mortality, melanomas are among the deadliest of cancers. The high mortality is due to its propensity to metastasize while still small and to its resistance to radio- and chemotherapy, making prevention and early treatment especially important. Therefore, understanding the causes of melanoma and the early events in tumor development/progression is essential.

A major obstacle to understanding the etiology of primary cutaneous melanoma is the limited number of animal models for this disease. The predominant animal model for human cancer, the mouse, does not readily develop melanoma. Much of what we know about the etiology of this disease and the contribution of UVB radiation (280–320 nm) and immune responses to melanoma growth comes from cell lines derived from only a handful of murine tumors. Therefore, the generation of new melanoma cell lines and methods to induce these tumors in mice would greatly advance our understanding of this disease.

We have shown that adult inbred C3H/HeN mice treated with a combination of ethanol, aloe emodin, and UV radiation develop high numbers of primary cutaneous melanomas (11) . The present study describes the production of cell lines, karyotype analysis, and molecular characterization of a tumor suppressor gene and oncogene from two tumors induced by ethanol and UV radiation. The availability of new murine melanoma cell lines for immunological and genetic studies should enhance our ability to understand the etiology of malignant melanoma.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice.
Specific pathogen-free female C3H/HeN (MTV-) mice were purchased from the Animal Production Area of the Frederick Cancer Research Facility (Frederick, MD) and maintained in a pathogen-free barrier facility in accordance with NIH and American Association for Assessment and Accreditation of Laboratory Animal Care International guidelines. The mice were housed in filter-protected cages and provided with NIH-31 open formula mouse chow and sterile water ad libitum. Ambient light was controlled to provide regular cycles of 12 h of light and 12 h of darkness. All procedures were approved by the Institutional Animal Care and Use Committee of The University of Texas M. D. Anderson Cancer Center.

UV Radiation.
UV radiation was administered in vivo using a bank of six unfiltered FS40 sunlamps (National Biological Corp., Twinsburg, OH). The energy emitted from these lamps consists of 0.5% UVC (<280 nm), 64.5% UVB (280–315 nm), and 35% UVA (315–400 nm) as measured by an Optronics model 742 scanning spectrophotometer interfaced with a Compaq 386 personal computer (Optronics Laboratories, Orlando, FL). The peak emission of the FS40 lamps was 313 nm. The average irradiance of the source was approximately 4.5 W/m2 at 20 cm distance, as measured by an IL1700 radiometer with a SEE280 filter and a W quartz diffuser (International Light Inc., Newburyport, MA). The cage tops reduced the incident light received by the animals to 2.6 W/m2.

Treatment of Mice.
The dorsal fur of the mice was shaved with electric clippers. Beginning when the mice were 10–12 weeks of age, the shaved dorsal skin, ears, and tails of the animals were treated with 25% U.S.P. grade ethanol/water (1 ml/mouse) applied using cotton Q-tip applicators. Thirty min later, the animals were exposed to 15 kJ/m2 UVB radiation as described previously (11) . The mice were treated thrice weekly for approximately 33 weeks.

Derivation and Maintenance of Tumor Cell Lines.
Tumors arising on the treated skin of the mice were aseptically excised before they reached 1 cm in diameter. A portion of each tumor was fixed in 10% neutral buffered formalin, embedded in paraffin, and routinely processed for H&E and Fontana silver stain for melanin, and diagnosis was made as described previously (11) . The diagnosis of melanoma was made independently by two pathologists [Dr. L. Clifton Stevens (The University of Texas M. D. Anderson Cancer Center) and Dr. H. Konrad Muller (The University of Tasmania)] from coded slides. A segment of each primary tumor was stored frozen in liquid nitrogen in a solution of 40% fetal bovine serum/RPMI 1640 containing 10% DMSO. After diagnosis of the tumor as a melanoma, fragments of the primary tumor, which was stored in liquid nitrogen, were thawed and transplanted s.c. into syngeneic SCID3 mice. The tumors that developed in the SCID mice from these fragments were excised and cultured in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum, vitamins, 0.1 mM nonessential amino acids, 10 mM HEPES buffer, sodium bicarbonate, 100 IU/ml penicillin, and 100 µg/ml streptomycin (DMEM) to establish cell lines. The K1735 and B16 mouse melanoma cell lines were kind gifts from Dr. I. J. Fidler (Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center). All cell lines were maintained in DMEM in a 7% CO2 balanced air atmosphere at 37°C. Subconfluent cultures were expanded by treatment with 0.25% trypsin/0.1% EDTA.

Western Blot Analyses.
Pelleted culture cells of the B16, K1735, SD0302 melanoma, and Pam212 keratinocyte cell lines were frozen in liquid nitrogen and extracted as described by Orlow et al. (12) . Protein from neonatal human foreskin melanocytes (FHMs) was a kind gift from Dr. Estella Medrano (Baylor College of Medicine, Houston, TX; Ref. 13 ). Protein concentrations were determined using the Bio-Rad Protein Assay (Pierce Chemicals, Rockford, IL), with a BSA standard, according to the manufacturer’s instructions. Fifty µg of protein from B16, K1735, and SD0302 and 15 µg of FHM were denatured by boiling for 5 min in 2% ß-mercaptoethanol,10% SDS, 0.3 M Tris (pH 6.8), 50% glycerol (w/v), and 0.005% (w/v) bromphenol blue and applied to 7.5% polyacrylamide gels as described previously (14) . Horseradish peroxidase-conjugated molecular weight markers were included in each analysis. Proteins were transferred to nitrocellulose membranes, blocked with 1% BSA, and incubated overnight at 4°C with a 1:1000 dilution in PBS/1% BSA of the following polyclonal rabbit antisera: anti-PEP13, which recognizes GP100/mel 17 (15) ; anti-PEP7H, which is specific for murine tyrosinase (16) ; anti-PEP1, which recognizes the COOH-terminal peptide of TRP-1 (16) ; and anti-PEP8 against the COOH-terminal peptide of TRP-2 (17) . The antibodies were a kind gift from Dr. Vincent J. Herring (National Cancer Institute, Bethesda, MD). The blots were washed in PBS/1% BSA and reincubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin at 4°C for 2 h. Antibody binding and molecular weight markers were detected using Supersignal West Pico chemiluminescent substrate (Pierce, Rockford, IL) according to the manufacturer’s instructions.

DNA analysis for Ras and p16INK4a.
The PixCell II Laser Capture Microdissection system (ARCTURUS Engineering, Mountain View, CA) was used to select and microdissect tumor and adjoining tissue from 5-µm, paraffin-embedded, H&E-stained sections of primary melanoma tumors and was used according to the manufacturer’s instructions. Briefly, selected populations of cells from a section of complex, heterogeneous tissue were identified by light microscopy. Transparent CapSure transfer film was applied to the surface of the selected cells, and a laser pulse was delivered to activate the film, resulting in adhesion of the selected cells to the film. The procured region was immediately placed in 250-µl Eppendorf tubes containing 50 µl of proteinase K digestion buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% Tween 20, and 0.1 mg/ml proteinase K] and incubated overnight at 37°C. The mixture was then boiled for 10 min to inactivate the proteinase K and stored at 4°C. DNA was obtained from cultured cell lines as follows. Subconfluent cultures were treated with 0.25% trypsin/0.1% EDTA. The detached cells were centrifuged for 5 min at 500 x g and washed once in PBS. The pelleted cells were incubated in proteinase K digestion buffer overnight at 37°C followed by inactivation of the proteinase K and storage at 4°C. DNA-containing tubes were briefly spun, and 10 µl of the supernatant were removed for analysis. DNA (100 ng) was amplified by PCR in a in 50-µl reaction mixture containing 1x PCR buffer, 200 µM each deoxynucleoside triphosphate, 1.5 mM MgCl2, 1 unit of Taq polymerase, and 20 pmol of each primer. The primers used were as follows: (a) p16INK4a exon 1, 5'-ATGGAGTCCGCTGCAGACAG (forward) and 5'-CTGAATCGGGGTACGACCGA (reverse); (b) p16INK4a exon 2, 5'-GTGATGATGATGGGCAACGT (forward) and 5'-TGGGCGTGCTTGAGCTGAGG (reverse); (c) N-ras 12/13 exon 1, 5'-ATGACTGAGTACAAACTGGT (forward) and 5'-CTCTATGGTGGGATCATATT (reverse); and (d) N-ras 61 exon 2, 5'-GTGGTGATTGATGGTGAGAC (forward) and 5'-TACACAGAGGAACCCTTCGC (reverse).

Sequences of primers for p16INK4a and N-ras were as described previously (7 , 18) . The reaction mixture was first incubated at 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for 45 s. Finally, the reaction mixture was incubated for 10 min at 72°C in a GeneAmp PCR System 9600 thermocycler (Perkin-Elmer, Branchburg, NJ). The PCR products were analyzed on 2% agarose gels (BioWhittaker Molecular Applications, Rockland, ME). Automated fluorescence DNA sequencing was performed by Lone Star Laboratory (Houston, TX) using an Applied Biosystems 377XL DNA sequencer (Foster City, CA) and ABI Prism Big Dye Terminator cycle sequencing kit.

Cell Harvesting and Chromosome Banding Analysis.
Freshly fed (within 24 h) and approximately 75% confluent melanoma cell cultures were used for chromosome preparation. The cell cultures were harvested for air drying preparations following the standard techniques described previously (19) . From each cell line, at least 20–25 Giemsa (G)-banded metaphase spreads were analyzed, and 10 complete karyotypes were prepared from each sample.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Histopathology.
The pigmented tumor, designated SM190, arose on the dorsal skin of a C3H mouse treated for 28 weeks with UV radiation plus 25% ethanol/water. Figs. 1Citation and 2ACitation illustrate the histopathology of the primary tumor and the adjacent nontumorous skin. Grossly, the tumor consisted of a 10-mm-wide nodule, 5 mm in depth, with an ulcerated surface, displacing the panniculus carnosus inferiorly. The upper one-third of the tumor was intensely pigmented (Fig. 1A)Citation , whereas the lower two-thirds were fish-fleshed in appearance. As Fontana silver staining revealed (Fig. 1)Citation , virtually all cells in the upper portion of the tumor contained melanin (lower magnification, Fig. 1ACitation ; upper portion and higher magnification, Fig. 1CCitation ). This melanin pigmentation is progressively lost as one goes deeper into the tumor (Fig. 1ACitation , lower portion), and differentiation is lost. In the better-differentiated portions of the tumor, only occasional anaplastic cells are observed, and the pigmentation is prominent even with the H&E stain (Fig. 2A)Citation . In most of the cells, the melanin was organized either as dense clumps or small grains (Fontana stain, Fig. 1CCitation ; H&E stain, Fig. 2ACitation ). In some areas of the central portion of the tumor, numerous dark pyknotic nuclei were visible (Fig. 2ACitation , arrows), consistent with a high apoptotic index. In the more peripheral portions of the primary tumor, there was progressively less melanin, the tumor became more nodular in pattern, and there was a tendency toward necrosis. The histopathology in these areas closely resembled that of primary melanomas we have described previously (11) , featuring pleomorphic or spindle-shaped cells with vesicular nuclei and multiple, prominent nucleoli. In these areas, cytoplasmic melanin was visible only as a "dusty pattern" after Fontana staining (11) .



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  		Fig. 1. Light microscopic images of primary tumor SM190 (Fontana silver stain). Low magnification views (x4 objective) demonstrate the degree and distribution of pigment in the tumor (A) versus chronically UV-irradiated skin immediately adjacent to the tumor (B). Bar, 100 µm. Closed arrow indicates an area of melanization in the basal layer of skin due to activity of melanocytes transferring melanin granules to keratinocytes. Open arrows indicate melanin in the dermis residing in melanophages. C, higher magnification view (x40 objective) of the well-differentiated, nonnecrotic region of tumor shown in A. D, higher magnification (x40) of the dermal/epidermal junction indicated by the closed arrow in B. E, x40 magnification of the region indicated by the open arrows in B.

 


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  		Fig. 2. Melanin production in primary and transplanted melanomas in vivo (stained with H&E). The primary tumor SM190 (A) arose in a C3H mouse chronically treated with UV radiation plus ethanol, as described in "Materials and Methods." The melanoma cell lines SM190.2 and SM190.626 were derived in our laboratory from this tumor. This view is a higher magnification (x40 objective) view of the Fontana-stained field shown in Fig. 1ACitation . Open arrows, pyknotic nuclei consistent with apoptosis. SM190.2 cells transplanted s.c. into a normal syngeneic mouse produced a pigmented tumor (B). Before tumor transplant, the recipient mouse was exposed 3x/week to 5 kJ/m2 UVB radiation for 2 weeks to provide a tissue environment that would be favorable to tumor growth. The tumor grew for 3 weeks and was removed for analysis. Numerous pigmented cells, including an occasional melanophage, resided in the central, apoptosis-rich interior of the rapidly growing tumor. Arrowheads, tumor cells with clear perinuclear areas. Five-µm paraffin sections stained with H&E. x40 magnification.

 
Compared with the highly melanized tumor (Fig. 1A)Citation , the adjacent, chronically UV-irradiated skin (Fig. 1B)Citation had a melanin content several orders of magnitude lower. This melanization occurred in two locations. Foci of melanocytes were observed at the epidermal-dermal junction (Fig. 1BCitation , solid arrow; Fig. 1DCitation illustrates the area identified by the arrow in Fig. 1BCitation at higher magnification). At high magnification (Fig. 1D)Citation , the melanocyte dendritic processes are well demarked by the Fontana stain. In the area above the basal layer, almost all of the granular cells have silver-stained granules indicative of transfer of melanin from melanocytes to keratinocyte precursors. In the upper dermis (Fig. 1DCitation , lower portion), there are only scattered granules of incontinent melanin pigment. This focus of melanocytes is clearly nonneoplastic. Melanin is also observed in the deeper dermis (Fig. 1BCitation , open arrows; Fig. 1ECitation is a higher magnification of the area indicated by the open arrows in Fig. 1BCitation ). Half a dozen melanophages are scavenging incontinent melanin pigment near a hair follicle (Fig. 1ECitation , top right corner). During chronic UV irradiation of the C3H mouse, melanin-containing hair follicles in the deep dermis undergo necrosis and inflammation, spilling silver-staining pigment into the dermis. This process is different from the increased cycling of the pilosebaceous units seen in early UV-associated changes, which involve apoptosis rather than necrosis and do not cause pigmentary incontinence in the dermis.

Melanoma Transplants.
Highly pigmented fragments of the SM190 tumor were transplanted into a syngeneic SCID mouse, where they formed pigmented tumors in the recipient. One of these tumors was excised when it reached 1 cm in diameter and was cultured to establish two cell lines, SM190.2 and SM190.626. A third cell line, SD0302, was established from a primary melanoma (0302) arising in a separate, essentially identical experiment. All three cell lines arose from primary tumors induced in C3H mice chronically treated with 25% ethanol plus UV radiation. The cell lines were maintained in culture for at least 5 passages and frozen in liquid nitrogen. To ensure that the cells could be recovered and were tumorigenic, they were thawed and maintained in culture for another 15 passages. The cells were injected into syngeneic SCID mice, in which all three cell lines formed solid tumors. The SM190.2 cell line was also transplanted into a normal C3H mouse that had been given a mild treatment of UV radiation. The injected tumor cells produced a solid, pigmented tumor (Fig. 2B)Citation that recapitulated the same pattern of central pigmentation and peripheral amelanosis observed in the primary melanoma. The UV radiation was administered to simulate a cytokine environment from which the tumor arose, but it was an amount of UV insufficient to suppress tumor immune surveillance (20) . Later experiments indicated that the UV irradiation was unnecessary because the tumor cells also grew in normal (nonirradiated) syngeneic animals (data not shown). The effect of amounts of UV radiation sufficient to suppress the growth of nonmelanoma skin cancer on the growth of these melanoma cell lines is currently unknown.

At high power, the central portions of the transplanted tumor (Fig. 2B)Citation differed from the primary tumor (Fig. 2A)Citation in that a significant number of cells differentiated into melanin-laden dendritic tumor cells (Fig. 2BCitation , arrows) or tumor cells with a clear perinuclear area (arrowheads). In this respect, the central portions of the transplanted tumor showed greater melanocyte differentiation than the primary neoplasm. The periphery of the transplanted tumor demonstrated dedifferentiation toward an amelanotic, almost sarcomatous appearance similar to that observed in the primary melanoma and our previously described murine melanomas.

Cell lines SM190.626 and SD0302 produced amelanotic, highly undifferentiated tumors when transplanted into syngeneic SCID mice (data not shown). Although the tumors produced by all three cell lines were locally invasive, they did not spontaneously metastasize to the draining lymph nodes or distant organs.

Histology of Cell Lines in Culture.
Cultures of the three cell lines were heterogeneous in cell size and appearance. The SM190.2 line contained small, stellate cells with long dendritic processes and larger epitheliod cells (Fig. 3A)Citation . Addition of 3,4-dihydroxyphenylalanine to the cultures increased the number of pigmented cells and the amount of pigment per cell, but the majority of the cells in this line were amelanotic in culture. The SD0302 cells were more homogeneous in size than the SM190.2 cells and were predominantly stellate with long intermingling dendritic processes (Fig. 3B)Citation . After addition of 3,4-dihydroxyphenylalanine to the culture, approximately 40% of the cells in the SD0302 cultures contained Fontana stain-positive melanin granules. SM190.626 cells (Fig. 3C)Citation were predominantly stellate with long dendrites, but unlike the SM190.2 line, these cells were completely amelanotic. The cultured tumor cells varied in their melanin content but were generally negative or only weakly positive for melanin in vitro. However, even the established K1735 melanoma cell line, derived from a C3H mouse, was melanin-poor under these conditions (Fig. 3D)Citation .



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  		Fig. 3. Melanoma cell lines from C3H mice. A, SM190.2; B, SD0302; C, SM190.626; D, K1735. The cells were grown on glass slides, fixed with 10% neutral buffered formalin, and stained with Fontana silver stain for melanin (black). Cultures were counterstained with eosin (pink). x20 magnification.

 
Tyrosinase and Melanocyte Markers.
The tumors were examined for the presence of enzymes involved in the pigment cell pathway. A large amount of protein (50 µg) from the B16, K1725, SM190, and SD0302 cell lines was loaded on the gel to increase the detection of potential tyrosinase proteins. Fig. 4Citation shows that antibodies against peptides in the pigment cell pathway reacted strongly with protein from the B16 melanoma but were negative for the same amount of protein from the K1735 melanoma. Like the SM190.2, SM190.626, and SD0302 cell lines, the K1735 tumor arose in the C3H strain of mice. Another protein, GP100, was detectable in human melanocytes and in the B16 tumor, where it had a molecular weight of 78,000, but not in K1735 or SD0302 melanomas. These results suggest that there are immunological differences between the proteins that synthesize the black (eumelanin) pigment, observed in C57/BL6 mice, and proteins that produce red (pheomelanin) pigment, present in C3H animals.



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  		Fig. 4. Strain differences in reactivity of anti-tryosinase antibodies. The cultured cell lines B16 melanoma (C57BL/6), K1735 melanoma (C3H), SD0302 melanoma (C3H), Pam212 keratinocytes (BALB/c), and FHMs were extracted and electrophoresed on 7.5% SDS polyacrylamide gels as described in "Materials and Methods." Fifty µg of protein were loaded in each lane. The proteins were transferred to nitrocellulose membranes, treated with the antibodies indicated, and visualized using chemiluminescent antirabbit antibody and autoradiography.

 
Karyotype Analysis of Cell Lines.
A typical G-banded karyotype of the SM190.2 cell line is shown in Fig. 5ACitation . The chromosome number in this cell line ranged between 60 and 78, with a mode at 60 chromosomes. There were no biarmed chromosomes representing Robertsonian translocations or unusually long acrocentric chromosome representing tandem translocation present in SM190.2 metaphases. The cell line was aueuploid with multiple copies of many chromosomes present. One homologue of chromosome pair number 4 showed an interstitial deletion in the long arm where the p16INK4a gene is mapped (identified by an arrow in Fig. 5ACitation ). The remaining chromosomes appeared normal in their characteristic G-banding patterns. Two or three copies of the X chromosome were present in most metaphases analyzed.



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  		Fig. 5. Giemsa-banded karyotypes of mouse melanoma cell lines sharing both numerical and structural abnormalities. A, most chromosomes were present in multiple copies except 7, 8, 13, 14, and X in cell line SM190.2. A chromosome 4 homologue showing an interstitial deletion in the long arm is indicated by an arrow. B, cell line SM190.626 had four biarmed markers (M1 to M4), of which M1 and M4 were present in two copies each, and M2 and M3 were present in one copy each (see "Karyotype Analysis of Cell Lines" for their tentative identification). C, the SD0302 cell line had two unusually long clonal acrocentric marker chromosomes designated M1 and M2. Each of these markers was present in multiple copies in each metaphase spread. A homologue pair of number 4 chromosome had an interstitial deletion in the long arm, indicated by the arrow. Six or seven unidentified markers (UM) ranging from very long to minute in size, are arranged at the bottom line of this karyotype.

 
The SM190.626 cell line showed a range between 72 and 81 with a modal number of 75 chromosomes. There were 4 biarmed markers present in metaphases of this cell line. A typical G-banded karyotype is shown in Fig. 5BCitation , where the biarmed marker chromosomes (M1 to M4) are identified by their characteristic G-banding patterns. Two copies of M1 and M4 and a single copy of M2 and M3 each were present in every cell analyzed. Tentative identifications of these markers were as follows: M1 = t(7q;7q); M2 = t(10q;14q); M3 = t(11q;17q); and M4 = t(15q;15q). A typical deletion in the long arm of chromosome 4 was not clearly visible in this cell line. The X chromosome was present mostly in 3 copies/metaphase spread.

In the third cell line, SD0302, the range of chromosome distribution was 92 and 112 with a peak at 98 chromosomes. A typical G-banded karyotype of this cell line is shown in Fig. 5CCitation . This cell line is also aneuploid, with no biarmed marker chromosome present. However, two clonal markers were present in each metaphase spread analyzed. A tentative classification of these markers was as follows: M1 = t(8q;10q); M2 = ins(10q). Two or three copies of each of these M1 and M2 were present in each metaphase spread. Six or seven unidentified markers (UM) are arranged on the bottom row of Fig. 5CCitation . One homologue of chromosome 4 containing an interstitial deletion is indicated by an arrow.

Molecular Analysis of Cell Lines.
We analyzed p16INK4a and N-ras genes in the pigmented primary melanomas and cell lines from the SM190 and 0302 tumors. The DNA from the primary tumors was isolated by Laser Capture microdissection from routinely processed 5-µm tissue sections stained with H&E. This approach enabled us to analyze the DNA from cells within a mixed population and minimize contamination with DNA from normal cells present in the tissue. No PCR amplification of DNA was observed for p16INK4a exon 2 using DNA from the primary SM190 tumor or its cell lines, suggesting that there was a partial deletion of the p16INK4a gene in this tumor (Table 1)Citation . Similarly, exon 2 but not exon 1 was absent in the K1735 cell line. Both exons of the p16INK4a gene were absent in B16 melanoma cells. The 0302 tumor and its cell line, SD0302, contained four nucleotide substitutions in the p16INK4a gene: A-C and C-A transversions in codons 18 and 21 of exon 1; and G-A and T-C transitions in codons 50 and 149 in exon 2. Identical nucleotide changes were observed in exon 1 in SM190 and K1735 tumors and cell lines. However, further analysis revealed that these four "substitutions" were present in tissues of untreated mice from the C3H strain but not C57BL/6, DBA, and 129SvJ strains of mice (21, 22, 23) and thus represent a previously unreported allotypic difference between the C3H and other strains of mice.4 The transversion at codon 18 resulted in a change in amino acid residues from histidine to proline. Such a change would be predicted to alter the secondary and tertiary structure and possibly the function of the p16INK4a protein. The nucleotide changes in codons 21 and 50 resulted in amino substitutions that were silent or conserved. Although the change in codon 149 produced amino acids that were both hydrophobic, a change from phenylalanine to leucine could impact protein structure because of the removal of a bulky phenolic ring of phenylalanine.


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  		Table 1 Mutations in the p16INK4a tumor suppressor gene and N-ras oncogene in melanoma tumors and cell linesa

 
The N-ras gene contained a C to T mutation at codon 19 in both 0302 and SM190 primary tumors and their cell lines. The SM190 primary tumor and cell lines and K1735 melanoma cell line also had a C to T transition on the nontranscribed strand at codon 13. The mutations in codons 13 and 19 occurred at dipyrimidine sites and are UV associated (24) . No mutations were observed in exon 2 of N-ras in any of the primary tumors or cell lines (data not shown). Together, these results demonstrate the similarities between our newly arising melanomas and established melanoma cell lines.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we generated primary melanomas in mice and characterized them with the goal of establishing melanoma tumor models and cell lines. We have shown previously that skin tumors diagnosed as melanomas by histopathological criteria were induced in C3H/HeN mice chronically treated with UV plus ethanol and UV plus aloe emodin (11) . There arose some question from that publication as to whether the tumors were true melanomas or whether they were sarcomas invading areas of UV-irradiated skin containing incontinent pigment. Furthermore, many tumors from our initial studies were large (1.5 cm2) when prosected, were scantily pigmented or amelanotic, and contained areas of necrosis. We therefore generated new melanomas using UV and ethanol, prosected the tumors soon after they became visible, and established transplantable cell lines to further characterize the tumor cells.

In the present study, we examined three cell lines generated from two melanomas that arose independently on separate animals. The SM190 primary tumor contained variable amounts of pigment. Highly pigmented fragments of this tumor were used to generate two cell lines, SM190.2 and SM190.626, which also produced tumors upon transplantation into syngeneic SCID mice, which were variable in their melanin content. Whether the mixed appearance of the tumor was due to independent amelanotic and melanotic tumors cannot be determined from these data. However, cloned melanotic cells can produce tumors that are both melanotic and amelanotic in vivo, even though they arise from a single progenitor cell (25 , 26) . The SM190.2 cell line also grew in immunocompetent C3H mice given a low dose of UV radiation and in an unirradiated, syngeneic animal. The UV radiation was given to create a tissue and growth factor environment closer to the one from which the primary tumor originated. The amount of UV radiation was insufficient to induce UV-specific suppressor T cells that permit the growth of nonmelanoma skin cancer (20) . The role of UV radiation in permitting the progression and growth of the primary melanomas or their cell lines is currently unknown.

The melanoma cell lines were characterized by aneuploidy, which is considered a driving force for tumorigenicity (27 , 28) . Interstitial deletion in mouse chromosome 4, which contains the p16INK4a gene, has already been reported in the UV-induced K1735 melanoma cell line (29) . Two of our cell lines showed a similar deletion in chromosome 4 in addition to multiple copies of many autosomes. Mutations and deletions of the tumor suppressor genes p16INK4a and p19ARF are frequently observed in human familial melanoma and are a key element, in combination with ras oncogene overexpression, for melanoma development in transgenic mouse models (7 , 30) . Molecular analyses confirmed that a portion of the p16INK4a gene was deleted in SM190 primary tumor and cell lines as well as the K1735 melanoma. Both exons of p16INK4a were disrupted or deleted in B16 melanoma cells. The finding that the p16INK4a gene in C3H mice differs from that in other strains of mice in four codons was unexpected (21, 22, 23) . At least one of these nucleotide substitutions results in a hisidine to proline amino acid change that would be predicted to alter the structure and perhaps the function of the protein. If the allotypic differences in the p16INK4a tumor suppressor gene decrease the function of the protein, they may render the C3H strain more susceptible to melanoma induction. However, other genes and other factors are clearly involved because an elevated incidence of melanoma has not been reported in C3H mice using UV radiation alone (8 , 10 , 26) . The N-ras gene was altered in both primary melanomas and K1735. The mutations found in exons of the N-ras gene were associated with UV radiation-induced thymine dimer formation (24) . These data support a role for the combination of p16INK4a and ras in our mouse model of primary melanoma and are consistent with the view that whereas UV radiation contributes to melanoma induction, it is only one of several factors in a complex process (10 , 31, 32, 33, 34, 35) .

Chromosome 14, which is implicated in the metastatic phenotypes of a variety of mouse tumors including melanomas and skin abnormalities (29 , 36, 37, 38) , was present in two normal copies in the SM190.2 cell line. However, chromosome 14 was involved in Robertsonian translocation in the SM190.626 cell line (Fig. 5B)Citation , and therefore, cells of this line should show metastatic phenotypes. Although we did not observe metastases in animals that received this tumor, we cannot rule out the presence of micrometastases. Nevertheless, our findings are consistent with those of other investigators who showed that primary melanomas are genetically heterogeneous and can contain cells that have acquired metastatic potential while still small (25 , 39, 40, 41) .

The failure of antibodies specific for proteins in the pigment pathway to react with tumors from C3H mice may be due to several factors. First, tyrosinase becomes undetectable when melanization declines (12) . Although melanin can be detected in the K1735 and SD0302 tumors by Fontana silver stain, we have observed that the production of pigment is low compared with the B16 melanoma. However, even with longer exposure times, the large amount of protein used, and visually evident pigment in the K1735 cells, no reactivity was observed, suggesting that the quality rather than the quantity of pigment may be responsible. C3H mice express pheomelanin (yellow/red melanin). Although tyrosinase is required for synthesis of both pheomelanin and eumelanin, expression of tyrosinase-related proteins 1 and 2 is only required for eumelanin synthesis (42) . The absence of tyrosinase reactivity in C3H-derived cell lines suggests that there are immunological differences in epitopes of the enzyme from this strain of mouse because we were also unable to detect reactivity of these antibodies in sections of skin from C3H mice (data not shown). Finally, culture conditions, substrate availability, hormones, and other growth factors can influence signaling and the tyrosinase pigment synthesis pathways, resulting in changes or loss of pigment-associated proteins and their detection by specific antibodies (43) .

In conclusion, this study confirms that chronic treatment with UV radiation plus ethanol can induce primary melanomas in C3H mice and that these tumors contain mutations in p16INK4a and ras similar to those in established melanoma cell lines. Whereas there are many differences in the histopathology of these melanomas and superficial spreading melanomas in humans, this model will be a valuable tool with which to study the role of chemicals and UV radiation in the induction and growth of primary melanoma.


   ACKNOWLEDGMENTS
 
We thank Dr. Vincent Hearing (Pigment Cell Biology Section of the Laboratory of Cell Biology, NIH, Bethesda, MD) for the kind gift of antibodies against the pigmentation pathway. The gift of human melanocyte protein from Dr. Estella Medrano is also appreciated. We thank Whitney T. Baker and Diana Brewer (Department of Immunology, The University of Texas M. D. Anderson Cancer Center) for excellent technical assistance. We also thank Dr. Tilahun Jiffar (Department of Immunology, The University of Texas M. D. Anderson Cancer Center) for Laser Capture microdissection of the tumor sections and Walter Pagel (Department of Scientific Publications, The University of Texas M. D. Anderson Cancer Center) for critical reading of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Dermatology, 4D Henry Ford Health Sciences Center, One Ford Place Room 4D49, Detroit, MI 48202. Phone: (313) 874–3385. Back

2 Present address: Abbott Laboratories, North Chicago, IL 60035. Back

3 The abbreviations used are: SCID, severe combined immunodeficient; FHM, foreskin human melanocyte. Back

4 The sequence for C3H/HeN strain has been submitted to the National Center for Biotechnology Information GenBank. Sequences for other strains of mice are from the National Center for Biotechnology Information GenBank: 129/SvJ, AF332190; C57BL6J x DBA, U66086, U66087, and AF004588 (Refs. 21 22 23 ). Back

Received 9/30/02. Accepted 4/23/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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