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Carcinogenesis |
in Their Epidermis Exhibit Reduced Papilloma Burden but Enhanced Carcinoma Formation after Tumor Promotion1
Department of Human Oncology, Medical School, University of Wisconsin [P. J. R., N. E. D., J. Z., S. E. B., A. K. V.], and Department of Pathology & Laboratory Medicine, Veterans Administration Hospital and Medical School, University of Wisconsin [T. D. O.], Madison, Wisconsin 53792
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PKC) may play in skin
growth, differentiation, and tumor promotion, transgenic mice were
generated that overexpressed an epitope-tagged protein kinase C
(T7-PKC) in their epidermis using the human keratin 14 promoter.
Three independent mouse lines that overexpressed the T7-PKC in their
epidermis were produced. The three independent lines 206, 224, and 215
exhibited a 3-, 6-, and 18-fold elevation, respectively, in the level
of PKC immunoreactive protein. Line 215 exhibited a 19-fold greater
phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate
(TPA) stimulated kinase activity than line 224. Line 206 exhibited a
low basal T7-PKC activity, which failed to be stimulated by
phosphatidylserine and TPA. All of the line 215 transgenic mice
(F0 to the F2 generation) displayed phenotypic
changes in the skin. The phenotypic changes progressed gradually,
starting around 4–5 months of age, with mild dryness of the tail
accompanied by hair loss and inflammation at the base of the tail.
Hyperproliferation and ulceration of the affected regions were observed
around 7–8 months of age. The hyperproliferative epidermis from the
affected regions exhibited an expansion of the suprabasal epidermal
cells. Inflammation and/or ulceration were also observed in the dorsal
skin, the ears, and around the eyes. The line 215 mice, which expressed
the highest level of PKC, were evaluated for sensitivity to mouse
skin tumor promotion by TPA. Tumors were elicited by the initiation
(7,12-dimethylbenz[a]anthracene, 100 nmol)-promotion
(TPA, 5 nmol/twice weekly) protocol. The papilloma burden was reduced
by 95–96% for male and female T7-PKC mice compared to wild-type
controls. However, carcinomas developed rapidly in the T7-PKC mice
treated with 7, 12-dimethylbenz[a]anthracene and TPA.
These carcinomas appeared to form independently of prior papilloma
development. These results demonstrate that PKC is an important
regulator of skin tumor development. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ßI, ßII, and 
) are
Ca2+-dependent and are activated by diacylglycerol/TPA and
PS. The novel PKCs (
, , 
, and 
) are Ca2+-
independent and require DAG/TPA and PS for activation. The atypical
PKCs (
and 
) require only PS for activation, although other
activators, such as ceramide, have been identified (4)
.
PKCµ, originally classified as a novel PKC, appears to be a unique
isoform (1)
.
Of these 11 PKC isoforms, PKC appears to play an important role in
cellular growth regulation. In several small cell lung carcinoma cell
lines, the catalytic fragment of PKC was constitutively expressed,
indicating that activated PKC may be important for the survival of
these cells (5)
. Overexpression of PKC in Rat-6 or
NIH-3T3 fibroblasts led to increased growth rates, anchorage
independence, and tumor formation in nude mice (6
, 7)
.
Additionally, PKC overexpression transformed nontumorigenic rat
colonic epithelial cells (8)
. Overexpression of PKC
also suppressed apoptosis of interleukin-3-dependent human myeloid
cells induced by removal of interleukin-3 (9)
.
The mouse skin tumor promoter TPA binds and activates PKC
(10)
. However, the role PKC plays in mouse skin tumor
promotion and epidermal cell growth and differentiation remains unclear
(11
, 12) . Current evidence indicates that treatment of the
mouse skin with TPA leads to a general reduction in PKC activity that
persists for at least 4 days (13
, 14)
. Examination of the
effects of acute TPA treatment on the protein level of different PKC
isoforms demonstrated decreases in PKCß and but has little or no
effect on the levels of PKC, , or (15
, 16)
. The
level of PKC activity for PKC, ß, and was found to be reduced
after acute or repeated TPA treatments, but PKC activity was not
examined (15)
. Analysis of PKC isoforms in
DMBA-TPA-induced papillomas demonstrated decreases in cytosolic levels
of PKC and ßII protein, but insignificant alterations in the
levels of PKC, , or protein (17)
. In cultured
mouse skin keratinocytes, induction of differentiation by elevation of
Ca2+ induces translocation of PKC, , and to the
membrane fraction, suggesting a role for activation of these isoforms
in keratinocyte differentiation (18)
.
To further define the in vivo role of PKC in mouse skin
carcinogenesis, we have generated transgenic mice expressing an
epitope-tagged PKC under the control of the human K14 promoter.
Overexpression of PKC in the untreated mouse epidermis led to
phenotypic abnormalities (such as inflammation, hyperkeratosis,
hyperplasia, cellular hypertrophy, and ulceration), especially of the
skin surrounding the tail base of older mice (
7–8 months of age).
Paradoxically, two-stage tumor promotion with DMBA and TPA in the
PKC transgenic mice exhibited significant reductions in papilloma
burden compared to wild-type controls. However, carcinomas developed
rapidly in the T7-PKC mice independently of papilloma formation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and
actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). DAKO immunoperoxidase LSABB+Kit was purchased from DAKO
(Carpinteria, CA). Immobilized protein A/G-agarose was purchased from
Pierce (Rockford, IL). Enhanced chemiluminescence and enhanced
chemifluorescence Western blotting detection reagents and the PKC
enzyme assay system were purchased from Amersham Life Sciences Inc.
(Arlington Heights, IL). FVB/N mice, 7–9 weeks of age, were purchased
from Taconic (Germantown, NY).
Transgene Construction and Generation of Transgenic Lines.
The BglII/SalI fragment from the pET-21c(+)
vector containing the T7-tag open reading frame was ligated to the
NH2 terminus of mouse PKC cDNA in the pRSV-PKC vector
to produce the pRSV-T7-PKC vector. The T7-PKC cDNA from
pRSV-T7-PKC was ligated into the BamHI site of the
pGEM3Z-K14 ß-globin vector by insertion of two Eco 47III
fragments of the T7-PKC cDNA previously linked to BglII
and BamHI sites to produce the pGEM3Z-K14-T7-PKC vector.
The functional elements of pG3Z-K14-T7-PKC were isolated by partial
digestion with HindIII and complete digestion with
EheI. The purified K14-T7-PKC expression cassette was
microinjected into the male pronuclei of one-cell fertilized embryos
(FVB/N x FVB/N mice) by the University of Wisconsin’s
Transgenic Mouse Facility. The transgene was detected by Southern blot
analyses using genomic DNA from tail biopsies digested with
EcoRV and using the radiolabeled
EcoRV/BamHI fragment from pGEM3Z-K14 ß-globin
vector for the probe. Founders bearing the transgene were bred to
wild-type FVB/N mice to generate F1 offspring. Transgenic
F1 mice were bred with other transgenic or wild-type FVB/N
mice as necessary to maintain and expand the colony.
Mice.
The mice were housed in groups of three to four mice in
plastic-bottomed cages in light-, humidity-, and temperature
(24°C)-controlled rooms; food and water were available ad
libitum. The animals were kept in a normal rhythm of 12-h
light/12-h dark periods. At 8–10 weeks of age, the dorsal skins of the
mice were shaved 3–4 days before treatment, and those mice in the
resting phase of their hair cycle were used for experimentation. The
solutions of TPA and DMBA were prepared in acetone and applied to the
shaved backs of individual mice in a volume of 0.2 ml.
Tumor Induction Experiments.
Mouse skin tumors were induced by the initiation-promotion regimen. The
line 215 T7-PKC mice were used for tumor promotion. For mouse skin
tumor initiation, a single 100-nmol dose of DMBA in 0.2 ml of acetone
or acetone alone was applied topically to the shaved backs of the mice.
Two weeks after initiation, 5 nmol of TPA in 0.2 ml of acetone or
acetone alone was applied twice weekly to the skin for the duration of
the experiment. The tumor incidence and burden were observed weekly
starting at 4 weeks of TPA promotion. The number of mice for each
experimental group was as follows: DMBA-TPA, 11 wild-type females, 15
transgenic females, 20 wild-type males, and 12 transgenic males; and
DMBA-acetone, 11 wild-type females, 16 transgenic females, 19 wild-type
males, 12 transgenic males; acetone-TPA, 10 wild-type females, 15
transgenic females, 19 wild-type males, and 11 transgenic males.
Carcinomas were recorded grossly as downward-invading lesions, a subset
of which was examined histologically, and malignancy was confirmed as
invading the panniculus carnosus. Carcinoma-bearing mice were killed
shortly after diagnosis.
Immunoblotting of PKC.
The mouse skin was excised and scraped to remove the s.c. tissue. The
skin was ground with a mortar and pestle under liquid N2.
The ground tissue was homogenized with 5 volumes of PKC extraction
buffer [20 mM Tris-HCl (pH 7.4), 0.3% Triton X-100, 2
mM EDTA, 10 mM EGTA, 0.25 M
sucrose, 1 mM DTT, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin]. The homogenate was centrifuged at 100,000 x g for 60 min at 4°C, and the supernatant was used
as the total PKC extract. Protein concentration in the total PKC
extract was determined, and 100 µg of total PKC extract protein were
fractionated on a 7.5% or 10% SDS-PAGE. The proteins were transferred
to 0.45 µm supported nitrocellulose membrane. The membrane was then
incubated with anti-T7 Tag (1:2000 dilution) or anti-PKC (1:100
dilution) antibody, the bound antibody was detected using the
appropriate secondary antibodies, and the detection signal was
developed with Amersham’s enhanced chemiluminescence or enhanced
chemifluorescence reagents. Immunoblotting of lysates from the
immunocomplex kinase assays (see below) was also performed with 100
µg of total protein.
T7-PKC Immunocomplex Kinase Assay.
The dorsal skin of the mice was shaved and depilated 24 h before
experimentation. The mice were euthanized, the dorsal skin was removed,
and the epidermis was scrapped off on ice with a razor. The epidermis
was placed in 0.5 ml of IP lysis buffer [50 mM HEPES (pH
7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5
mM MgCl2, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 1 mM phenylmethylsulfonyl fluoride, 200
µM Na3VO4, 200 µM
NaF, 1 mM EGTA, 100 µM benzamidine, 5 µg/ml
antipain, 5 µg/ml pepstatin, 40 µM MG132, and 40
µM calpain inhibitor I], homogenized using a glass
Teflon tissue homogenizer, agitated for 30 min at 4°C, and
centrifuged at 14,000 rpm in a microcentrifuge for 15 min, and the
supernatant of the lysate was used for IP. The lysate was preabsorbed
with 5 µl of protein A/G-agarose for 10 min at 4°C. Five µg of
anti-T7 Tag antibody and 10 µl of protein A/G-agarose were added to
the lysate, and the volume of the lysate was adjusted to 1 ml with
lysis buffer. The mixture was incubated for 2–4 h at 4°C with
agitation. The immunoprecipitate was pelleted at 14,000 rpm in a
microcentrifuge, washed, and resuspended in 300 µl of assay buffer
[50 mM Tris (pH 7.4), 5 mM EDTA (pH 8.0), 10
mM EGTA (pH 7.9), 0.3% ß-mercaptoethanol, 5 µg/ml
aprotinin, 5 µg/ml leupeptin, and 50 µg/ml phenylmethylsulfonyl
fluoride]. Twenty-five µl of the immunoprecipitate were assayed in
kinase buffer containing 50 mM Tris (pH 7.4), 8
mM MgCl2, 0.136 mM ATP, 100
µM EGFR peptide (ERKRTLRRL), 3 mM DTT, 34
µg/ml of L--phosphatidyl-L-serine, 3
µg/ml TPA, and 1 mM EGTA. The reaction was incubated at
37°C for 15 min, stopped with 10 µl of 300 mM
H3PO4, spotted onto filter discs, washed with
75 mM H3PO4, and counted.
Determination of the level of T7-PKC kinase activity of the
epidermis and tumors from T7-PKC mice at the end of the tumor
promotion experiment was performed as described above for the T7-PKC
immunocomplex kinase assay, but with the following modifications. The
skin was not depilated. Skin papillomas and carcinomas were excised
before scraping off the uninvolved epidermis. The excised papillomas,
carcinomas, and epidermis were separately homogenized and extracted in
0.5–1.0 ml of IP lysis buffer. For each treatment group, the epidermis
from three mice was combined for extraction. Two to four papillomas or
one to two carcinomas were excised, combined, and extracted in the IP
lysis buffer. One hundred µg of the total protein extract, before IP,
were used for immunoblot analysis.
Histology and Immunohistochemistry.
The tissue to be examined was excised promptly after euthanasia and
immediately placed in 10% neutral buffered formalin. The tissue was
fixed for 1 h in formalin and then embedded in paraffin. Four-µm
sections were cut for H&E staining or immunostaining. Deparaffinized
slides were used for immunostaining with the DAKO immunoperoxidase
LSAB+Kit. Endogenous peroxidase activity was blocked with 30%
H2O2. Nonspecific protein binding was blocked
with normal swine serum. The slides were incubated overnight with the
appropriate primary antibody and developed with biotinylated secondary
antibody, streptavidin-conjugated horseradish peroxidase, and
3,3'-diaminobenzidine.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression Vector. to the basal epidermal cells of the
mouse skin, we used the human K14 promoter. This promoter has been used
successfully to direct expression of several different genes to the
epidermis (19)
. A mouse PKC cDNA containing the T7
bacteriophage epitope tag at its 5' terminus was inserted into the
BamHI site of the pG3Z-K14 ß-globin vector to create the
pG3Z-K14-T7-PKC vector (Fig. 1A)
. The pG3Z-K14-T7-PKC vector expressed active T7-PKC assayed by
immunocomplex kinase assays of CV-1 cells transiently transfected with
the T7-PKC vector (data not shown).
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Mouse Lines. expression cassette was removed from the
pG3Z-K14-T7-PKC vector by complete digestion with the endonuclease
EheI and partial digestion with the endonuclease
HindIII (Fig. 1A)
. The linear K14-T7-PKC
expression cassette was purified from the contaminating vector DNA by
agarose gel electrophoresis, electroelution, and ion-exchange
chromatography. Once purified, the linear cassette was microinjected
into the pronuclei of fertilized eggs of FVB/N mice (University of
Wisconsin Biotechnology Center Transgenic Animal Facility), and the
offspring were analyzed for integration of the K14-T7-PKC expression
cassette. Genomic DNA isolated from tail biopsies of weanling mice was
digested with the endonuclease EcoRV, and a Southern
analysis was performed. A 1-kb fragment of the K14 promoter ligated
to the ß-globin intron isolated from the pG3Z-K14 ß-globin vector
with EcoRV and BamHI was used as a probe. Nine
mice exhibited transgene integration after analysis of 24 potential
founder mice (Fig. 1B)
.
These founder mice were bred to wild-type FVB/N mice to produce
F1 offspring. F1 mice from each line that was
positive for the K14-T7-PKC transgene were examined for expression
of the transgene. F1 mice were euthanized, the dorsal skin
was shaved, and a Triton X-100-soluble extract was made from the dorsal
skin. Immunoblots of these extracts were examined for T7-PKC
expression by probing with either anti-T7 tag or anti-PKC
antibodies. The blots probed with the anti-T7 antibody demonstrated
that three of these lines were expressing the T7-PKC transgene (Fig. 1C)
. Examination of PKC levels with the anti-PKC
antibody demonstrated that the PKC levels were significantly
elevated in these transgenic lines (Fig. 1C)
. The increase
in PKC immunoreactive protein levels was 3-, 6-, and 18-fold for
line 206, 224, and 215, respectively. The T7-PKC expressed in these
F1 mice was assayed for enzymatic activity. The epidermis
was isolated from euthanized F1 mice, Triton X-100-soluble
extracts were made, and the T7-PKC transgene was immunoprecipitated
with the anti-T7 antibody. The immunoprecipitates were assayed for
kinase activity by measuring the incorporation of 32P into
an EGFR peptide (ERKRTLRRL) in the presence of PS and TPA. The level of
overexpressed PKC protein was the highest in line 215, which
positively correlated with the level of PKC activity (Fig. 1D)
. The amount of activated T7-PKC kinase activity
detected in line 215 was approximately 19-fold greater than that
observed in the line 224 mice. Line 206 displayed a low constitutive
T7-PKC activity but exhibited no response to the presence of PS and
TPA (Fig. 1D)
.
The pattern of expression of T7-PKC in different tissues was also
examined. Organs from a line 215 transgenic T7-PKC mouse were
isolated, and Triton X-100-soluble extracts were made. Immunoblotting
these extracts with the anti-T7 antibody demonstrated T7-PKC
expression in the thymus and trachea in addition to the skin (Fig. 1E)
.
Epidermal Expression of T7-PKC.
The expression pattern of the T7-PKC transgene was examined in the
skin of T7-PKC mice. Formalin-fixed dorsal skin samples were taken
from a wild-type mouse and a line 215 T7-PKC mouse. The tissues were
hybridized with a polyclonal rabbit anti-PKC antibody. The wild-type
dorsal skin sample exhibited light immunoreactivity to the anti-PKC
antibody throughout the epidermis. Light and infrequent nuclear
staining was observed (Fig. 2A)
. Staining with the anti-PKC antibody was more intense in the dorsal
skin of the transgenic T7-PKC mouse than their wild-type
littermates. PKC exhibited strong staining in the basal cells of the
epidermis and was focally present in the suprabasal layers (Fig. 2B)
.
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may cross-talk to other PKC
isoforms by modulating their levels and their associated signals
(20
, 21)
. In this experiment (Fig. 3)
, we determined the levels of other PKC isoforms in the epidermis of
T7-PKC mice to examine whether the elevated PKC levels in the
epidermis altered the level of expression of other PKC isoforms. A
modest increase in the levels of PKC, ßII, and immunoreactive
proteins in the T7-PKC mice was observed (Fig. 3)
. There were no
observed changes in the immunoreactive protein levels of the other PKC
isoforms examined (Fig. 3)
.
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Mice. mice exhibited several phenotypic alterations.
The line 215 mice exhibited the greatest penetrance, with all of the
transgenic mice exhibiting phenotypic alterations. The mice were
phenotypically normal at birth. The phenotypic alterations began around
4–5 months of age for the F1 and F2 mice with
a mild hyperkeratosis in the tail epidermis (12 of 26 mice) around 4
months that did not persist. This was accompanied by persistent
inflammation at the base of the tail in 26 of 26 (100%) mice and
inflammation of the ears in 20 of 26 (77%) mice starting about 4–5
months of age. The inflammation of the tail base and ears was followed
by the formation of ulcerative lesions at these sites around 7–8
months of age in 17 of 26 (65%) transgenic mice observed (Fig. 4, A and B)
. Mild, transient inflammation around the eyes was
also observed in 7 of 26 mice (27%) in line 215 T7-PKC with a
variable age of onset. Although not observed in the F1
mice, ulceration in the proximal dorsal skin that became evident at
5–6 months of age occurred in 7 of 20 (35%) of the F2
transgenic mice. Line 224 and 206 exhibited phenotypic abnormalities
similar to that observed in line 215 mice. However, the severity of the
abnormalities was greatly diminished.
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mice were examined histologically. These
samples were fixed in formalin, sectioned, and stained with H&E. These
mice consistently exhibited hyperkeratosis in the dorsal skin, but with
no other apparent abnormalities (Fig. 4D)
. The grossly
affected skin regions displayed several significant alterations.
Hyperplasia, characterized by acanthosis, was found in the affected
regions along with cellular hypertrophy. Focal regions of necrosis were
also evident at sites of ulceration. Mixed inflammatory cell
infiltrates were observed in the dermal layer with infiltration into
the epidermis and keratinizing layers in affected regions. Prominent
amongst the inflammatory cell infiltrates were neutrophils and mast
cells (Fig. 4F)
.
Tumor Promotion.
The effect of PKC overexpression on mouse skin carcinogenesis in
line 215 T7-PKC mice was determined. The mice were initiated by
applying 100 nmol of DMBA to the skin in the acetone. Two weeks after
initiation, the mice were promoted twice weekly with 5 nmol of TPA in
acetone. Control mice were treated with acetone only. At the beginning
of the experiment, the 8–10-week-old mice exhibited no phenotypic
abnormalities. Treatment with DMBA and TPA elicited an average of 20
papillomas/mouse in both the wild-type females and males (Fig. 5,A and B)
. However, the T7-PKC mice averaged
less than 1 papilloma/mouse for both male and female mice after
treatment with DMBA and TPA. This was an average 95% reduction in
papilloma burden for female and male T7-PKC mice. The papillomas
that did form in any of the T7-PKC mice were small, usually less
than 2 mm in diameter (Fig. 5, A and B)
. Both,
T7-PKC and wild-type mice exhibited no differences in weight gain
during the course of the experiment. The total survival for the
wild-type mice was 92% and 88% for the T7-PKC mice at the end of
tumor promotion.
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mice developed
carcinomas independently of papilloma development (Fig. 5, C and D
; Table 1
). After 22 weeks of tumor promotion, 27% of the female and 50% of the
male T7-PKC mice treated with DMBA and TPA developed carcinomas in
the absence of prior formation of papillomas. Wild-type mice treated
with DMBA and TPA developed carcinomas by this time (30% and 15% for
females and males, respectively); however, all of these carcinomas
developed from existing papillomas (Table 1)
. Additionally, 25% of the
female T7-PKC treated with DMBA alone developed carcinomas by the
twenty-first week of promotion (Table 1)
.
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kinase activity present in papillomas and
carcinomas that developed on the T7-PKC mice was measured to
determine whether modulation of the T7-PKC kinase activity was
necessary for the formation of these lesions. The epidermis from
uninvolved skin was removed from three T7-PKC mice from each
treatment group in the tumor promotion experiment, combined, and
extracted with IP lysis buffer. Papillomas or carcinomas were also
excised and extracted with IP lysis buffer. The levels of precipitable
T7-PKC kinase activity in the epidermal extracts were very similar
between each of the treatment groups at 72 and 120 h after the
last treatment (Table 2)
. Both the basal (without PS/TPA) and the stimulated (with PS/TPA)
T7-PKC kinase activity extracted from papillomas or carcinomas of
DMBA and TPA-treated mice were greatly reduced compared to the levels
found in the surrounding epidermis (Table 2)
. This reduction in kinase
activity positively correlated with the reduced level of T7-PKC
protein present in the total epidermal extracts used for the
immunoprecipitations (Fig. 6)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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into the mouse
epidermis led to inflammation of the tail base and ears around 4–5
months of age sometimes followed by ulceration of the tail base at
around 7 months of age (Fig. 4)
. Histological examination of the
affected mouse tail base revealed hyperplasia and inflammatory cell
infiltration. Histological examination of the grossly unaffected dorsal
skin from these mice revealed hyperkeratosis, but no apparent
hyperplasia. This hyperkeratosis without apparent hyperplasia was also
observed in K10-c-Ha-ras transgenic mice (22)
.
Dorsal epidermis from mice exhibiting inflammation and ulceration also
displayed hyperplasia and inflammatory cell infiltrates. The majority
of infiltrates appeared to be neutrophils and mast cells.
This delayed manifestation of phenotypic alterations is different from
the time frame observed with the introduction of other transgenes using
the human K14 promoter (23
, 24)
. Introduction of TGF-
into the mouse epidermis driven by the K14 promoter led to hyperplasia
of the suprabasal epidermal cells and hyperkeratosis in mice around
3–5 weeks of age. This phenotype disappeared in the TGF- mice as
they became adults. This loss of phenotype occurred despite maintenance
of transgene expression (23)
. Expression of an activated
erB-2 receptor from the K14 promoter elicited severe phenotypic
alterations in newborn mice. Most of the founder mice died immediately
after birth, with the survivors exhibiting hyperplastic and dysplastic
epidermal cell growth and papilloma formation (24)
.
Several transgenic mouse lines have demonstrated delayed development of
phenotypic alterations. For example, epidermal thickening in the ear,
tail, footpads, rectum, foreskin, scrotum, and vagina was observed in
adult TGF- mice (23)
. Furthermore, mice expressing the
complete human papilloma virus type-16 genome from the K14 promoter
exhibited progressive skin abnormalities with full expression of the
phenotype around 5 months of age (25)
. Mice expressing an
activated c-Ha-ras gene from the K10 promoter or
v-fos from the human K1 promoter also exhibited a delayed
appearance of phenotypic abnormalities (22
, 26)
. The focal
nature of the alterations in the T7-PKC mice has also been observed
with other transgenes directed to the epidermis. For instance, the
TGF- mice exhibited focal epidermal thickening in adult mice
(23)
. Additionally, mice expressing the activated
c-Ha-ras from the K10 promoter (K10 c-Ha-ras)
developed papillomas at base of the tail, behind the ears, and on the
footpads (22)
. Expression of the complete human papilloma
virus type-16 genome from the K14 promoter resulted in mice with ear
and face hyperplasia, dysplasia, and papillomatosis with complete
penetrance (25)
. The delayed and focal nature of the
phenotypic alterations in the T7-PKC mice suggests that secondary
events need to occur in these regions to allow manifestation of this
phenotype. The areas affected by expression of the T7-PKC transgene,
i.e., the base of the tail and the ears, are sites that are
prone to irritation. Irritation by scratching or other contact was also
suggested to be the cause of the focal, late-onset skin alterations
observed in K10-c-Ha-ras and K14-TGF- transgenic mice
(22
, 23)
.
Transgenesis in the mouse epidermis has demonstrated that several genes
are important in epidermal growth regulation. Introduction of elevated
levels of TGF-, activated erB-2, activated c-Ha-ras,
v-fos, or T7-PKC led to papilloma formation associated
with hyperplasia and/or hyperkeratosis, usually at sites of mechanical
irritation (22
, 23
, 26
, 27)
. Overexpression of
keratinocyte growth factor also led to a thickened epidermis, but no
papilloma formation (27)
. Several of these genes have been
shown to be important in the regulation of cell growth in culture and
interact along the same signal transduction pathways. PKC can
transform fibroblasts and epithelial cells, and this correlates with
its ability to activate the c-Ras/Raf1/mitogen-activated protein kinase
signaling pathways (28, 29, 30)
. Additionally, in these same
cells, a dominant negative Ras failed to inhibit transformation by
PKC (28
, 30)
. Thus, PKC may be acting downstream of
the recruitment of Raf-1 to the membrane by Ras. In Rat-6 fibroblasts,
elevation of PKC decreased c-fos mRNA levels, suggesting
that PKC may be important in c-fos regulation
(31)
. Additionally, activated Ras can induce TGF-
production in keratinocytes (32)
. Thus, the similarity of
the phenotypes observed by overexpression of these genes in the mouse
epidermis may be due to the fact that they affect similar signal
transduction pathways.
Overexpression of PKC in the epidermis had an unexpected effect on
mouse skin tumor promotion by TPA. In vitro models of
cellular transformation indicated that PKC was a potent transforming
protein when overexpressed in both fibroblasts and epithelial cells.
PKC alone induced complete transformation of these cells, allowing
tumor formation when these cells were injected s.c. in athymic mice
(6, 7, 8)
. This suggested that PKC would act to enhance
the effects of tumor promotion. However, we observed a dramatic
reduction in the papilloma burden when the line 215 T7-PKC mice were
initiated with 100 nmol of DMBA and promoted with 5 nmol of TPA.
Treatment of the mice with DMBA or TPA alone led to a few, small
papillomas in T7-PKC mice, but the papilloma burdens were less than
1 papilloma/mouse. In papillomas obtained from T7-PKC mice 72 or
120 h after the last TPA treatment, the level of T7-PKC
activity and protein was greatly reduced compared to the uninvolved
epidermis. Thus, it appears that elevated levels of PKC activity may
inhibit papilloma development, and this increased activity may need to
be reduced for papillomas to develop. The low levels of immunoreactive
PKC in papillomas from wild-type mice further indicate that a
reduction in PKC levels is important for papilloma formation during
tumor promotion.
The reasons for this paradoxical effect of PKC on mouse skin
tumor promotion are unclear. Other proteins that have been termed
oncogenes based on in vitro studies have also demonstrated
unexpected results when tested in vivo. Homozygous deletion
of c-fos did not affect the development of papillomas in
skin tumor promotion experiments, but it did block the progression of
papillomas to carcinomas (33)
. Furthermore, E2F-1 null
mice exhibited increased tumor incidences and defects in thymocyte
development associated with alterations in apoptosis leading to
hyperproliferation (34
, 35)
. Thus, PKC may be able to
stimulate or inhibit cell growth depending on the cellular context.
Surprisingly, in the absence of prior papilloma growth, the T7-PKC
mice started developing carcinomas between 11 and 12 weeks of tumor
promotion in DMBA and TPA-treated mice. The appearance of the T7-PKC
mice resembled mice in a complete carcinogenesis experiment
(36)
. Additionally, a few of the T7-PKC mice initiated
with DMBA and promoted with acetone developed carcinomas.
The reduction in the T7-PKC levels in the carcinomas indicates that
elevated levels of PKC are not necessary for the maintenance of the
carcinoma. However, the positive correlation between elevated levels of
PKC and carcinoma formation indicates that PKC can induce the
molecular changes necessary for carcinoma formation after treatment of
the skin with a single dose of DMBA, alone or in conjunction with
repeated TPA treatments.
Several proteins appear to have important roles in carcinoma
development, some of which appear to be influenced by PKC. As described
above, homozygous deletion of c-fos prevents the progression
of papillomas to carcinomas (33)
. Additionally,
v-fos cooperates with v-ras to induce primary
mouse keratinocytes to form squamous cell carcinomas in nude mice
(37)
. Connections between c-Fos and PKC have been
previously identified. PKC can induce the activity of activator
protein 1 response elements in reporter gene studies (38
, 39)
. PKC also appears to be a part of the signaling cascade
from c-Ha-ras that leads to activation of the
c-fos promoter (20)
. Thus, the interplay
between c-fos and PKC may be important for the
development of carcinomas in the T7-PKC mice. Regulation of p53 also
appears to be critical for development of carcinomas in mouse skin.
Mutations at the p53 locus have been identified in mouse skin
carcinomas, but rarely in papillomas (40
, 41)
.
Furthermore, homozygous deletions of p53 in knockout mice did not
enhance papilloma development by the DMBA/TPA tumor promotion protocol
but did lead to enhanced conversion to squamous cell carcinomas
(42)
. Although the interaction specifically between PKC
and p53 has not been examined, PKC does appear to play a role in the
regulation of p53. PKC can phosphorylate p53 in vitro at
sites that are phosphorylated in vivo, and this may regulate
p53 DNA binding (43
, 44)
. TPA-induced growth arrest in
transformed rat fibroblasts positively correlated with enhanced p53 DNA
binding and required active, wild-type p53 (43)
.
Therefore, p53 may be an important downstream target for PKC-induced
carcinoma development. The proteins TGF-ß1 and TGF-ß2 appear to be
important in suppressing malignant progression. Papillomas that
progress with a high frequency to carcinomas do not express either
TGF-ß1 or TGF-ß2. Squamous cell carcinomas do not exhibit any
TGF-ß1 or TGF-ß2 expression, although these proteins are expressed
in normal mouse skin (45)
. Additionally, keratinocytes
with the TGF-ß1 gene deleted can cooperate with v-Ha-ras
to rapidly form squamous cell carcinomas in athymic mice
(46)
. Treatment of primary mouse keratinocytes with TPA
elevates the level of TGF-ß2 expression (32)
.
Additionally, rat fibroblasts transformed with PKC exhibited
elevated levels of TGF-ß2 and TGF-ß3 (47)
. Thus,
regulation of TGF-ß expression by PKC may be important for
carcinoma development in T7-PKC mice.
In summary, PKC appears to be an essential enzyme for the genesis of
mouse skin cancer. Elevation of PKC levels in the epidermis may
disrupt cellular homeostasis and may push the cells to a hyperplastic,
undifferentiated state. The delayed and focal appearance of
abnormalities suggests that a secondary stimulus, such as mechanical
irritation, may be necessary to elicit the phenotypic changes. Because
PKCs are regulated at multiple levels (presence of agonist,
phosphorylation, and cellular localization), the need for a secondary
stimulus is predictable. The counterintuitive effects of PKC
overexpression on tumor promotion demonstrate the importance of
in vivo testing of the oncogenic capacity of genes proposed
to be transforming based largely on cell culture studies. These mice
will be useful for examining the mechanisms of tumor suppression and
the development of carcinomas mediated by PKC in the mouse skin
carcinogenesis model and identifying PKC effectors important for
these responses.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by NIH Grant CA 35368. 
2 To whom requests for reprints should be
addressed, at Department of Human Oncology, K4/532 Clinical Sciences
Center, 600 Highland Avenue, Madison, WI 53792. Phone: (608) 263-9136;
Fax: (608) 262-6654.
3 The abbreviations used are: PKC, protein kinase
C; PS, phosphatidylserine; TPA,
12-O-tetradecanoylphorbol-13-acetate; DMBA,
7,12-dimethylbenz[a]anthracene; FVB/N,FVB/NTacfBR; IP,
immunoprecipitation; K14, Keratin 14; TGF, transforming growth factor;
EGFR, epidermal growth factor receptor.
Received 6/30/99. Accepted 12/ 2/99.
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REFERENCES |
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) or male (
) was
extracted in IP lysis buffer, and a 100 µg aliquot of protein from
cleared lysates were used for IP with the T7 antibody. The
immunoprecipitated T7-PKC
), DMBA and acetone (
), DMBA and acetone (
). The
error bars indicate the SE of the papilloma burden for each
data point. By the Wilcoxan rank-sum test, the papilloma burden was
significantly (P < 0.001) different after 10
weeks of promotion between the DMBA-TPA-treated wild-type and T7-PKC