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Basic Fibroblast Growth Factor and Ultraviolet B Transform Melanocytes in Human Skin

2001; Elsevier BV; Volume: 158; Issue: 3 Linguagem: Inglês

10.1016/s0002-9440(10)64041-2

1525-2191

Carola Berking, Richelle Takemoto, Kapaettu Satyamoorthy, Rosalie Elenitsas, Meenhard Herlyn,

Cutaneous Melanoma Detection and Management

Ultraviolet (UV) light is an epidemiological risk factor for melanoma, but its specific contribution to melanoma induction is not known. The first critical step of melanoma development, ie, the uncontrolled proliferation of melanocytes, may be induced by a combination of UV damage and an imbalance of growth factor production by cells in the immediate area of the melanocyte. Among several candidates, basic fibroblast growth factor (bFGF) is the major autocrine growth factor in melanoma and associated with tumor progression. Overexpression of bFGF via adenoviral gene transfer in human skin xenografted to severe combined immunodeficiency mice led to black-pigmented macules within 3 weeks of treatment. Immunofluorescence analysis demonstrated pathological hyperpigmentation, proliferation and hyperplasia of activated melanocytes, but no malignant transformation. Similar changes were observed in skin reconstructs. When bFGF was combined with UVB, pigmented lesions with hyperplastic melanocytic cells were detected, including a lesion with high-grade atypia resembling lentiginous forms of malignant melanoma. Donor-matched control grafts revealed no melanocytic changes. bFGF was overexpressed in dermal fibroblasts demonstrating the co-carcinogenic influence of paracrine-acting growth factors by cells of the microenvironment. This is the first report suggesting that an imbalance of physiological growth factor production in the skin may cause melanoma in combination with UVB. Ultraviolet (UV) light is an epidemiological risk factor for melanoma, but its specific contribution to melanoma induction is not known. The first critical step of melanoma development, ie, the uncontrolled proliferation of melanocytes, may be induced by a combination of UV damage and an imbalance of growth factor production by cells in the immediate area of the melanocyte. Among several candidates, basic fibroblast growth factor (bFGF) is the major autocrine growth factor in melanoma and associated with tumor progression. Overexpression of bFGF via adenoviral gene transfer in human skin xenografted to severe combined immunodeficiency mice led to black-pigmented macules within 3 weeks of treatment. Immunofluorescence analysis demonstrated pathological hyperpigmentation, proliferation and hyperplasia of activated melanocytes, but no malignant transformation. Similar changes were observed in skin reconstructs. When bFGF was combined with UVB, pigmented lesions with hyperplastic melanocytic cells were detected, including a lesion with high-grade atypia resembling lentiginous forms of malignant melanoma. Donor-matched control grafts revealed no melanocytic changes. bFGF was overexpressed in dermal fibroblasts demonstrating the co-carcinogenic influence of paracrine-acting growth factors by cells of the microenvironment. This is the first report suggesting that an imbalance of physiological growth factor production in the skin may cause melanoma in combination with UVB. Loss of growth control is a hallmark of cancer and frequently associated with aberrant growth factor production. Many tumor cell populations release mitogenic factors that sustain autonomous growth via autocrine stimulation and generate a microenvironment favoring tumor survival and invasion via paracrine effects.1Favoni RE de Cupis A The role of polypeptide growth factors in human carcinomas: new targets for a novel pharmacological approach.Pharmacol Rev. 2000; 52: 179-206PubMed Google Scholar Although many of these growth factors have been characterized and described in detail in tumor progression and metastasis, especially in breast cancer, prostate cancer, and melanoma, their roles in early stages of tumor development have been addressed only marginally. In melanoma, several growth factors are expressed, including basic fibroblast growth factor (bFGF), melanoma growth stimulatory activity/Gro, interleukin (IL)-8, platelet-derived growth factor-A, IL-6, vascular endothelial growth factor, and granulocyte/macrophage-colony stimulating factor.2Lazar-Molnar E Hegyesi H Toth S Falus A Autocrine and paracrine regulation by cytokines and growth factors in melanoma.Cytokine. 2000; 12: 547-554Crossref PubMed Scopus (361) Google Scholar In contrast, normal melanocytes produce none or only low levels of these factors and during normal skin development and homeostasis, depend on the production of bFGF, endothelin-1 and -3, stem cell factor (SCF), hepatocyte growth factor (HGF), and melanocyte-stimulating hormone by keratinocytes and fibroblasts.3Halaban R The regulation of normal melanocyte proliferation.Pigment Cell Res. 2000; 13: 4-14Crossref PubMed Scopus (149) Google Scholar Disruption of this homeostatic balance might have an impact not only on melanocyte development and distribution, but also on nevus and melanoma development.4Herlyn M Berking C Li G Satyamoorthy K Lessons from melanocyte development for understanding the biological events in naevus and melanoma formation.Melanoma Res. 2000; 10: 1-10Crossref Scopus (81) Google Scholar A potent environmental candidate for inducing an imbalance of growth factor production in skin is ultraviolet (UV) light, whose association with nevus and melanoma development has been documented by epidemiological studies.5Elwood JM Jopson J Melanoma and sun exposure: an overview of published studies.Int J Cancer. 1997; 73: 198-203Crossref PubMed Scopus (635) Google Scholar, 6Green A Whiteman D Frost C Battistutta D Sun exposure, skin cancers and related skin conditions.J Epidemiol. 1999; 9: S7-S13Crossref PubMed Scopus (76) Google Scholar Limited experimental data have demonstrated UV induction of melanoma in animal models (Xiphophorus hybrid fish and opossum) as well as in a human skin graft/immunodeficient mouse model when combined with 7,12-dimethyl(a)benzanthracene.7Setlow RB Woodhead AD Grist E Animal model for ultraviolet radiation-induced melanoma: platyfish-swordtail hybrid.Proc Natl Acad Sci USA. 1989; 86: 8922-8926Crossref PubMed Scopus (159) Google Scholar, 8Robinson ES VandeBerg JL Hubbard GB Dooley TP Malignant melanoma in ultraviolet irradiated laboratory opossums: initiation in suckling young, metastasis in adults, and xenograft behavior in nude mice.Cancer Res. 1994; 54: 5986-5991PubMed Google Scholar, 9Atillasoy ES Seykora JT Soballe PW Elenitsas R Nesbit M Elder DE Montone KT Sauter E Herlyn M UVB induces atypical melanocytic lesions and melanoma in human skin.Am J Pathol. 1998; 152: 1179-1186PubMed Google Scholar In addition to its direct DNA-damaging effects, UVB has been shown to stimulate expression of IL-1, IL-3, IL-6, tumor necrosis factor-α, granulocyte/macrophage-colony stimulating factor, endothelin-1, IL-8, IL-12, and vascular endothelial growth factor in keratinocytes and IL-1α and bFGF expression in HeLa cells.10Schwarz T Luger TA Effect of UV irradiation on epidermal cell cytokine production.J Photochem Photobiol B. 1989; 4: 1-13Crossref PubMed Scopus (163) Google Scholar, 11Imokawa G Yada Y Miyagishi M Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes.J Biol Chem. 1992; 267: 24675-24680Abstract Full Text PDF PubMed Google Scholar, 12Kondo S Kono T Sauder DN McKenzie RC IL-8 gene expression and production in human keratinocytes and their modulation by UVB.J Invest Dermatol. 1993; 101: 690-694Abstract Full Text PDF PubMed Google Scholar, 13Enk CD Mahanty S Blauvelt A Katz SI UVB induces IL-12 transcription in human keratinocytes in vivo and in vitro.Photochem Photobiol. 1996; 63: 854-859Crossref PubMed Scopus (38) Google Scholar, 14Brauchle M Funk JO Kind P Werner S Ultraviolet B and H2O2 are potent inducers of vascular endothelial growth factor expression in cultured keratinocytes.J Biol Chem. 1996; 271: 21793-21797Crossref PubMed Scopus (164) Google Scholar, 15Kramer M Sachsenmaier C Herrlich P Rahmsdorf HJ UV irradiation-induced interleukin-1 and basic fibroblast growth factor synthesis and release mediate part of the UV response.J Biol Chem. 1993; 268: 6734-6741Abstract Full Text PDF PubMed Google Scholar UVA could induce IL-6 and tumor necrosis factor-α expression in keratinocytes and dermal fibroblasts.16Avalos-Diaz E Alvarado-Flores E Herrera-Esparza R UV-A irradiation induces transcription of IL-6 and TNF-alpha genes in human keratinocytes and dermal fibroblasts.Rev Rheum Engl Ed. 1999; 66: 13-19PubMed Google Scholar In UVB-irradiated murine skin in vivo, the epidermal expression of bFGF increased, whereas interferon-β decreased, alterations that were associated with enhanced cutaneous angiogenesis.17Bielenberg DR Bucana CD Sanchez R Donawho CK Kripke ML Fidler IJ Molecular regulation of UVB-induced cutaneous angiogenesis.J Invest Dermatol. 1998; 111: 864-872Crossref PubMed Scopus (126) Google Scholar bFGF, also called FGF-2, is one of 21 members of the FGF gene family known to modulate cell growth, differentiation, motility, and angiogenesis.18Bikfalvi A Klein S Pintucci G Rifkin DB Biological roles of fibroblast growth factor-2.Endocrinol Rev. 1997; 18: 26-45Crossref PubMed Scopus (859) Google Scholar bFGF binds to low-affinity receptors on the cell surface and in the extracellular matrix. These low-affinity receptors that are heparan sulfate proteoglycans are required for binding of FGF to the four different types of high-affinity receptors.19Klagsbrun M Baird A A dual receptor system is required for basic fibroblast growth factor activity.Cell. 1991; 67: 229-231Abstract Full Text PDF PubMed Scopus (498) Google Scholar In melanoma, bFGF is the most important autocrine growth factor. Inhibition of bFGF production by antisense oligodeoxynucleotides led to inhibition of melanoma proliferation in vitro and in vivo.20Becker D Meier CB Herlyn M Proliferation of human malignant melanomas is inhibited by antisense oligodeoxynucleotides targeted against basic fibroblast growth factor.EMBO J. 1989; 8: 3685-3691Crossref PubMed Scopus (245) Google Scholar, 21Wang Y Becker D Antisense targeting of basic fibroblast growth factor and fibroblast growth factor receptor-1 in human melanomas blocks intratumoral angiogenesis and tumor growth.Nat Med. 1997; 3: 887-893Crossref PubMed Scopus (280) Google Scholar Through its mitogenic effects on endothelial cells and fibroblasts, bFGF production by melanoma can also promote angiogenesis and fibrous stroma formation via a paracrine mode.22Shih IM Herlyn M Autocrine and paracrine roles for growth factors in melanoma.In Vivo. 1994; 8: 113-123PubMed Google Scholar Although expression of bFGF is absent in normal melanocytes, it is moderate to high in compound and dysplastic nevi and always present in melanomas.23Scott G Stoler M Sarkar S Halaban R Localization of basic fibroblast growth factor mRNA in melanocytic lesions by in situ hybridization.Invest Dermatol. 1991; 96: 318-322Abstract Full Text PDF PubMed Google Scholar, 24al-Alousi S Barnhill R Blessing K Barksdale S The prognostic significance of basic fibroblast growth factor in cutaneous malignant melanoma.J Cutan Pathol. 1996; 23: 506-510Crossref PubMed Scopus (22) Google Scholar, 25al-Alousi S Carlson JA Blessing K Cook M Karaoli T Barnhill RL Expression of basic fibroblast growth factor in desmoplastic melanoma.J Cutan Pathol. 1996; 23: 118-125Crossref PubMed Scopus (34) Google Scholar, 26Albino AP Davis BM Nanus DM Induction of growth factor RNA expression in human malignant melanoma: markers of transformation.Cancer Res. 1991; 51: 4815-4820PubMed Google Scholar This change in bFGF expression early in melanoma development suggests an alteration in the growth control mechanisms during melanocyte transformation. However, bFGF alone cannot induce complete melanocyte transformation, as demonstrated by different groups. Infection of murine melanocytes with a retrovirus carrying the cDNA for bFGF caused autonomous growth and suppressed differentiation properties in vitro, but was insufficient to form malignant tumors in vivo.27Dotto GP Moellmann G Ghosh S Edwards M Halaban R Transformation of murine melanocytes by basic fibroblast growth factor cDNA and oncogenes and selective suppression of the transformed phenotype in a reconstituted cutaneous environment.J Cell Biol. 1989; 109: 3115-3128Crossref PubMed Scopus (136) Google Scholar, 28Ramon y Cajal S Suster S Halaban R Filvaroff E Dotto GP Induction of different morphologic features of malignant melanoma and pigmented lesions after transformation of murine melanocytes with bFGF-cDNA and H-ras, myc, neu, and E1a oncogenes.Am J Pathol. 1991; 138: 349-358PubMed Google Scholar Transfection of human melanocytes with bFGF via retroviral gene transfer still required exogenous bFGF for growth, whereas adenoviral gene transfer of the bFGF gene in human melanocytes reduced dependence on growth factors in vitro, induced anchorage-independent growth in vitro, and increased survival and proliferation in vivo.29Coleman AB Lugo TG Normal human melanocytes that express a bFGF transgene still require exogenous bFGF for growth in vitro.J Invest Dermatol. 1998; 110: 793-799Crossref PubMed Scopus (16) Google Scholar, 30Nesbit M Nesbit HK Bennett J Andl T Hsu MY Dejesus E McBrian M Gupta AR Eck SL Herlyn M Basic fibroblast growth factor induces a transformed phenotype in normal human melanocytes.Oncogene. 1999; 18: 6469-6476Crossref PubMed Scopus (99) Google Scholar These observations led to the hypothesis that melanocytes may be activated by bFGF, but require additional stimulation by a cooperating factor for complete transformation. In this study, the effects of bFGF on melanocytes in vivo with and without exposure to UVB were analyzed in human skin grafted to immunodeficient mice. To achieve high and sustained levels of bFGF in the skin, adenoviral gene transfer for bFGF was used. A highly mitogenic effect with hyperpigmentation and melanocytic hyperplasia was found by bFGF overexpression alone. When combined with UVB irradiation a lentiginous melanoma-like lesion developed within 2 months of treatment. This is the first report suggesting that human melanoma in vivo can be experimentally induced by a growth factor and UVB. Normal human keratinocytes and melanocytes were isolated from the epidermis, and fibroblasts from the dermis of neonatal human foreskins. Keratinocytes were cultured in serum-free medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with human recombinant epidermal growth factor and bovine pituitary extract. Melanocytes were cultured in MCDB153 (Sigma, St. Louis, MO) supplemented with 2% fetal bovine serum (FBS), 10% chelated FBS, 2 mmol/L glutamine, 20 pmol/L cholera toxin (Sigma), 150 pmol/L recombinant human bFGF, 100 nmol/L recombinant human endothelin-3 (Peninsula, Belmont, CA), and 10 ng/ml recombinant human SCF (R&D Systems, Minneapolis, MN). Fibroblasts were cultured in Dulbecco's modified Eagle's medium with glutamine (Life Technologies, Inc.), 8 mmol/L Hepes (Sigma), and 10% FBS (Hyclone, Logan, UT). The adenoviral vector bFGF/Ad5 carrying the gene for the 18-kd form of the bFGF protein has been described.30Nesbit M Nesbit HK Bennett J Andl T Hsu MY Dejesus E McBrian M Gupta AR Eck SL Herlyn M Basic fibroblast growth factor induces a transformed phenotype in normal human melanocytes.Oncogene. 1999; 18: 6469-6476Crossref PubMed Scopus (99) Google Scholar The control adenoviral vector LacZ/Ad5 (Vector Core, University of Pennsylvania, Philadelphia, PA) induces expression of the reporter gene β-galactosidase from Escherichia coli. The adenoviral vector for HGF was kindly provided by Dr. J. M. Wilson (Institute for Human Gene Therapy, The Wistar Institute, Philadelphia, PA).31Phaneuf D Chen SJ Wilson JM Intravenous injection of an adenovirus encoding hepatocyte growth factor results in liver growth and has a protective effect against apoptosis.Mol Med. 2000; 6: 96-103Crossref PubMed Google Scholar The adenoviral vectors for platelet-derived growth factor-A and insulin-like growth factor-1 were generated from d17001 and AdEasy-1 viruses, respectively, with deleted E1 and E3 regions and the transgenes driven by the CMV promoter (Satyamoorthy K, Li G, Vaidya B, Patel D, Herlyn M, unpublished). The vectors were prepared, purified, and titered to 1 to 5 × 1010 plaque-forming units (p.f.u.)/ml. Human skin grafts were injected intradermally with the adenoviral vectors using a 26-gauge needle at a concentration of 5 × 108 p.f.u. in a total volume of 100 μl sterile phosphate-buffered saline (PBS). The needle was inserted 2 mm apart from the edge of the graft and directed toward the center of the graft during injection. Generally, 100 μl were injected at one site into foreskin grafts and 50 μl were injected at two sites into trunk skin grafts, in which the fluid penetration was usually slower. Injections were performed once per week by the same person (CB). Human foreskins from newborns and abdominal or breast skin from adult donors, who underwent plastic surgery (Cooperative Human Tissue Network, Philadelphia, PA), were kept in sterile transport media (RPMI-1640 or Hanks' balanced salt solution supplemented with antibiotics) and grafted within 48 hours of excision as described with modifications.9Atillasoy ES Seykora JT Soballe PW Elenitsas R Nesbit M Elder DE Montone KT Sauter E Herlyn M UVB induces atypical melanocytic lesions and melanoma in human skin.Am J Pathol. 1998; 152: 1179-1186PubMed Google Scholar Female and male C.B-17 SCID mice were bred at the Animal Facility of the Wistar Institute and housed under pathogen-free conditions in groups of up to five animals per isolator cage. At 6 to 10 weeks of age, a 1 to 3 cm2 skin segment behind the shoulder of the animal was excised, leaving the panniculus carnosus muscle intact. The wound was immediately covered with full-thickness human skin that was held in place by the bandage alone or by 6-0 nonabsorbable polyviolene sutures. The bandage consisted of nonadhesive Vaseline dressing, sterile sponges, and surgical tape and was changed after 2 weeks. Grafts were well healed after 4 to 6 weeks and used for the experiments. The Wistar Institutional Animal Care and Use Committee approved all protocols. At the end of each experiment, mice were sacrificed by CO2 inhalation and skin grafts were excised. Half of the grafts were fixed in 10% neutral-buffered formalin (Fisher Scientific, Pittsburgh, PA) for 6 to 12 hours at room temperature and embedded in paraffin. The other half was dehydrated by increasing concentrations of sucrose solutions (5%, 10%, and 20%) at 4°C overnight, embedded in OCT medium (Miles, Elkhart, IN), snap-frozen and stored at −70°C until cryosectioning at 6 to 8 μm. Formalin-fixed sections were stained with hematoxylin and eosin (H&E) for histopathological evaluation. The DNA-binding fluorochrome Hoechst 33258 (Sigma) was used to distinguish human from murine cells. Immunohistochemistry was performed on serial sections using an avidin-biotin-peroxidase system kit (Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine tetrahydrochloride (Sigma) or 3-amino-9-ethylcarbazole (Vector) as chromogens. Antigens in the formalin-fixed tissues were retrieved by trypsin digestion at 37°C or microwave heat treatment in citrate buffer. Cryostat sections of 6 to 8 μm were air-dried and fixed in ice-cold acetone for 10 minutes. Before incubation with the primary antibodies in a humidified chamber at 4°C overnight or at room temperature for 1 to 2 hours, nonspecific binding was blocked with 10% normal horse or 10% normal goat serum. Primary monoclonal antibodies used in this study were: mouse anti-bFGF (bFGF-8, IgG1);30Nesbit M Nesbit HK Bennett J Andl T Hsu MY Dejesus E McBrian M Gupta AR Eck SL Herlyn M Basic fibroblast growth factor induces a transformed phenotype in normal human melanocytes.Oncogene. 1999; 18: 6469-6476Crossref PubMed Scopus (99) Google Scholar mouse anti-human TRP-1/gp75 (clone TA99, IgG2a; kind gift from Dr. V. Setaluri, Winston-Salem, NC); mouse anti-human Ki-67 (clone MIB-1, IgG1; Immunotech, Westbrook, ME); and mouse anti-human HMB45 (IgG1; Biogenex, San Ramon, CA). A mouse IgG1 isotype antibody (P3) was used as negative control for each staining. Between each incubation step, slides were rinsed twice in PBS for 3 to 5 minutes. A biotin-labeled anti-mouse secondary antibody was applied for 30 minutes at room temperature followed by incubation with a preformed avidin-biotinylated enzyme complex for 30 minutes. After color development by addition of the chromogen and counterstaining with Mayer's hematoxylin (Sigma), sections were mounted and evaluated under a light microscope. For immunofluorescence detection of the proliferation marker Ki-67 or the melanoma/activated melanocyte marker HMB-45, a biotin-labeled goat anti-mouse IgG1 secondary antibody (Jackson Immunoresearch, West Grove, PA) was used followed by incubation with streptavidin-conjugated Cy3 (Jackson Immunoresearch). For immunofluorescence detection of the melanocyte-specific antigen TRP-1, a horse anti-mouse IgG2a secondary antibody directly conjugated with fluorescein isothiocyanate (Jackson Immunoresearch) was used. Double-immunofluorescence staining was performed for Ki-67 (red) and TRP-1 (green), and cells were counterstained with Hoechst 33258 (blue). Sections were scored in a blinded manner by counting five fields (∼1,000 epidermal basal cells) at ×200 magnification in each of three randomly selected sections using a fluorescence microscope (Leika, Wetzlar, Germany). Cells stained for TRP-1 (green), for Ki-67 (red), and for both TRP-1 and Ki-67 (yellow) were counted. All data are expressed as mean ± SD of the mean of observations. Individual groups were compared with Student's unpaired t-test. P < 0.05 was considered significant. UV light was provided by two Westinghouse FS72T12/UVB lamps (UV Resources International, Lakewood, OH) with a peak output at 313 nm and a range of 280 to 370 nm. The light was filtered through cellulose triacetate Kodacel TA 407 sheets (Eastman Kodak, Rochester, NY) to exclude wavelengths <295 nm. The UV dose was continuously monitored with a PMA 2100 radiometer (Solar Light, Philadelphia, PA) and ranged between 30 and 50 mJ/cm2 for UVB and 0.1 and 0.2 J/cm2 for UVA in the in vivo experiments. During irradiation, mice were separated from each other and allowed to move freely in the cage. Irradiation was performed three times weekly for ∼10 minutes each time throughout a period of 2 to 10 months. Skin reconstructs were prepared essentially as described with modifications.32Meier F Nesbit M Hsu MY Martin B Van Belle P Elder DE Schaumburg-Lever G Garbe C Walz TM Donatien P Crombleholme TM Herlyn M Human melanoma progression in skin reconstructs: biological significance of bFGF.Am J Pathol. 2000; 156: 193-200Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar Human fibroblasts (FF2441) were added to neutralized bovine type I collagen (Organogenesis, Canton, MA) to a final concentration of 0.8 to 1 mg/ml of collagen in MEM (Biowhittaker, Walkersville, MA), 1.66 mmol/L l-glutamine (Life Technologies, Inc.), 10% FBS, and 0.21% sodium bicarbonate (Biowhittaker). Three milliliters of fibroblast-containing collagen (2.5 × 104 cells/ml) were added to each insert of a 6-well tissue-culture tray (Organogenesis) after precoating with 1 ml of acellular collagen. Mixtures were allowed to constrict in Dulbecco's modified Eagle's medium with 10% FBS for 5 to 7 days. The day before seeding, keratinocytes or melanocytes were infected with bFGF/Ad5, and controls with LacZ/Ad5 at 20 p.f.u./cell for 4 hours in protein-free, serum-free medium and then incubated overnight in complete serum-free medium. Keratinocytes were mixed with melanocytes at a ratio of 5:1 or 2.5:1 in low-calcium epidermal growth medium containing Dulbecco's modified Eagle's medium, F-12 Ham's (Life Technologies, Inc.), 1% newborn calf serum (Hyclone), 4 mmol/L glutamine, 1.48 × 10−6 mol/L hydrocortisone, 4 pmol/L progesterone, 20 pmol/L triiodothyronine, 0.1 mmol/L O-phosphorylethanolamine, 0.18 mmol/L adenine (Sigma), 5 mg/ml insulin, 5 mg/ml transferrin, 5 mmol/L ethanolamine, 5 g/ml selenium (Biowhittaker) and 50 μg/ml gentamicin (Mediatech, Hemdon, VA). A total of 5 to 6 × 105 cells was seeded on each contracted collagen gel. Cultures were maintained submerged in low-calcium epidermal growth medium for 2 days and in normal calcium (1.88 mmol/L) epidermal growth medium for another 2 days, and then raised to the air-liquid interface for 10 to 12 days with feeding from below with normal calcium high-serum (20%) epidermal growth medium. Six human skin grafts were injected intradermally with bFGF/Ad5 once weekly receiving up to seven treatments (Table 1). In the third week, one abdominal skin graft developed a brown macule (Figure 1A), one foreskin graft showed a small black spot centrally (not shown), and one foreskin graft turned from pink to an almost complete black pigmentation (Figure 1B). Other bFGF/Ad5-injected skin grafts showed no pigmentation changes, although thickening of the skin was observed (Figure 1C). Eight skin grafts injected with bFGF/Ad5 once only and evaluated 3 days later revealed no change in pigmentation (not shown).Table 1Clinical and Histopathological Characteristics of Human Skin Grafted to SCID Mice and Injected with bFGF/Ad5*At the start of treatment all skin xenografts were pale to pink and showed no pigmented lesions.Treatment groupSkin type and age of donor(years)Clinical appearance*At the start of treatment all skin xenografts were pale to pink and showed no pigmented lesions. in weeks 1 to 3†After beginning of treatment.Clinical appearance*At the start of treatment all skin xenografts were pale to pink and showed no pigmented lesions. in weeks 4 to 6†After beginning of treatment.Total bFGF/Ad5 injections‡Intradermally at 5 × 108p.f.u. in 100 μl of sterile PBS once weekly.Total UVB irradiations§UVB irradiations were done three times weekly at a dose of 30 to 50 mJ/cm2.Biopsy†After beginning of treatment.Histological melanocytic changes¶Other histological changes, such as increase in vessels, extracellular matrix, or stromal cells are not listed.bFGFBreast (unknown)Normal pinkNormal pink70Month 7IncreaseForeskin, neonatalNormal pink1 Brown macule60Month 8HyperplasiaForeskin, neonatal70% black70% black40Month 7HyperplasiaAbdomen (54)1 Brown macule1 Brown macule20Month 3HyperplasiaForeskin, neonatal1 Black spot—20Week 2IncreaseAbdomen (39)Normal pink—10Day 7NoneForeskins, neonatal (n = 8)Normal pink—10Day 3NonebFGF+ UVB§UVB irradiations were done three times weekly at a dose of 30 to 50 mJ/cm2.Foreskin, neonatalBlack spots and streaks6 Black macules941Month 3IncreaseForeskin, neonatalDark brown70% dark brown/black8117Month 10.5IncreaseAbdomen (52)1 Black macule5 Black macules726Month 2Lentiginous form of melanomaAbdomen (52)1 Dark brown macule4 Black spots790Months 5 and 7HyperplasiaForeskin, neonatalEntirely blackEntirely black618Months 1 and 2HyperplasiaAbdomen (39)3 Black macules4 Black macules336Months 3 and 6Increase* At the start of treatment all skin xenografts were pale to pink and showed no pigmented lesions.† After beginning of treatment.‡ Intradermally at 5 × 108p.f.u. in 100 μl of sterile PBS once weekly.§ UVB irradiations were done three times weekly at a dose of 30 to 50 mJ/cm2.¶ Other histological changes, such as increase in vessels, extracellular matrix, or stromal cells are not listed. Open table in a new tab Another six human skin grafts were injected weekly with bFGF/Ad5 and irradiated with UVB (Table 1). Pigmented lesions were detected in all grafts in the third week of treatment. Adult abdominal skin developed black or brown macules, which increased in size and number with further injections (Figure 1; D, E, I, J, and K), whereas foreskins tended to become entirely black (Figure 1M). All pigment changes gradually disappeared 1 to 2 months after discontinuation of bFGF/Ad5 injections and despite continuation of UVB irradiations (Figure 1, F and N). Control skin grafts injected with adenoviral vectors for Lac Z and irradiated with UVB did not develop pigmented lesions during the observation period of 3 weeks to 8 months (Figure 1, L and O, and Table 2). Control skin grafts of the same donors UVB-irradiated only (Figure 1G) also showed no changes except for some tanning as expected and previously reported.9Atillasoy ES Seykora JT Soballe PW Elenitsas R Nesbit M Elder DE Montone KT Sauter E Herlyn M UVB induces atypical melanocytic lesions and melanoma in human skin.Am J Pathol. 1998; 152: 1179-1186PubMed Google Scholar Overexpression of other growth factors such as HGF, insulin-like growth factor-1, and platelet-derived growth factor-A had no detectable effect on the pigment cell system of the skin grafts (Table 2).Table 2Clinical and Histopathological Characteristics of Human Skin Grafted to SCID Mice and Injected with Different Adenoviral Vectors*Intradermally at 5 × 108 p.f.u. in 100 μl of sterile PBS once weekly.Treatment group and number of skin grafts (n)Skin type of donors†Foreskins were from newborns; all other skin specimens were from adult donors. (number of grafts)Total injections*Intradermally at 5 × 108 p.f.u. in 100 μl of sterile PBS once weekly.Total UVB irradiations‡UVB irradiations were done three times weekly at a dose of 30 to 50 mJ/cm2.Clinical appearance until biopsyBiopsy§After beginning of treatment.Histological melanocytic changes¶Other histological changes, such as acanthosis of the epidermis, increase in vessels, extracellular matrix, or stromal cells are not listed.LacZ (20)Foreskin (15)1–20Normal pinkDay 3–Month 5N/DForeskin (2)6, 745, 16TanMonths 4.5, 2N/D, increaseAbdomen (2)6, 7108, 16TanMonths 12, 2N/DBreast (1)10Normal pinkWeek 3N/DPDGF-1 (8)Foreskin (3)10PinkDay 3N/DBreast (3)9, 10, 1027, 89, 89TanMonths 3, 13, 15N/DFace (1)958TanMonth 12N/DHGF (10)Foreskin (6)