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Regular Articles |
From The Wistar Institute,*
Philadelphia; and the
Department of Dermatology,
University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.2
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.3
Disruption of this
homeostatic balance might have an impact not only on melanocyte
development and distribution, but also on nevus and melanoma
development.4
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.5,6
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.7-9
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-
,
granulocyte/macrophage-colony stimulating factor, endothelin-1, IL-8,
IL-12, and vascular endothelial growth factor in keratinocytes and
IL-1 and bFGF expression in HeLa cells.10-15
UVA could
induce IL-6 and tumor necrosis factor- expression in keratinocytes
and dermal fibroblasts.16
In UVB-irradiated murine skin
in vivo, the epidermal expression of bFGF increased, whereas
interferon-ß decreased, alterations that were associated with
enhanced cutaneous angiogenesis.17
bFGF, also called FGF-2, is one of 21 members of the FGF gene family known to modulate cell growth, differentiation, motility, and angiogenesis.18 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.19 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.20,21 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.22 Although expression of bFGF is absent in normal melanocytes, it is moderate to high in compound and dysplastic nevi and always present in melanomas.23-26 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.27,28 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.29,30 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
Adenoviral Vectors
The adenoviral vector bFGF/Ad5 carrying the gene for the 18-kd form of the bFGF protein has been described.30 The control adenoviral vector LacZ/Ad5 (Vector Core, University of Pennsylvania, Philadelphia, PA) induces expression of the reporter gene ß-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).31 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 x 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 x 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 Skin Grafting
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.9 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.
Histology, Immunohistochemistry, and Immunofluorescence
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);30 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 x200 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 Irradiation
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 Reconstruction
Skin reconstructs were prepared essentially as described with modifications.32 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 x 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 x 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 x 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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. 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 LacZ 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.9
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)
.
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bFGF Induces Dermal Thickening and Angiogenesis
Thickening of the skin after two or more bFGF/Ad5 injections was
observed in the foreskin grafts and remained even after discontinuation
of injections (Figure 1, C and N)
. Three of six foreskin grafts became
hypervascular after 1 to 2 weeks of treatment, which was noticed by
easy bleeding during subsequent injections.
Intradermal Injection of bFGF/Ad5 Induces bFGF Expression in Fibroblasts
Immunohistochemical detection of bFGF protein to assess the
transduction efficiency of the intradermal bFGF/Ad5 injections
indicated abundant expression of bFGF in fibroblasts in the upper
dermis (Figure 2, A and B)
. Generally, no
bFGF expression was detected in the epidermis except for one confined
area in one section where single keratinocytes showed strong bFGF
expression. This area was hyperplastic and parakeratotic and appeared
to be the entrance site of the injection needle through the epidermis
(not shown). Controls showed no or only weak bFGF expression (Figure 2C)
.
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Capillaries and small vessels were markedly increased in the
dermis of some of the bFGF/Ad5-injected foreskin grafts (Figure 2D)
.
The clinically observed thickening of the skin during the treatment was
reflected by an increase in extracellular matrix and fibroblasts in the
dermis (Figure 2E)
.
bFGF Induces Pathological Hyperpigmentation and Proliferation of Activated Melanocytes
A strong increase in pigment in the epidermis was noted in the
bFGF/Ad5-injected skin grafts (Figure 2, E and F)
. Accumulation of
black pigment in confined areas of the epidermis (Figure 2F)
correlated
macroscopically with black macules. In other sections,
hyperpigmentation was present throughout the human epidermis (Figure 2E)
. Pigment-laden melanophages in the subepidermis were commonly seen
and remained present after the epidermal hyperpigmentation had
disappeared weeks after discontinuation of the bFGF/Ad5 injections
(data not shown).
In the bFGF/Ad5-treated skin grafts, there was a striking increase in
the number of melanocytes in the epidermis, as confirmed by
immunofluorescence staining for TRP-1. The melanocytes showed prominent
dendricity and were located close to each other along the basement
membrane of the epidermis (Figure 3, A and B)
; in some areas, incipient cluster formation was noted (Figure 3C)
. Proliferating melanocytes were identified by dual staining for
TRP-1 and Ki-67 (Figure 3, B and C)
. The increased number of
melanocytes and proliferating melanocytes compared to controls was also
demonstrated by counting the TRP-1 and/or Ki-67 positively stained
cells in randomly chosen fields of different histological sections.
There were significantly more proliferating melanocytes in the
bFGF/Ad5-treated skin grafts compared to the controls
(P < 0.006) with up to 10 cells per counting
field in contrast to 0 to 1 cell in the controls (Figure 4)
. The number of melanocytes varied
considerably among the different skin grafts ranging from 20 per
counting field in both groups to 90 in the bFGF group only.
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. Skin
grafts injected with control adenoviral vectors and/or irradiated with
UVB were negative for HMB-45 (not shown) or showed only single positive
cells when injected with LacZ/Ad5 and irradiated with UVB
(Figure 3F)
. bFGF Induces Melanocyte Hyperplasia and, in Combination with UVB, Lentiginous Melanoma
In sections of the bFGF/Ad5-treated skin grafts, melanocytes
displayed a hyperplastic morphology that was still present months after
the last treatment (Figure 5A)
.
Hyperplasia with additional atypia was detected in one bFGF/Ad5-treated
abdominal skin graft that concomitantly had been irradiated with UVB
for 2 months. In this pathologically pigmented lesion, the
hyperplastic, high-grade atypical melanocytic cells were distributed in
a lentiginous growth pattern resembling lentiginous forms of malignant
melanoma in humans (Figure 5; B, C, and E
). Control grafts from the
same donor, either not treated or UVB-irradiated only showed no
melanocytic abnormalities (Figure 5F)
.
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Like the pigmentation changes observed in the human skin in
vivo, increased pigmentation was observed in human skin
reconstructs in vitro after overexpression of bFGF in
keratinocytes (Figure 6
, top). Compared
to LacZ controls, pigmentation of the epidermis was stronger
and, as expected, darkest in reconstructs with the highest number of
initially seeded melanocytes (ratio 1:2.5). The same pigmentation
difference was seen when melanocytes were transduced with bFGF/Ad5
(Figure 6
, bottom). Clusters of black pigment were observed
histologically in the epidermis and on top of the keratin layer. The
number of dermal fibroblasts was strikingly increased, demonstrating a
paracrine mitogenic effect by the bFGF overexpression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-melanocyte-stimulating hormone.33,34
The more
widespread hyperpigmentation in the foreskin grafts as opposed to the
more confined black macule formation in the trunk skin grafts might
have been because of a more widespread penetration of the injected
fluid in foreskin tissue than in the more compact trunk skin tissue.
The hyperplasia of melanocytes after bFGF treatment suggests an
activated stage of melanocytes superior to mere proliferation, but
inferior to malignant transformation, which parallels the in
vitro bFGF transduction studies of murine and human cells where
melanocytes displayed a transformed phenotype but no
malignancy.27,30
The requirement for additional
cooperating factors that confer a malignant phenotype to melanocytes
has been suggested.35
We focused on UV light as an
additional melanocyte-stimulating factor, because it has been
associated with melanoma development, although its carcinogenic effects
on melanocytes in humans have remained only speculative.36
Histopathological analysis suggested that the combination of bFGF
overexpression in the skin and UVB irradiation in our model led to
melanocyte transformation in vivo and a consequent
lentiginous form of malignant melanoma. Experimental induction of human
melanoma by UVB has already once been reported before, however, only in
combination with topical treatment of the chemical carcinogen
7,12-dimethyl(a)benzanthracene, which is not found in the natural
environment.9
The present study suggests that 1) melanoma
can be induced by naturally occurring factors in the immediate micro-
and macroenvironment of epidermal melanocytes, specifically by bFGF and
UVB light; 2) although each factor alone can activate melanocytes, both
are needed to induce malignant transformation; and 3) factors
beneficial or necessary for melanocyte proliferation and survival can
become carcinogenic when released in overdose. An acute overdose of UV
light is clinically reflected by sunburn that is a clearly documented
risk factor for melanoma development.37,38
However, the
conditions in vivo that can lead to an imbalance and/or
overdose of growth factor production with an activating potential on
melanocytes remain to be elucidated. Numerous stress-inducing factors
in addition to UV light, such as external heat, shock, local trauma, or
inflammatory diseases can induce local overproduction of cytokines and
growth factors. Recently, it has been demonstrated that bFGF expression
is significantly increased in rat skin during wound healing after
burning.39
On the other hand, there are no epidemiological
data showing that certain inflammatory diseases or trauma are risk
factors for melanoma development. It is not known how genetic factors
predispose to exogenous influences by growth factors and UV light,
apart from the known increased risk in DNA damage repair
enzyme-deficient patients.
The observed effects on the melanocytes in the human epidermis in this
study are all consistent with the known effects of bFGF on melanocytes
in vitro and may have been mediated via paracrine secretion
of bFGF by the adenoviral vector-transduced fibroblasts in the dermis.
However, the bFGF protein lacks a hydrophobic secretory signal sequence
and only a few cell types including melanoma cells have been shown to
secrete it, presumably via a pathway independent of the endoplasmic
reticulum-Golgi complex.30,40,41
bFGF can be concentrated
intracellularly and can be biologically active without secretion.
Hence, it is also possible, that the strong activating effects on the
melanocytes in the epidermis were not directly mediated by bFGF but by
other secondarily induced factors, for example, HGF, SCF,
-melanocyte-stimulating hormone, endothelin-1, or endothelin-3.
Human dermal fibroblasts are known to produce HGF, SCF, and bFGF and
have been suggested to play a role in regulating cutaneous pigmentation
during inflammation and aging.42
This suggestion is
supported by the observation that two primary mediators of inflammation
and injury, IL-1 and tumor necrosis factor-, can stimulate HGF
and SCF secretion by fibroblasts. HGF production can also be induced by
epidermal growth factor, platelet-derived growth factor, and bFGF,
illustrating the complex network of growth factors mediating cell-cell
and cell-stroma interactions.43
The importance of
epidermal-dermal interactions mediated by diffusible factors for tissue
regeneration and maintenance of homeostasis of rapidly renewing
epithelia was recently suggested by Maas-Szabowski and
colleagues,44
who demonstrated a double-paracrine pathway
between fibroblasts and keratinocytes, ie, the induction of
keratinocyte growth factor in the fibroblasts by IL-1 secretion by the
keratinocytes. We show here that an imbalance of these
paracrine-mediated mesenchymal-epithelial interactions may be also
important in early steps of melanoma development. However, we cannot
exclude that other major factors for melanoma development were missing
in this model, because melanocyte transformation was only observed in
one of four skin grafts treated with a similar regimen, ie, at least 7
weeks of bFGF overexpression and 2 months of UVB irradiation. Two of
these skin grafts were even from the same donor indicating the relative
rarity of melanocyte transformation in vivo independent from
the individual genetic predisposition.
In light of this functional paracrine communication between cells of the epidermis and dermis, our human skin in vivo model demonstrates also that adenoviral gene transduction of cells in the dermis can efficiently and transiently target cells in the epidermis and vice versa, as shown in the reconstruct experiments. This strategy can be considered in the field of cutaneous gene therapy, eg, for wound healing. Although adenoviral gene transduction of cells in the dermis can direct local, highly efficient expression of the protein of interest, injections of recombinant proteins immediately into the tissues are often ineffective because of rapid degradation and short half-lives.
In conclusion, the experimental induction of a melanoma lesion in human skin in vivo by overexpression of bFGF in the dermis and concomitant UVB irradiation throughout 2 months strongly suggests the importance of local growth factor production and UVB light in the pathogenesis of malignant melanoma.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants CA80999, CA25874, and CA10815 (to M. H.) and a postdoctoral research fellowship BE2189/1-1 from the Deutsche Forschungsgemeinschaft (to C. B.)
Accepted for publication December 12, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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