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Carcinogenesis |
Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957 [J. D., D. K. B., E. W., L. B., S. C., S. M., K. K.], and Department of Cell and Molecular Biology, Ciemat Instituto, 28040 Madrid, Spain [A. R., J. J.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50%) of older BK5.IGF-1 mice. Treatment of
BK5.IGF-1 transgenic mice with multiple topical applications of the
phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, in
the absence of tumor initiation led to the development of additional
skin papillomas. Furthermore, treatment of BK5.IGF-1 transgenic mice
with an initiating dose of
7,12-dimethylbenz[a]anthracene only led to the
formation of additional papillomas in the absence of promotion. In
two-stage carcinogenesis experiments, BK5.IGF-1 transgenic mice
developed 7-fold more papillomas than nontransgenic littermates.
Phosphatidylinositol-3-kinase and protein kinase B (Akt) activities
were elevated (3–4-fold), and mitogen-activated protein kinase
activity was elevated 1.7-fold in the epidermis of transgenic mice
compared with nontransgenic mice. In addition, UV light-induced
epidermal apoptosis was significantly suppressed in BK5.IGF-1
transgenic mice. These data suggest that persistent activation of IGF-1
receptor signaling pathways in basal epithelial cells leads to
spontaneous tumor promotion and that up-regulation of both mitogenic
and cell survival signaling pathways may play an important role in the
action of IGF-1 in this model system. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In recent studies from our laboratory, the expression of human IGF-1 was successfully targeted to skin epidermis of transgenic mice using the HK1 promoter (16) . HK1 targets expression to suprabasal cells with some expression also in basal cells of the epidermis (17 , 18) . Deregulated expression of IGF-1 in the epidermis of these mice resulted in characteristics that were immediately obvious at birth. Neonatal HK1.IGF-1 transgenic mice exhibited a thick, wrinkled skin, and when examined histologically, the epidermis showed a marked hyperplasia and hyperkeratosis. All of these phenotypic traits in the HK1.IGF-1 transgenic mice subsided with age; however, adult HK1.IGF-1 mice exhibited a potentiated response to proliferation induced by the phorbol ester tumor promoter, TPA. Moreover, squamous papillomas arose in HK1.IGF-1 transgenic mice treated only with TPA. In addition, transgenic mice exposed to an initiation-promotion protocol (using DMBA-TPA) developed tumors considerably faster and in far greater numbers than similarly treated nontransgenic mice. Collectively, these data demonstrated for the first time that signaling through the IGF-1r played an important role in the development of skin tumors in an in vivo model of epithelial tumorigenesis.
In other studies, signaling through the IGF-1 receptor has been shown to be essential for the development and growth of the skin, because mice lacking the IGF-1r have hypoplastic skin (19) . Transgenic animals overexpressing IGF-1 (driven by a metallothionein promoter) have been described previously (20) ; however, a significant skin phenotype was not reported in these animals. Two additional studies have described transgenic mice expressing IGF-2 (21 , 22) . In one of these studies, IGF-2 expression was driven by the MUP (21) . Although these mice developed hypoglycemia and hypoinsulinemia, in addition to a variety of tumors in older animals, a significant skin phenotype was not reported. In the second study, overexpression of IGF-2 in only the suprabasal layer of epidermis using a BK10 promoter resulted in overgrowth of the skin and mild epidermal hyperplasia and hyperkeratosis (22) . These latter results, in combination with the results reported in our previous study with HK1.IGF-1 transgenic mice (16) , suggested that overexpression of an IGF-1r ligand in the suprabasal compartment of the epidermis results in excessive growth of the skin accompanied by an increase in epidermal proliferation.
To further explore the role of IGF-1 during carcinogenesis, we have now created transgenic mice in which human IGF-1 expression is targeted specifically to the basal layer of mouse epidermis using BK5 (23 , 24) . Our results show that persistent expression of IGF-1 in the basal layer of epidermis yields both similar, as well as distinct, phenotypic characteristics compared with HK1.IGF-1 transgenic mice. The current data provide new evidence that unconstrained signaling through the IGF-1r can act primarily as a tumor promoter and that cell survival signaling pathways in addition to mitogenic signaling pathways may contribute to this effect of IGF-1 in mouse skin.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor Induction Experiments.
BK5.IGF-1 transgenic mice (6–20 weeks of age), along with age-matched
littermate controls, were shaved 2 days prior to initiation. Mice were
initiated with 25 nmol DMBA in 0.2 ml of acetone, applied as a single
topical application to the shaved dorsal skin of both transgenic and
nontransgenic mice. Two weeks later, TPA promotion was begun by topical
application of 5 nmol TPA in 0.2 ml of acetone twice a week. Individual
groups of both transgenic and control mice received treatment regimens
of either acetone/TPA, DMBA/acetone, or DMBA/TPA. Mice were examined
weekly for the presence of papillomas. Tumor multiplicity
(papillomas/mouse) and tumor incidence were recorded weekly.
Ha-ras Mutation Analysis.
DNA was isolated from skin tumors that had been snap frozen and was
analyzed for point mutations in codons 12, 13, and 61 of the
Ha-ras gene. Briefly, individual tumors were ground
in liquid nitrogen using a mortar and pestle, and genomic DNA was
extracted, as has been described (16)
, followed by
treatment with RNase A and successive phenol and chloroform/isoamyl
alcohol extractions. Purified DNA was amplified by PCR with the
following primer sets: codon 61 (268-bp fragment), 5'-TGT GGA TTC TCT
GGT CTG AGG AGA G-3' and 5'-CAT AGG TGG CTC ACC TGT ACT GATG-3'; and
codons 12 and 13 (214-bp fragment), 5'-CCT TGG CTA AGT GTG CTT CTC ATT
GG-3' and 5'-ACA GCC CAC CTC TGG CAG GTA GG-3'. Amplification products
were gel purified and then sequenced using the Thermo Sequenase
Radiolabeled Terminator Cycle Sequencing kit (Amersham Pharmacia
Biotech).
Histological Analysis and Indirect Immunofluorescence Staining
for IGF-1.
For histological analysis, dorsal skin samples and tumors were fixed in
formalin and embedded in paraffin prior to sectioning. Sections of 4
µm were cut and stained with H&E. For the analysis of epidermal LI,
mice received an i.p. injection of BrdUrd in PBS (100 µg/g body
weight) 30 min prior to sacrifice, and paraffin sections were stained
using an anti-BrdUrd antibody, as described previously
(27)
.
To determine the responsiveness of the BK5.IGF-1 mice to TPA, female mice (four/group, 6–8 weeks of age) were shaved on the dorsal side and after 2 days were treated topically with 1.7 nmol TPA or the acetone vehicle (0.2 ml), twice a week for 2 weeks. The mice were sacrificed 48 h after the last treatment. Dorsal skin was removed, fixed in formalin, and embedded in paraffin and then subsequently processed for conventional H&E staining and BrdUrd labeling, as described above. The determinations of epidermal thickness and LI were performed as described previously (28) .
The expression and localization of IGF-1 was determined using indirect immunofluorescence on sections of dorsal skin and skin tumors. The tissues were fixed in formalin and embedded in paraffin; 4-µm sections were adhered to slides. After deparaffinization, the slides were microwaved twice for 5 min each time to enhance the staining of IGF-1. Sections were incubated with 10% nonimmunized rabbit serum for 30 min to block the nonspecific Fc receptor in tissue and then washed three times with PBS (pH 7.5) containing 1% BSA (BSA/PBS). Sections were then incubated with either a 1:100 dilution of the primary sheep antihuman IGF-1 (Chemicon International, Inc., Temecula, CA) in BSA/PBS or preimmune sheep serum as negative controls for 1 h. After three washes with BSA/PBS, the sections were incubated with the secondary fluorescence (FITC)-conjugated affinity pure F(ab')2 fragment rabbit antisheep IgG (Jackson Immuno Research Lab, West Grove, PA; 1:200) for 40 min. The sections were then covered with Vectashield mounting solution (Vector Labs, Inc., Burlingame, CA) before the coverslips were attached. Immunospecificity of IGF-1 IgG was confirmed by preabsorption with the antigen.
Preparation of Epidermal Lysates.
Transgenic and nontransgenic mice were killed by cervical dislocation.
The dorsal skins were treated with a depilatory agent (1 min) followed
by washing. The skin was excised, and the epidermis was scraped off
with a razor blade into lysis buffer. For immunoprecipitation, the
lysis buffer was 1% Triton X-100, 10% glycerol, 50 mM
HEPES (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1.5 mg
MgCl2, 1 mM PMSF, 20
µg/ml leupeptin, 20 µg/ml aprotinin, 2
mM
Na3VO4, 1
mM NaVO3, 100
mM NaF, 10 mM
p-nitrophenyl phosphate, 5 µg/ml
N
-p-tosyl-L-lysine
chloromethyl ketone, and 10 µg/ml
N-tosyl-L-phenylalanine chloromethyl
ketone. Samples were homogenized with a Polytron PT10 homogenizer
(3 x 10 s bursts at setting 6) and then
centrifuged at 12,000 x g for 15 min at
4°C. The supernatant was used immediately for immunoprecipitation.
For analysis of pMAP, the lysis buffer was 50 mm Tris (pH 7.4), 1%
Triton X-100, 150 mM NaCl, 1 mM EDTA,
1 mM PMSF, 1 mM
Na3VO4, 1
mM NaF, and 1 µg/ml each of aprotinin,
leupeptin, and pepstatin. The lysates were homogenized using a needle
(18-gauge) and syringe and were centrifuged at 14,000 x g for 15 min at 4°C. The supernatant was used immediately
for Western blot analysis.
Immunoprecipitation.
Mouse epidermal lysates (1 mg protein) were incubated with 4 µg of
polyclonal anti-IGF-1r for 2 h at 4°C and then incubated with
protein G plus agarose for 1 h. The immunocomplex was precipitated
by brief centrifugation and washed three times with lysis buffer.
Immunoprecipitates were subjected to Western blot analysis.
Western Blot Analysis.
For the analysis of the IGFr and its phosphorylation status,
immunoprecipitates were electrophoresed in 9% SDS polyacrylamide gels
according to the method of Laemmli (29)
. In all cases,
electrophoresis was performed under reducing conditions. Separated
proteins were electrophoretically transferred onto nitrocellulose
membranes and blocked with 5% nonfat milk. Blots were incubated with
either 1 µg/ml rabbit polyclonal IGF-1rß or PY99 antibody (Santa
Cruz Biotechnology, Santa Cruz, CA) overnight in 5% nonfat milk in TBS
with 0.1% Tween 20 (TTBS). Blots were washed three times with TTBS for
15 min each, and the protein bands were visualized by enhanced
chemiluminescence (ECL; Amersham, Arlington Heights, IL).
To determine the phosphorylation status of MAPK, 50 µg of protein from epidermal lysates were electrophoresed and transferred onto polyvinylidene difluoride membranes. After blocking with 5% nonfat milk in PBS with 0.05% Tween 20 (TPBS), the blots were incubated with antibodies specific for either phosphorylated MAP (New England Biolabs) or MAPK (Upstate Biotechnologies, Inc.) for 2 h in TPBS plus 5% nonfat milk at 4°C. Blots were then washed three times with TPBS for 10 min each and then incubated with horseradish peroxidase-conjugated secondary antibody (Sigma) in TPBS plus 5% nonfat milk for 1 h at 4°C. After incubation, the blots were washed three times for 10 min each in TPBS and once in PBS for 10 min.
MAPK and PKB (Akt) Assays.
Kits for both kinase assays were purchased from UBI (Lake Placid, NY),
and assays were performed according to the manufacturer’s
instructions. Briefly, 0.25–1 mg of epidermal lysate was
immunoprecipitated with 4 µg of an anti-Akt rabbit polyclonal
antibody or anti-phosphospecific MEK rabbit polyclonal antibody (UBI)
in RIPA buffer containing 50 mM NaF, 10 mM
NaPP, 50 mM
MoNa2O4, 1 mM
NaVO3, 1 mM PMSF, 20 µg/ml
leupeptin, and 20 µg/ml aprotinin at 4°C. Immunoprecipitation
reactions were incubated with protein A agarose and collected by brief
centrifugation (14,000 x g), followed by
three washes with RIPA buffer. Samples were incubated for 20 min at
30°C in the presence of an PKB (Akt)-specific substrate/or MAPK
substrate peptide, along with specific kinase inhibitors (included in
both kinase kits). Reactions were stopped with the addition of 40%
trichloroacetic acid, and the reaction volume was spotted on
phosphocellulose paper. Incorporated radioactivity was assayed using
liquid scintillation counting.
PI3K Assay.
The PI3K assay was performed as described previously (30)
.
Briefly, 2 mg of epidermal homogenate were immunoprecipitated with 4
µg of anti-PI3K antibody (UBI) overnight at 4°C in 20
mM Tris (pH 7.4), 50 mM NaCl, 50 mM
NaF, 20 mM NaPP, 1% Triton X-100, 0.2 mM
Na3VO4, 0.2 mM
PMSF, 20 µg/ml leupeptin, and 20 µg/ml aprotinin.
Immunoprecipitates were incubated with 60 µl of a 50% slurry of
protein A agarose suspended in 0.1 Tris-HCl (pH 7.4), 5 mM
LiCl, and 0.1 mM
Na3VO4 at 4°C for 2 h and collected by centrifugation (5 s). Immunoprecipitates were washed
three times with 1% NP40 Tris buffer, three times with 0.1
M Tris-HCl (pH 7.4), 5 mM LiCl, and 0.1
mM Na3VO4 and
twice with TNE buffer [10 mM Tris-HCl (pH 7.4), 150
mM NaCl, 5 mM EDTA, 0.1 mM
Na3VO4). Samples were
resuspended in 50 µl of TNE, 10 µl (20 µg) of
phosphatidylinositol, and 10 µl of 10 mM
MgCl2. Reactions were started by the addition of
100 µCi [
32-P]ATP and incubated at 37°C
for 10 min. Reactions were stopped with 20 µl of 6 N HCl
and extracted with 400 µl of CHCl3:methanol
(1:2). The organic phase was washed with 2 N KCl (three
times), and radioactivity was assayed by liquid scintillation counting.
Measurement of Epidermal Apoptosis.
BK5.IGF-1 transgenic mice and nontransgenic mice, 7–8 weeks of age,
were housed under yellow lights and irradiated one time under a bank of
8 x 100-W FS20 fluorescent lamps (Westinghouse)
emitting predominantly UVB light with a peak wavelength at 313 nm.
The light was filtered through UVT cast acrylic (Polycast Technology
Corp., Stamford, CT), which excludes stray light <280 nm. The fluence
rate was measured with an IL1400A Radiometer/Photometer coupled to an
SEL240/UVB-1/TD detector (International Light, Inc., Newburyport, MA).
Animals were exposed to UVB in an irradiation chamber of our design,
which was manufactured to maximize incident fluence uniformity
(StarchArt Corp., Smithville, TX). Individual mice were confined in a
cassette constructed from cast acrylic (see above) containing 10 small
ventilated chambers that allowed minimal movement. Six such cassettes
were oriented along the circumference of a "carousel" that rotated
the animals at 6.5 rpm through the outer portion of the circular
transmittance field emitted by the lights. The average fluence rate at
the level of the dorsa (20 cm) was 5
J/m2/s, and the total incident dose for each
treatment was determined by integrating the fluence over the time of
exposure. The animals were divided into groups of three mice and
received a single incident dose of 5 kJ/m2. At
various time points after treatment (0, 12, 24, 36, and 48 h),
mice were sacrificed, and the dorsal skin was removed, fixed in
formalin, and embedded in paraffin prior to sectioning. The individual
apoptotic cells in these sections were identified by the terminal
deoxytransferase-mediated dUTP nick end-labeling technique (In
Situ Cell Death Detection kit, Fluorescein; Boehringer Mannheim,
Indianapolis, IN).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In addition to the BK5 promoter,
proper transcript processing was aided by the inclusion of rabbit
ß-globin intron 5' of the IGF-1 cDNA as well as SV40 early gene
polyadenylation signal sequences 3' of the gene (25)
. Six
founders with varying degrees of phenotypic presentation were
generated. Two lines were established, one representing the most severe
phenotype (BK5.IGF-1.II) and one representing a more moderate phenotype
(BK5.IGF-1.EE), which passed the transgene in Mendelian fashion to
offspring and were bred for later experiments. By 3 days of age,
transgenic mice from line II could be easily distinguished from control
littermates based on gross physical characteristics, eliminating the
need for genotyping. Animals from line EE were genotyped by PCR as
described previously (16)
using human specific IGF-1
oligonucleotide primers. All mice used in the current study were
hemizygous and were derived from line II.
Mice from line II had the most severe skin phenotype, obvious
from the excessive thickening and wrinkling of the skin at 3 days of
age (Fig. 1B
, center). Line II mice were also smaller at 3
days of age than both the nontransgenic littermate (Fig. 1B
, left) and an age-matched HK1.IGF-1 transgenic mouse shown for
comparison (Fig. 1B
, right). Nevertheless, both the
BK5.IGF-1 mice and the previously described HK1.IGF-1 mice
(16)
exhibited very similar gross phenotypic
characteristics at day 3. In this regard, BK5.IGF-1 mice exhibited
excessive growth of ear tissue at this age; however, this was less
severe than seen in HK1.IGF-1 transgenic mice. The slight differences
in gross phenotype between the two types of IGF-1 transgenic mice at
this age could potentially be attributable to differences in the timing
and/or level of expression of the transgene during development.
Notably, the gross phenotype of BK5.IGF-1 mice persisted as the animals
aged. In fact, the gross phenotype in line II (Fig. 1C
and Fig. 1D
, center; Fig. 1E
, right) became more severe
with age, and these mice could be easily distinguished by their
enlarged, thickened ears and excessive skin proliferation, especially
around the eyes and extremities. Expression of IGF-1 in the basal layer
of the epidermis also resulted in an altered hair coat characterized by
a ruffled and shaggy appearance (Fig. 1D and E)
.
In addition to the obvious physical characteristics of BK5.IGF-1
line II transgenic mice, squamous papillomas (some of which converted
to squamous cell carcinomas) arose spontaneously in older animals (>6
months of age). In this regard, a group of 16 line II mice and 16
age-matched nontransgenic littermates were housed for 1 year. Fifty
% (8 of 16) of the transgenic mice had one or more skin tumors,
whereas none of the nontransgenic littermates developed spontaneous
skin tumors. These spontaneous tumors arose at various locations; few
developed on dorsal skin. A BK5.IGF-1 mouse with a spontaneous
papilloma is shown in Fig. 1E
, right. Sections stained with
H&E from a papilloma (Fig. 2A)
and a squamous cell carcinoma
(Fig. 2C)
obtained from BK5.IGF-1 mice are shown in Fig. 2
. Indirect immunofluorescence staining for human IGF-1 from
corresponding sections of the papilloma (Fig. 2B)
and
squamous cell carcinoma (Fig. 2D)
show the persistent
expression of transgene in these lesions. All spontaneous tumors in the
transgenic mice were histologically similar to those induced in ICR
mice using an initiation-promotion regimen (16)
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. Skin from transgenic mice exhibited epidermal hyperplasia and
hyperkeratosis that was accompanied by an increase in the LI. The LI in
the epidermis of adult BK5.IGF-1 mice was 7.2 ± 1.4
compared with 2.3 ± 0.9 for age-matched nontransgenic
littermates (Fig. 3D
versus Fig. 3C
,
respectively). Overall, the skin was also much thicker in BK5.IGF-1
mice compared with nontransgenic mice. This is evident in Fig. 3B
, where only the top of the hypodermis is visible, whereas
at the same magnification, both the hypodermis and muscle layers are
visible in skin from nontransgenic mice (Fig. 3A)
. The skin
phenotype shown in Fig. 3B
coincided with localized
expression of the transgene, as determined by indirect
immunofluorescence detection of IGF-1 protein (Fig. 3H)
.
Notably, the skin phenotype in these transgenic mice persisted and
progressively worsened with age.
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, keratin 6, a putative marker of
epidermal proliferation, was found elevated in focal areas of the
interfollicular epidermis. This observation was consistent with the
increased LI noted in the epidermis of transgenic mice. No differences
were noted in the expression of the other keratins examined.
Analysis of TPA Responsiveness in BK5.IGF-1 Transgenic Mice.
In previous studies with HK1.IGF-1 transgenic mice (16)
,
we found they were hypersensitive to the proliferative effects of the
skin tumor promoter, TPA. To determine whether BK5.IGF-1 transgenic
mice were also hypersensitive to TPA, the mice were treated four times
over a 2-week period with 1.7 nmol of TPA. Forty-eight h after the last
dose of TPA, changes in epidermal thickness and LI were measured. In
BK5.IGF-1 mice, TPA treatment led to a potentiated hyperplasia compared
with nontransgenic mice as shown in Fig. 4, A and B
. Also shown in Fig. 4
is the
immunofluorescence staining for transgene (C and
D). Note that the transgene expression was relatively
unaffected by either the promoter treatment or the ensuing hyperplasia.
This result is consistent with the previous report of Casatorres
et al. (24)
, showing that keratin 5 expression
was unaffected by TPA.
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2 times higher (14.7 ± 1.4 versus
7.1 ± 1.1) in TPA-treated transgenic compared with
nontransgenic mice. Thus, these data indicate that BK5.IGF-1 transgenic
mice, like HK1.IGF-1 transgenic mice (16)
, are
hypersensitive to the effects of TPA.
Responsiveness of K5.IGF-1 Mice to Two-Stage Carcinogenesis.
To determine the responsiveness of BK5.IGF-1 transgenic mice to
two-stage carcinogenesis, three groups of transgenic and nontransgenic
mice were treated as follows: (a) acetone at initiation
followed 2 weeks later by twice-weekly applications of 5 nmol TPA;
(b) DMBA initiation (25 nmol) followed 2 weeks later by
twice-weekly applications of acetone; and (c) DMBA
initiation (25 nmol) followed 2 weeks later by twice-weekly
applications of 5 nmol TPA. The experiment was continued for 30 weeks,
during which time the incidence and multiplicity (papillomas per mouse)
of tumors were scored in each group. The results from this experiment
are shown in Table 1
. Note that the true spontaneous tumors that arose in older mice did not
appear prior to 6 months of age; the tumors in the treated groups arose
much earlier and exclusively on the treated, dorsal skin.
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2 and Mann-Whitney
U tests, respectively; P < 0.05).
A very interesting result was obtained in the two groups of mice that
were initiated with DMBA but did not receive tumor promoter treatment.
Fifty % (average of 1.4 papillomas/mouse) of the BK5.IGF-1 transgenic
mice developed papillomas, whereas none of the nontransgenic mice
developed papillomas (also significantly different;
P < 0.05). Again, the tumors that developed
in BK5.IGF-1 mice treated with DMBA alone arose much earlier than the
spontaneous tumors in older, untreated mice. Finally, the BK5.IGF-1
transgenic mice were dramatically more sensitive to the
initiation-promotion regimen. In this regard, the BK5.IGF-1 transgenic
mice had 21.0 papillomas/mouse (100% incidence), whereas the
nontransgenic mice had only 2.8 papillomas/mouse (69% incidence). Note
that the papillomas arising in BK5.IGF-1 transgenic mice grew faster
and were considerably larger than those produced in nontransgenic mice
at the end of the experiment (28 weeks). This latter observation is
similar to our earlier results reported with HK1.IGF-1 mice
(16)
.
Twelve papillomas induced in BK5.IGF-1 mice by treatment with TPA only
were analyzed for mutations in the Ha-ras gene. Eleven of
these tumors (92%) had mutations in codons 12, 13, or 61 of this gene
as follows: G35 
A (six tumors);
G38 T (two tumors);
C181 A (one tumor); and
A182 G (two tumors).
Analysis of the IGF-1r, PI3K, PKB (Akt), and MAPK Status in Skin of
BK5.IGF-1 Mice.
To confirm that the IGF-1r was activated in a persistent manner in the
epidermis of transgenic mice, we analyzed its phosphorylation status by
Western blot analysis. For these experiments, adult (7–9 weeks of age)
BK5.IGF-1 transgenic mice and corresponding age-matched littermates
were used. Tissue lysates were prepared from the epidermis as described
in "Materials and Methods" and immunoprecipitated with an antibody
against the endogenous mouse IGF-1r. The Western blot analysis for
IGF-1r levels and IGF-1r phosphorylation status in the epidermis of
nontransgenic and transgenic mice is shown in Fig. 5A
. Receptor levels appeared to be very similar in both
transgenic and nontransgenic mice. In contrast, the IGF-1r (IGF-1r ß
chain at Mr 94,000) was
hyperphosphorylated in the epidermis of BK5.IGF-1 transgenic mice. Note
the presence of another band (Mr
165,000) in the PY99 blot of Fig. 5A
. The apparent
molecular mass of this band is similar to that of IRS-1, and it too was
dramatically hyperphosphorylated in the epidermis of BK5.IGF-1
transgenic mice.
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, the phosphorylation of MAPK was elevated in the
epidermis of transgenic versus nontransgenic mice. In
addition, epidermal PI3K and PKB (Akt) activities were elevated (3.8-
and 3.3-fold, respectively) in transgenic mice compared with
nontransgenic littermates (Fig. 5C)
. These differences were
statistically significant (P < 0.05). MAPK
activity also was significantly elevated in transgenic
versus nontransgenic mice (1.7-fold; P < 0.05). The increased activity of MAPK correlated with the
higher level of phosphorylation of MAPK proteins, as shown in Fig. 5B
.
Analysis of Apoptosis in Epidermis of BK5.IGF-1 Mice.
In light of the data indicating that PI3K and PKB (Akt) activities were
elevated in the epidermis of BK5.IGF-1 transgenic mice, the response to
an apoptosis-inducing agent, UV light, was evaluated. For these
experiments, mice were exposed to UV irradiation (5
kJ/m2) and then sacrificed at various times after
treatment. In general, epidermal thickness and LI values (Fig. 6, A and B)
increased over the 48-h time course
after exposure to UV light. The exception to this statement was the
value for LI at 12 h after UV exposure, which was decreased
compared with the 0-h time point. Both of these markers of epidermal
proliferation were significantly higher (P 
0.05) at
all time points in the BK5.IGF-1 mice. Thus, BK5.IGF-1 transgenic mice
also exhibited an exaggerated proliferative response after UV
irradiation compared with nontransgenic mice. At 0 h (no UV
treatment), there was no significant difference in epidermal apoptotic
index between transgenic and nontransgenic mice. UV irradiation induced
a significant increase in the number of apoptotic cells in the
epidermis of nontransgenic mice at all time points, with a peak at
24 h. UV light exposure also induced an increased apoptotic
response in epidermis of BK5.IGF-1 transgenic mice. However, a
suppression of apoptosis was observed in BK5.IGF-1 mice compared with
nontransgenic mice at all time points after UV irradiation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The current results represent the first report that overexpression of IGF-1 alone leads directly to the development of skin tumors in transgenic mice. Previously, we reported that HK1.IGF-1 transgenic mice (16) did not develop any spontaneous skin tumors in the absence of TPA treatment. In light of the current data, this is likely attributable to the fact that transgene expression and skin phenotype subsided in older HK1.IGF-1 transgenic mice. Several other transgenic mouse models have been reported where IGF-1 or IGF-2 expression was targeted to or occurred in epidermis (20 , 22) . Spontaneous skin tumors were not observed in these transgenic mice, which may be attributable to a lower level of expression and/or milder skin phenotypes in these mice. Rogler et al. (21) reported that transgenic mice in which IGF-2 expression was driven by the MUP promoter developed hepatocellular carcinomas and lymphomas, in addition to several other tumors, at a higher frequency than controls after 18 months of age. However, no tumors were reported in the skin of these IGF-2 transgenic mice. The MUP promoter is expressed at high levels in liver and preputial glands, and these mice had high serum levels of IGF-2. Because IGF-2 binds to and activates the IGF-1r, it is likely that the mechanism for tumorigenesis in the MUP.IGF-2 transgenic mice involves signaling through the IGF-1r. The fact that MUP.IGF-2 transgenic mice did not develop spontaneous skin tumors like BK5.IGF-1 transgenic mice suggests that the tissue-specific expression is critical for producing spontaneous tumors in IGF-1 transgenic mice.
In the current study, we found that BK5.IGF-1 transgenic mice were hypersensitive to two-stage carcinogenesis, similar to HK1.IGF-1 transgenic mice (16) . In addition, when BK5.IGF-1 transgenic mice were treated with only the tumor promoter TPA, tumors developed much earlier and at a higher incidence than spontaneous tumors that developed in older BK5.IGF-1 transgenic mice. These results are also similar to those reported for HK1.IGF-1 transgenic mice (16) . However, a major new finding of our current study was that treatment of BK5.IGF-1 transgenic mice with DMBA alone led to tumor development. In contrast, HK1.IGF-1 transgenic mice treated with DMBA alone did not develop any skin tumors (16) . Thus, based on these observations, it appeared that constitutive activation of the IGF-1r in epidermal basal cells substituted for both the initiation and promotion stages of skin carcinogenesis in BK5.IGF-1 transgenic mice. However, further observations discussed below suggest that the main effect of constitutive IGF-1 expression may be related primarily to tumor promotion.
In our previous study with HK1.IGF-1 transgenic mice, we hypothesized
that constitutive signaling through the IGF-1r and activation of the
Ha-ras signal transduction cascade could substitute for
Ha-ras activation that occurs as a result of carcinogen DNA
adduct formation and subsequent mutation during the process of tumor
initiation. This potential mechanism could explain the observation that
tumors arose in skin of IGF-1 transgenic mice treated with TPA only. A
similar mechanism was postulated to explain skin tumor formation after
treatment with TPA in mice overexpressing transforming growth
factor- in skin epidermis (31
, 32)
. Skin tumors
produced in transforming growth factor- transgenic mice by treatment
with TPA did not possess any Ha-ras mutations, supporting
this hypothesis (31
, 32)
. However, analysis of papillomas
produced in BK5.IGF-1 transgenic mice by TPA treatment alone revealed
that essentially all had mutations in Ha-ras. These data
suggest that enhanced IGF-1 signaling may not be substituting for
initiation but rather enhancing the ability of TPA to select for
already existing initiated cells (with Ha-ras mutations)
that are present in the epidermis. It is interesting to note that the
spectrum of mutations in Ha-ras from the TPA-induced
papillomas included all codons, and the A182T
mutation seen in TPA-induced papillomas from other studies
(33, 34, 35, 36)
was not observed in any of the 12 tumors analyzed
in the current study. We do not know whether this is attributable to
the genetic background of the BK5.IGF-1 transgenic mice (ICR), because
of the fact that tumor promotion by TPA in the presence of constitutive
IGF-1r signaling is qualitatively different from that in the absence of
constitutive IGF-1r signaling or because of the small sample size. In
SENCAR mice, we have analyzed 50 TPA-induced papillomas
(36)
5
and find that the spectrum of mutations in these tumors includes codons
12, 13, and 61 in the following proportions, 15%:26%:59%. Therefore,
additional studies will be required to determine the exact mechanism
and implications of these data.
The fact that persistent IGF-1r signaling alone can promote papilloma development in mice initiated with DMBA is also very interesting. The epidermal hyperplasia and the increase in LI of the BK5.IGF-1 mice indicate that IGF-1r signaling induces epidermal cell proliferation. Both of these changes are hallmarks of tumor promotion in mouse skin (37) . In addition, the BK5.IGF-1 transgenic mice showed an exaggerated hyperplasia and LI after TPA treatment compared with nontransgenic mice. Thus, IGF-1r signaling may promote tumor development, in part through stimulation of mitogenic pathways. The signaling pathways responsible for the mitogenesis mediated by IGF-1 appear to include the ras pathway, although the PI3K pathway also has been implicated in mitogenic responses to IGF-1 (3 , 5 , 38) .
To further explore the signaling pathways involved in mediating the
phenotype in BK5.IGF-1 transgenic mice, we analyzed MAPK activity (both
ERK1 and ERK2) and both PI3K and PKB (Akt) activities in epidermal
tissue preparations. As shown in Fig. 5
, both PI3K and Akt activities
were significantly elevated in epidermis of transgenic mice compared
with nontransgenic mice. MAPK activity also was constitutively elevated
in the epidermis of transgenic mice. These data suggest that both the
MAPK and PI3K signaling pathways may be important for the proliferative
phenotype seen in BK5.IGF-1 transgenic mice, including the effects on
tumor promotion.
Signaling through the IGF-1r can also mediate effects on apoptosis
(inhibition) and differentiation (induction; Refs. 39, 40, 41
and reviewed in Refs. 3
and 5
). As noted in
"Results," we examined the expression of keratins 1, 6, 13, and 14
in the epidermis of BK5.IGF-1 transgenic mice and found that keratin 6,
which is associated with proliferation in the interfollicular
epidermis, was focally elevated in the epidermal compartment. No
alterations in expression of keratins 1, 13, and 14 were observed.
Preliminary experiments using keratinocyte cultures from BK5.IGF-1
transgenic mice have suggested a delay in
Ca2+-induced maturation compared with
keratinocytes from nontransgenic littermates; however, further work
will be required to substantiate this observation. We also examined the
apoptotic response in the skin of BK5.IGF-1 transgenic mice exposed to
UV light (Fig. 6)
. Although there was no difference in apoptotic rate
between transgenic and nontransgenic mice, there was a significantly
reduced epidermal apoptotic response after UV exposure in BK5.IGF-1
transgenic mice compared with nontransgenic mice. This result suggests
that constitutive signaling through the IGF-1r could counteract
p53-mediated apoptosis in skin keratinocytes. p53 has been known to
mediate UV-induced apoptosis in mouse skin keratinocytes because lack
of p53 suppresses the apoptotic response after UVB exposure in
p53-/- mice (39)
. Thus, increased
cell survival in addition to increased proliferation may contribute to
the phenotype observed in BK5.IGF-1 transgenic mice and contribute to
the response to both initiators and promoters.
Recently, transgenic mice expressing Bcl-XL in epidermis under control of the keratin 14 promoter were reported to develop more papillomas than corresponding nontransgenic mice when initiated with DMBA and promoted with TPA (40) . In addition, transgenic mice expressing Bcl-2 under control of the human keratin 1 promoter reportedly developed skin papillomas faster than nontransgenic mice in a two-stage carcinogenesis protocol (41) . An inhibition of apoptosis in response to treatment with either DMBA or UV light was reported in both of these transgenic models. In our current study, papillomas arose earlier, faster, and in greater numbers in BK5.IGF-1 transgenic mice initiated with DMBA and promoted with TPA than corresponding nontransgenic mice. These data are consistent with the hypothesis that activation or enhancement of cell survival signaling pathways in the epidermis may play an important role in the development of both spontaneous and induced skin tumors in the BK5.IGF-1 mice.
In conclusion, persistent IGF-1r signaling in mouse skin epidermis appears to act primarily as a tumor promoter in the context of the two-stage carcinogenesis model. The mechanism for the tumor promoting action of IGF-1r signaling may involve both mitogenic and cell survival pathways (the latter activated via PI3K), although further work is necessary to substantiate this hypothesis.
|
FOOTNOTES |
|---|
1 This work was supported by USPHS Grant CA37111
(to J. D.), University of Texas M. D. Anderson Cancer Center Core
Grant CA16672, and National Institute of Environmental Health Sciences
Center Grant ES07784. E. W. is supported by Training Grant ES07247. 
2 To whom requests for reprints should be
addressed at, Department of Carcinogenesis, University of Texas M. D.
Anderson Cancer Center, Science Park, Research Division, P. O. Box
389, Smithville, TX 78957. Phone: 512-237-9414; Fax: 512-237-2522;
E-mail: sa83107{at}odin.mdacc.tmc.edu
3 Present address: Department of Oncology,
Bristol-Myers Squibb Pharmaceutical Research Institute, P. O. Box
4000, Princeton, NJ 08543-4000.
4 The abbreviations used are: IGF-1r, insulin-like
growth factor-1 receptor; IGF-1, insulin-like growth factor; MUP, major
urinary protein; HK, human keratin; BK, bovine keratin; LI, labeling
index; DMBA, 7,12-dimethylbenz[a]anthracene; TPA,
12-O-tetradecanoylphorbol-13-acetate; BrdUrd,
bromodeoxyuridine; PMSF, phenylmethylsulfonyl fluoride; MAP,
mitogen-activated protein; MAPK, MAP kinase; PI3K, phosphatidylinositol
3-kinase; PKB, protein kinase B; UBI, Upstate Biotechnology, Inc.; ERK,
extracellular signal-regulated kinase; MEK, MAP/ERK kinase.
5 J. D. Giovanni and L. Beltrán, unpublished
data.
Received 9/24/99. Accepted 1/19/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in transgenic mice: induction of spontaneous and 12-0-tetradecanoylphorbol-13-acetate-induced papillomas via a mechanism independent of Ha-ras activation or overexpression. Mol. Carcinog., 10: 15-22, 1994.[Medline]
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