From the National Center for Toxicogenomics and the
¶ Laboratory of Signal Transduction, NIEHS, National
Institutes of Health,
Research Triangle Park, North Carolina 27709
Received for publication, June 25, 2002, and in revised form, October 25, 2002
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ABSTRACT |
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This study identifies genes expressed early in
12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin
carcinogenesis in genetically initiated Tg·AC v-Ha-ras
transgenic mice. Keratinocyte progenitor cells from TPA-treated Tg·AC
mice were isolated with fluorescence-activated cell sorting and
expression was analyzed using cDNA microarray technology. Eleven
genes were identified whose expression changed significantly in
response to carcinogen treatment. Deleted in split hand/split
foot 1 (Dss1) is a gene associated with a
heterogeneous limb developmental disorder called split hand/split foot
malformation. cDNA microarray expression analysis showed that the
mouse homologue of Dss1 is induced by TPA. Dss1
overexpression was detected by Northern blot analysis in early
TPA-treated hyperplastic skins and in JB6 Cl 41-5a epidermal cells.
Interestingly, Dss1 expression was also shown to be
elevated in skin papillomas relative to normal skins, and further
increased in squamous cell malignancies. Functional studies by
ectopically constitutive expression of Dss1 in JB6 Cl 41-5a
preneoplastic cells strongly increased focus formation and
proliferation of these cells and enhanced efficiency of neoplastic transformation of the cells in soft agar. These results strongly suggest that Dss1 is a TPA-inducible gene that may play an
important role in the early stages of skin carcinogenesis.
Skin carcinogenesis is a complex multistage process that
progresses through distinct stages of initiation, promotion,
progression, and malignancy (1-3). The Tg·AC mouse is a genetically
modified (transgenic) form of the FVB/N mouse strain that
carries a genomic copy of the v-Ha-ras gene fused to a fetal
The epidermis is a stratified, rapidly renewing tissue in which
terminally differentiated cells are continuously lost from the skin
surface and replaced by an intricate and highly regulated proliferative
process. Skin cells are regenerated through the proliferative capacity
of keratinocyte stem cells (KSCs) and transit amplifying (TA) cells in
the basal layer. KSCs are a minor subpopulation of relatively quiescent
cells that have broad proliferative potential and an unlimited capacity
for self-renewal (8, 9). It has been proposed that carcinogens generate
mutations in the population of stem cells which are transformed them
into initiated preneoplastic cells (10). It has also been reported that
tumor promoters such as TPA preferentially stimulate initiated
keratinocytes and lead to clonal expansion of the mutant cell
population (11). Recent evidence supports the proposal that KSCs are a
major target in skin tumorigenesis (12-15), but the molecular
mechanism(s) have not been determined.
KSCs can be isolated from heterogeneous tissue samples and used for
investigations of the mechanisms of epidermal tissue homeostasis, wound
repair, and for studying the role of stem cells in skin carcinogenesis.
However, it is difficult to obtain KSCs because there are few reliable
and specific molecular markers that discriminate KSCs from TA cells,
which have more restricted proliferative potential within the
germinative/basal layer. Recently, Li et al. (16) identified
and characterized a candidate marker for human KSCs, integrin
Previous studies identified ~30 TPA-inducible genes that play
important roles in skin tumor formation and metastasis. These genes
include transin (19), c-myc and c-fos
(20), mal1 (21), CD44 (22), urokinase
plasminogen activator (23), MMP-9 (24), and
serine protease BSSP (25). Many of these genes were
identified using in vitro methods and cultured cell lines or
two-step in vivo carcinogenesis with mouse skin as a target.
These studies should be interpreted with some caution because the exact
nature of the target cells may not be known with precision. This study identifies a novel gene that is induced in TPA-treated cells using a
different approach than previous studies. The candidate integrin Dss1 was originally identified on human chromosome
7q21.3-q22.1 as a gene deleted in patients with the heterogeneous limb developmental disorder SHFM1. Dss1 encodes a 70-amino acid,
highly acidic peptide (26). Up-regulation of Dss1 was
detected in TPA-treated mice using cDNA microarray and verified by
semiquantitative RT-PCR, Northern blot, and in situ
hybridization. Functional analysis of Dss1 gene in TPA
susceptible JB6 Cl 41-5a preneoplastic epidermal cells suggests that it
is required for cell proliferation and neoplastic transformation.
Animals and Cell Culture
Eight- to 10-week-old male homozygous Tg·AC mice were obtained
from Taconic Laboratory of Animals and Services (Germantown, NY).
Animal studies were carried out in compliance with NIH Guidelines for
Humane Care and Use of Laboratory Animals. TPA-susceptible JB6 Cl 41-5a
and TPA-resistant JB6 Cl 30-7b BALB/c mouse epidermal cell clonal
variants, generated by Nancy Colburn et al. (27), were from
American Type Culture Collections (Manassas, VA) and grown at 37 °C
in a 5% CO2 atmosphere in Eagle's minimal essential medium (Eagle's MEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS) containing 2 mM glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin sulfate (Invitrogen).
Mouse fibroblast cells Rat-1 and monkey transformed kidney cells COS-1
were cultured in Dulbecco's modified Eagle's medium
(Dulbecco's MEM) containing 10% FBS. NIH/3T3 cells were maintained as
described previously (28). Cell lines used in this study were free of
mycoplasma infection.
Topical Treatment with TPA
Five micrograms of TPA (Sigma) in 200 µl of acetone was
applied topically to groups of 5 homozygous male Tg·AC mice twice weekly for 2 weeks. Untreated control mice were sacrificed on day 1 (NS). Four dosing protocols were used as follows. Mice were dosed on
day 1 and sacrificed on day 5 (designated as TPA1); mice were dosed on
days 1 and 5 and sacrificed on day 8 (designated as TPA2); mice were
dosed on days 1, 5, and 8 and sacrificed on day 12 (designated as
TPA3); mice were dosed on days 1, 5, 8, and 12 and sacrificed at least
48 h after the last dose (designated as TPA4). Papillomas and
malignant tumors (one spindle cell tumor and two squamous cell
carcinomas) were identified, removed, and characterized as described
previously (7).
Gene Expression Profiling
AtlasTM Mouse 1.2 MicroArray carrying 1176 cDNAs
was obtained from Clontech (Palo Alto, CA). The
keratinocytes were harvested from the dorsal skin of Tg·AC mice (29),
and the integrin Vector Constructions
The full-length Dss1 cDNA was amplified by RT-PCR
using Tg·AC mice skin total RNA. The Dss1 forward and
reverse primers were 5'-CAC CAT GTC TGA AAA GAA GCA GCC-3' and 5'-TGA
TGT CTC CAT CTT GTA GCC GTG CTT-3', respectively. The PCR amplified
Dss1 cDNA was cloned into V5-His-tagged
pcDNA3.1D/V5-His-TOPOc mammalian expression vector
(Invitrogen, Carlsbad, CA). Dss1 cDNA fragment
was inserted into retroviral vector by digesting pcDNA3.1D/Dss1-V5-His plasmid with HindIII and
NotI and ligated into the HindIII-NotI
sites of the pLNCX2 (Clontech) using
the LigaFastTM Rapid DNA Ligation System (Promega, San
Luis, CA). A construct expressing Dss1 sense or antisense
RNA was generated as follows; pcDNA3.1D/Dss1-V5-His was digested
with BamHI and XbaI, and the Dss1
cDNA-containing fragment was cloned into the
BamHI-XbaI sites of the T3/T7-U19 plasmid
(Ambion, Austin, TX). The pEGFP-C3 vector (Clontech) was used to express the full-length Dss1
protein fused to the C terminus of the enhanced green fluorescent
protein (EGFP) in the JB6 Cl 41-5a epidermal cells. The
pcDNA3.1D/Dss1-V5-His plasmid was digested sequentially with
KpnI and ApaI and ligated into the
KpnI-ApaI sites of the pEGFP-C3 plasmid using the
LigaFastTM Rapid DNA Ligation System (Promega). The
pEGFP-C3 plasmid expressing the native EGFP protein was used as a
control. All the construct sequences were verified using an automated
Applied Biosystems sequencer and the BigDyeTM Terminator
Kit (PerkinElmer Life Sciences, Foster City, CA). Plasmid DNAs were
purified using purification kits from Qiagen (Stanford Valencia, CA)
and were endotoxin-free when used for transfection in mammalian cells.
Cell Transduction
Cells were transfected with vector (mock),
pcDNA3.1D/Dss1-V5-His, or pEGFP-C3/Dss1 plasmid DNA using
LipofectAMINE PLUSTM reagents (Invitrogen) or by infection
with virions packaged with ecotropic packaging cells
RetroPackTM PT-67 (Clontech). The
recombinant pLNCX2 retroviral vector carries mouse
Dss1 gene driven by the cytomegalovirus promoter and
neoR gene driven by the long terminal repeat
promoter. Plasmid-transfected and virus-infected cells were cultured
for at least 2 weeks in medium containing 400 µg/ml Geneticin (G418)
(Invitrogen). Cells were analyzed by Western blot or RT-PCR to confirm
the expression of Dss1.
RT-PCR Analysis
The single-stranded cDNA was prepared from total RNA using
the Moloney murine leukemia virus reverse transcriptase SuperScript II
(Invitrogen) with oligo(dT) primer and used as a template for PCR. The
primers used for PCR were as follows: Dss1 sense (5'-GAC GAC
GAG TTC GAG GAG TTT CCC G-3') and antisense (5'-GTG GTA GAG TCC ATG ATT
GAG GTT CC-3') are from Gensetoligos Corp. (La Jolla, CA);
pLNCX2 sequencing/PCR forward (2882-2906) (5'-AGC TGG TTT AGT GAA CCG TCA GAT C-3') and reverse primers (3057-3032) (5'-ACC TAC
AGG TGG GGT CTT TCA TTC CC-3') are from Clontech;
Northern Blot Analysis
Total RNA was prepared using a TRIzol reagent kit (Invitrogen)
and digested with RNase-free DNase 1 (Ambion). Eight micrograms of
isolated RNAs were separated electrophoretically on a 1% agarose gel
containing glyoxal and transferred onto a BrightStar-Plus nylon
membrane (Ambion). The membrane was UV-cross-linked and probed with
[ In Situ Hybridization
The in situ hybridization assay was performed as
previously described (30). Briefly, the cutaneous tumors were removed
from Tg·AC mice and fixed overnight in 10% neutral buffered
formalin. The tissues were paraffin-embedded, and sections (6 µm)
were cut onto SuperFrost plus microscope slides (Daigger, Vernon Hills, IL). The sections were deparaffinized and rehydrated by successive washes in xylene and graded alcohols to 2× SSC, then applied with ~2 × 106 cpm of 35S-labeled
Dss1 sense or antisense riboprobes. The riboprobes were prepared from T7/T3-U19-Dss1 plasmid linearized with EcoRI
(antisense) or HindIII (sense) using in vitro
Strip-EZTM T7 or T3 RNA transcription kit (Ambion).
Following 40 °C overnight hybridization, the tissues were washed in
2× SSC plus 50% formamide at 40 °C, then in 2× SSC, 1× SSC,
0.5× SSC, and 0.5× SSC, 30 min each wash at room temperature. To
remove unbound probe, the tissues were incubated with 20 µl of RNase
(10 mg/ml). After several washes, the slides were dehydrated in graded
alcohols and completely air-dried. The slides were then dipped into
NTB-3 autoradiographic emulsion (Eastman Kodak), exposed for 10 days at
room temperature in the dark, dried in a light-tight container, and
developed in Kodak D19 fixer and developer. The sections were
counterstained with hematoxylin, covered with coverslips, and
photographed under dark-field illumination (model BX51, Olympus Optical
Co., Tokyo, Japan).
Subcellular Localization
The eighty nanograms of different green fluorescent protein
(GFP) constructs, pEGFP-C3 and pEGFP-C3/Dss1, were transiently transfected into the JB6 Cl 41-5a cells cultured at 37 °C in
eight-well culture slides (Falcon, Bedford, MA) at a cell density of
2 × 105 cells/well using LipofectAMINE
PLUSTM reagents (Invitrogen) according to the protocol of
the manufacturer. The GFP fluorescence was observed 48 h after
transfection. Cells were washed twice with ice-cold 1×
phosphate-buffered saline buffer (150 mM NaCl, 10 mM Na2HPO4, 10 mM
KH2PO4, pH 7.4) and fixed in 2%
paraformaldehyde in 1× phosphate-buffered saline for 10 min at room
temperature. After washing five times for 2 min each, cells were
mounted with the Prolong antifade medium (Molecular Probes, Eugene,
OR). For nuclear localization, a DNA-bound nucleic dye
4,6-diamidino-2-phenylindole (DAPI) (Vector, Burlingame, CA) was used.
Cells were observed by fluorescence microscopy using a Leica DMRBE
microscope (Wetzlar GmbH) equipped with a 63× objective and 100-watt
mercury source. The images were taken with a Chroma GFP filter set for
EGFP (excitation maximum 488 nm, emission maximum 507 nm), a DAPI
filter set for chromatin (excitation 351/364 nm, emission 410/505 nm),
a SPOT RT cooled charge-coupled device (CCD) camera (Diagnostic
Instruments, Inc., Sterling Heights, MI), and MetaMorph 5.0 software
(Universal Imaging Corp., Downingtown, PA). Individual images were
pseudocolored and overlaid.
For immunocytochemical analysis, cells (1 × 106) were
transiently transfected with 4 µg of pcDNA3.1D/Dss1-V5-His in a
10-cm tissue culture plate using LipofectAMINE PLUSTM
reagents (Invitrogen). Cells were incubated for 48 h, and 2 × 105 cells were seeded on eight-well culture slides until
cells attached. Cells were fixed with methanol for 10 min, probed with
normal mouse IgG (negative control) or anti-V5 tag mouse monoclonal
antibody for 30 min, and stained with the M.O.M. Immunodetection Kit
(Vector, Burlingame, CA) according to instructions from the
manufacturer. The photographs were taken under the BX51 light
microscope (Olympus Optical Co., Tokyo, Japan).
Western Blot Analysis
Cells were lysed as described previously (32). Briefly, cells
were lysed in lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 4 mM EDTA, 2 mM EGTA, 10 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride
(Sigma). The whole-cell lysates were sonicated on ice and centrifuged
at 100,000 × g for 1 h at 4 °C. Protein
concentration was determined by Bradford assay (Bio-Rad). Proteins were
separated electrophoretically on 10 or 12% SDS-polyacrylamide gels and
transferred onto polyvinylidene difluoride membranes (Amersham
Biosciences). The membranes were probed with primary antibodies
including anti-V5 mouse monoclonal antibody (1:5000) (Invitrogen) and
anti-EGFP rabbit polyclonal antibody (1:2000)
(Clontech). These antibodies were detected using
horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG
(1:3000) (Amersham Biosciences) and enhanced chemiluminescence (ECL)
(Amersham Biosciences). The membranes were striped and rehybridized
with an anti- Transformation Assays
Focus-forming Activity--
Cells were seeded overnight at a
density of 2 × 105 cells/well in six-well plates.
Cells were transfected with 1 µg of vector or
pcDNA3.1D/Dss1-V5-His and selected in medium containing G418 selection for 14-21 days. Foci were fixed with methanol/acetic acid
(v/v = 1/3), stained with 0.4% crystal violet (methanol/acetic acid), and counted as described previously (33).
Characterization of Cell Growth--
Growth curves were
generated as described previously (33). In brief, cells (1 × 104) were grown as described above. The medium was changed
every 3-4 days. Cell number was counted in triplicate on a
hemocytometer every other day for 8 days.
Anchorage-independent Growth Assay--
Colony formation in soft
agar was assayed as described previously (33). In a 60-mm tissue
culture dish, 1 × 104 cells were resuspended in
0.33% Noble agar (Difco, Kansas City, MO) in Eagle's MEM with 10%
FBS and layered over 5 ml 0.5% agar in Eagle's MEM with 10% FBS.
Cells were grown at 37 °C in a 5% CO2 atmosphere, and
colonies with more than 8 cells were counted 14-18 days after seeding.
In Vivo Gene Expression Profiles in Keratinocyte Progenitor Cells
of TPA-treated Tg·AC Mice--
The goal of this study was to
identify novel genes induced in the skin by TPA using an in
vivo mouse model system. Gene expression profiles were determined
using a mouse cDNA array spotted with 1176 genes. Tg·AC mice were
treated with TPA, and keratinocyte progenitor cells carrying the cell
surface markers, integrin Dss1 Is a TPA-responsive Gene Induced Early in Skin
Tumorigenesis--
Previous studies demonstrated that chronic topical
application of TPA to the skin of Tg·AC mice induces epidermal
hyperplasia (30). Dss1 expression was analyzed in
hyperplastic skin in Tg·AC mice exposed to various doses of TPA, as
described under "Materials and Methods." Dss1 expression
increased in hyperplastic skin in a dose- and
time-dependent manner (Fig.
2A). Similar results were obtained previously for TPA-induced expression of PCNA (30).
The dose response and kinetics of TPA-induced transcriptional
activation of Dss1 were investigated using JB6 Cl 41-5a
preneoplastic epidermal cells. Cells were grown in 5% FBS/Eagle's MEM
containing 0, 0.1, 1.0, 10, and 100 ng/ml TPA; viable cells were
harvested at 18 h, and total RNA was prepared for Northern blot
analysis. Fig. 2B (a) shows that TPA induced
Dss1 1.7-fold at 1.0 ng/ml, and maximal induction
(~2-fold) was reached at 10-100 ng/ml TPA. A kinetic analysis at 0, 1, 2, 4, 8, 12, 18, 24, and 36 h after treatment with 10 ng/ml TPA
showed that Dss1 was induced 2.5-fold 1 h after TPA
treatment and reached a maximal level of 3-5-fold 12-18 h after
treatment. Dss1 expression appeared to decrease slightly
8 h after treatment and began to decline from the maximal level
18 h after treatment (Fig. 2B, b).
Tissue Distribution of Dss1--
Tissue distribution of
Dss1 mRNA was examined in Tg·AC adult mice using
Northern blot analysis. Dss1 mRNA was transcribed in
adult mouse tissues including heart, ovary, stomach, and skin. Dss1 was expressed at a higher level in heart than other
tissues. In kidney, liver, lung, and spleen, Dss1 expression
was barely detectable and Dss1 mRNA was not detected in
brain and small intestine (Fig. 3).
Similar results were observed in other strains of mice including BALB/c
and C57BL/6 (data not shown).
Subcellular Localization of Dss1--
Tagging expressed proteins
with the GFP from the jellyfish Aequorea victoria is a
highly specific and sensitive technique for studying the intracellular
dynamics of proteins and organelles (34, 35). We have constructed a
vector encoding an EGFP-Dss1 fusion protein to directly examine the
subcellular localization of Dss1 in epidermal cells. The plasmid DNA
pEGFP-C3 or pEGFP-C3/Dss1, which expressed EGFP-Dss1 fusion protein,
was transiently transfected into JB6 Cl 41-5a cells using the
LipofectAMINE DNA transfection method. The pEGFP-C3 plasmid containing
the cDNA encoding for EGFP alone was used as a control. After
48 h of transfection, cells were collected for preparation of the
whole-cell lysates. As shown in Fig.
4A, Western blot
analysis showed that EGFP-Dss1 fusion protein was efficiently expressed
in JB6 Cl 41-5a cells using an anti-EGFP rabbit polyclonal antibody,
when compared with mock EGFP control protein. Fig. 4B also
showed the photographs obtained by fluorescence microscope. EGFP-Dss1
fusion protein had a diffuse and uniform green fluorescent distribution
throughout the nucleus (I and IV), and was also
detected in cytoplasm (IV). Just after taking the EGFP-Dss1
images, a DNA-bound fluorescent dye DAPI was added and the nucleus was
stained into blue color (II and V). The light
blue areas (III and VI) were obtained upon merging of the green (I and IV) and blue
(II and V) images of identical cell. No cells
were observed and exhibited a plasma membrane localization of Dss1.
This distribution is similar to that seen in cells expressing GFP alone
(data not shown). A similar expression pattern and distribution was
also observed by immunocytochemical staining using anti-V5-tagged mouse
monoclonal antibody to detect the V5-Dss1 native fusion protein (Fig.
4C, II). The negative control was probed with
normal mouse IgG and showed the specificity of anti-V5-tagged mouse
monoclonal antibody in immunocytochemical analysis (Fig. 4C,
I).
Dss1 Overexpression in TPA-induced Skin Tumors--
TPA induced an
increase in Dss1 transcription level not only in in
vivo keratinocyte progenitor cells and in early hyperplastic mouse
skin, but also in in vitro JB6 Cl 41-5a cells.
Dss1 expression was also examined in TPA-induced skin
tumors. Interestingly, Dss1 RNA transcription was higher in
TPA-mediated Tg·AC mouse skin tumors, including eight papillomas
(2.5 ± 0.4-fold) and three malignant tumors (one spindle cell
tumor and two squamous cell carcinomas) (6.2 ± 1.3-fold) than in
normal skin (Fig. 5A). In addition, in situ hybridization assay was also employed to
detect the expression of the Dss1 messenger RNA in
TPA-induced skin tumors. As shown in Fig. 5B,
Dss1-specific signals were overexpressed and localized in
the squamous region of the papillomas (II) and malignancies
(squamous cell carcinomas) (V), with some expression in the
adjacent epidermis and hair follicles. However, normal-appearing skin
adjacent to the papillomas and malignancies did not contain detectable
Dss1 message (data not shown).
Dss1 Overexpression Enhances Neoplastic Transformation in
Preneoplastic Mouse Epidermal Cells--
Dss1 was
constitutively expressed in preneoplastic epidermal cells to determine
whether increased expression of Dss1 potentially stimulates
tumorigenesis in the skin. One plasmid construct,
pcDNA3.1D/Dss1-V5-His, was prepared to constitutively express
Dss1. Western blot analysis confirmed that this construct
expressed Dss1 efficiently in COS-1, JB6 Cl 30-7b, JB6 Cl
41-5a, Rat-1, and NIH/3T3 cells (Fig.
6A).
Constitutive expression of Ras family proteins and other oncogenic
proteins increase foci-forming capability and decrease growth contact
inhibition of normal untransformed cells (33). Fig. 6B shows
that constitutive expression of Dss1 increases foci formation ~2-fold in mouse epidermal cell lines including JB6 Cl
41-5a and JB6 Cl 30-7b; however, expression of Dss1 did not change the growth properties of NIH/3T3 cells. These results
demonstrate that Dss1 alters normal contact inhibition in
mouse epidermal cell lines, suggesting that Dss1 may have
some oncogenic properties.
Transformed cells have a growth advantage in monolayer
culture and acquire capacity for anchorage-independent growth (33). The
effects of Dss1 expression on these growth characteristics were measured in transfected JB6 Cl 41-5a, JB6 Cl 30-7b, and NIH/3T3 cells. Dss1-expressing and vector only control cells were
grown for 48 h and selected for 10 days with G418. Cells were
seeded at a density of 1 × 104 in 60-mm soft agar
plates to assay for anchorage-independent growth. The colony-formation
efficiency increased ~5- or 8-fold when Dss1 was expressed
in JB6 Cl 41-5a or JB6 Cl 30-7b cells, respectively; however, the
colony-forming efficiency did not increase in
Dss1-transfected NIH/3T3 cells (Fig. 6C). The
background colony formation was higher in JB6 Cl 41-5a cells than in
JB6 Cl 30-7b cells.
The role played by Dss1 in neoplastic transformation was
also tested using a retrovirally based method to express Dss1.
Dss1 and neoR were inserted into a
bicistronic construct using an ecotropic retroviral vector
pLNCX2 and transduced into JB6 Cl 41-5a cells. Cells were
selected for G418 resistance for 14 days, and one drug-resistant pooled
clone was identified (>100 colonies) (pLNCX2/Dss1-GR). Eight individual clones (designated as pLNCX2 and
pLNCX2/Dss1-C1
Constitutive Dss1 expression was also correlated with
increased growth rate. As shown in Fig. 7C, growth curves of
clones overexpressing Dss1 experienced an initial lag after
plating, but grew at a significantly faster rate than control cells by 2 days after seeding. The growth rate was also enhanced in COS-1, JB6
Cl 30-7b, and NIH/3T3 cells overexpressing Dss1 (data not shown). In addition, cells stably overexpressing Dss1 have
higher colony-forming efficiency than control cells. The colony-forming efficiency was 1.6-9-fold higher than control cells (Fig.
7D). These results indicate that cells that overexpressed
Dss1 develop in vitro characteristics of typical
of transformed cells.
This study uses a novel approach to identify Dss1 as a
potentially important gene in early skin tumorigenesis in mice. The cDNA was amplified using a PCR-based SMART technique, and the gene
expression profiles were generated using a mouse cDNA array membrane carrying 1176 genes. Expression was analyzed in keratinocyte progenitor cells from TPA-treated or control Tg·AC mice. Keratinocyte progenitor cells were isolated using FACS to select cells that express
the progenitor cell markers, integrin A previous report indicates that skin tumorigenesis may be initiated by
cellular transformation of KSCs (36). Elevation of p63 is homologous to p53 and plays a role in limb, craniofacial, and
epithelial development. In addition, p63 has been implicated in cell
regeneration and stem cell division (39-41). Heterozygous germ line
mutations in p63 cause ectrodactyly, ectodermal dysplasia, and facial
clefts syndrome (42). p63 is also associated with proliferative
potential in normal and neoplastic keratinocytes (43) as well as
recently identified as a marker for keratinocyte stem cells (44).
p63 and Dss1 are both involved in the autosomal dominant disease SHFM1 (26), which is a form of ectrodactyly characterized by deep median clefts, missing digits, and lobster claw-like appearance of the distal extremities (26, 45). Like p63, it
would be of more interests to know whether Dss1 has the same biological
functions with p63.
Our findings using immunocytochemical staining and
GFP-protein fusion fluorescence analysis reveal that Dss1 is
distributed in a uniform and diffuse pattern in the nucleus and is also
detected in the cytoplasm (Fig. 4). Cells that exhibit a nuclear
pattern of expression appear to be in a normal morphology. However, the cells that express EGFP in both the nuclear and cytoplasmic
compartments seem to have a slightly different morphology, suggesting
that they may be under stress or in an altered state of growth or
differentiation. In contrast, a previous study using MCF7 breast cancer
cells suggests that Dss1 is a nuclear protein that interacts directly
with the protein product of breast cancer susceptibility gene
Brca2 (46). It is possible that cytoplasmic Dss1 could be
activated and transported into the nucleus where it could interact with
nuclear proteins such as BRCA2. Further studies will be necessary
before the significance of this unique cellular distribution can be
fully understood.
A previous report indicated that Dss1 might be a transcription factor
expressed during embryogenesis in regions of rapid cell growth such as
limb bud, branchial arch, genital bud, and skin but not in regions of
cell differentiation like digital condensations (26). Thus, it is
possible that Dss1 promotes proliferation of these cells
during embryogenesis. A recent study has established a direct link
between BRCA2 and Dss1 using yeast two-hybrid systems, and also
recognized the important growth roles controlled by Dss1-like protein
in yeast. Loss of function of Dss1-like protein by deletion of
Dss1 in Schizosaccharomyces pombe resulted in a
defect in completion of cell division, eventually leading to an
accumulation of cells with greater than 2× DNA contents (46). We have
found that the elevated Dss1 expression in genetically modified JB6 Cl
41-5a individual stable clones, which were infected by
pLNCX2/Dss1 retroviral vector, produced by ecotropic
packaging cells RetroPackTM PT-67, and selected by G418,
significantly promoted cell proliferation under standard in
vitro tissue culture conditions (Fig. 7C). This result
was in good agreement with the levels of Dss1 transcription obtained in RNA Northern blot analysis (Fig. 7B). Similarly,
an enhancement in the rate of cell growth was also observed in COS-1, JB6 Cl 30-7b, and NIH/3T3 cells that were stably transfected with pcDNA3.1D/Dss1-V5-His plasmid DNA (data not shown). Conversely, we
also found the rate of cell growth to be selectively inhibited in
LipofectAMINE-pretreated Dss1-overexpressing Tg·AC 43 skin malignant tumor cells by addition to the culture medium of a specific antisense oligonucleotide to block Dss1 synthesis. This treatment consistently showed a decrease of proliferation rate from 100% down
to ~25-30%, suggesting that more than 70% of growth
inhibition was mediated by inactivation of Dss1-initiated cell growth
pathways.2 Cumulatively,
these results demonstrate that Dss1 play a crucial role in regulating
cell proliferation, although a Dss1 homologue SEM1 was also recently
implicated in the differentiation of Saccharomyces cerevisiae (47). It raises a possibility that Dss1 has
pleiotropic effects in a variety of cell types.
As seen in Fig. 6, Dss1 was able to successfully express in COS-1
cells, JB6 Cl 30-7b cells, JB6 Cl 41-5a cells, Rat-1, and NIH/3T3 (Fig.
6A). In addition, it appears to markedly increase focus-forming activity in epidermal cell lines (i.e. JB6 Cl
30-7b and JB6 Cl 41-5a) but not in fibroblast cell lines
(i.e. NIH/3T3) (Fig. 6B). More importantly,
overexpression of Dss1 increased colony-forming efficiency of JB6 Cl
41-5a and JB6 Cl 30-7b cells but not NIH/3T3 cells in soft agar (Fig.
6C). Thus, the ability of Dss1 to regulate
cellular transformation may be specific for epithelial cells. To
further confirm the functional roles played by Dss1 in enhancing
neoplastic transformation, retroviral vector pLNCX2/Dss1-infected JB6 Cl 41-5a individual stable clones
were employed. We showed that stable integration of Dss1
full-length cDNA into JB6 Cl 41-5a cells (Fig. 7A)
resulted in increased Dss1 mRNA levels (Fig.
7B) and acquisition of susceptibility to transformation in
soft agar (Fig. 7D). Thus, elevated Dss1
expression was sufficient to enhance the transformation activity in JB6
Cl 41-5a epidermal cells, consistent with the results as described
above. The transcriptional levels of Dss1 in one Dss1 pooled
stable clone (pLNCX2/Dss1-GR) and seven individual stable
clones (pLNCX2/Dss1-C1 Previous studies indicated that activator protein-1 (AP-1) is required
in an activated form for TPA-induced neoplastic transformation (27) and
inhibition of AP-1 by a c-Jun trans-activation domain deletion mutant
(TAM67) or AP-1 trans-repressing retinoids block TPA-induced cell
transformation in JB6 Cl 41-5a cells (48). Expression of TAM67 in
transgenic mice blocked TPA-induced AP-1 activity and papilloma
formation (49). Yang et al. (50) recently also indicated
that Pdcd4 is a novel transformation suppressor that inhibits AP-1
trans-activation. It is interesting to speculate that TPA, AP-1, and
Dss1 might coordinately regulate cell signaling and growth in
epithelial cells.
In summary, our studies provide the first functional evidence to
demonstrate that Dss1 may play a role in mediating
TPA-induced skin carcinogenesis and cellular transformation of
keratinocyte progenitor cells that are involved in this process.
Dss1 is induced in early hyperplastic skins and in
TPA-induced skin tumors. In addition, constitutive expression of
Dss1 in preneoplastic epidermal cells promotes cell growth
and enhances the process of neoplastic transformation in these cells.
Thus, Dss1 may be a useful biomarker of skin carcinogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-globin gene promoter (4). Tg·AC mice have already
entered the initiation stage of cancer development and have a higher
sensitivity to many types of environmentally inducible cancer than wild
type mice. Tg·AC mice develop hyperplasia in skin keratinocytes after
exposure to tumor promoters such as
TPA1 (4), full thickness
wounding (5), ultraviolet radiation (6), or carcinogens such as
7,12-dimethylbenz[a]anthracene (7). These hyperplasias
eventually develop into benign papillomas, some of which become
malignant tumors such as squamous cell carcinomas or spindle cell
tumors (7). The in vivo Tg·AC mouse model is a valuable
tool to study the early stages of skin carcinogenesis.
6bri 10G7dim. Tani et
al. (17) also successfully separated KSCs from TA cells using
in vivo cell kinetic analysis and FACS by enriching murine
dorsal KSCs for the cell surface marker integrin
6bri CD71dim. In addition,
Trempus et al. (18) in our laboratory have demonstrated that
the keratinocyte population expressing surface markers of integrin
6 and CD34, a hemopoietic stem and progenitor cell
marker, resides in the hair follicle bulge of mouse and human scalp,
and shows that follicular bulge cells are quiescent and highly
clonogenic, two hallmarks of stem cells.
6+ CD34+ keratinocyte progenitor
cells were isolated from hyperplastic skin of TPA-treated animals and
their gene expression analyzed using cDNA microarray. This method
employs FACS, switching mechanism at the 5' end of RNA templates
(SMART) cDNA amplification, and mouse cDNA array technology.
Eleven TPA-responsive genes were identified in the Tg·AC mouse; nine
genes were significantly up-regulated by TPA, and two genes were
remarkably down-regulated by TPA. Dss1 was selected
from the nine TPA-up-regulated genes for further characterization.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6+ CD34+
keratinocyte progenitor cells were isolated using FACS (18). Total RNAs
were extracted from TPA-treated or -untreated keratinocyte progenitor
cells using StrataPrepR Total RNA Miniprep Kit (Stratagene,
La Jolla, CA). Ten nanograms of total RNA was reverse-transcribed and
amplified using the Atlas SMARTTM system
(Clontech). Five hundred nanograms of purified
SMART cDNA were labeled for 30 min at 50 °C with
[
-33P]dATP (10 µCi/µl; >2500 Ci/mmol; Amersham
Biosciences) using Klenow DNA polymerase
(Clontech) and random hexamer priming. Array membrane was prehybridized with prewarmed ExpressHyb for 30 min with
continuous agitation at 68 °C, and hybridized overnight with [
-33P]dATP-labeled probes (3.7 × 106
cpm/ml). The membrane was washed four times in 2× saline sodium citrate (SSC), 1% SDS for 30 min at 68 °C, and two times in 0.1× SSC, 0.5% SDS. Array membrane was scanned with a phosphorimager (Typhoon 8600, Amersham Biosciences), and signals were quantified using
ImageQuant 5.1 software (Amersham Biosciences).
2-microglobulin sense (5'-GAC TGG TCT TTC TAT ATC CTG
G-3') and antisense (5'-CTT TCT GCG TGC ATA AAT TG-3') are from Sigma;
and
-actin sense and antisense primers were obtained from
Clontech. PCR cycling was as follows: denaturation
(94 °C, 45 s), annealing (58 °C, 45 s), and extension
(72 °C, 2 min) for 30 cycles. Reaction was carried out in a
PerkinElmer 9600 thermal cycler (PerkinElmer Life Sciences), and PCR
products were analyzed using 2% agarose gels.
-32P]UTP-labeled Dss1 antisense RNA
(1 × 106 cpm/ml). The riboprobe was prepared using
in vitro Strip-EZTM T7 RNA transcription kit
(Ambion) with EcoRI-linearized T3/T7-U19-Dss1 as a template.
Autoradiographs were developed using Amersham Biosciences hyperfilmTM MP at
80 °C. The integrity of total RNA is
good, and the ratio of 28 and 18 S ribosomal RNAs is ~2:1 in all
samples. The signals were quantified using ImageQuant 5.1 software
(Molecular Dynamics).
-tubulin mouse monoclonal antibody (1:1000)
(Zymed Laboratories Inc., San Francisco, CA) for
control to confirm loading efficiency.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6 and CD34, were isolated by
FACS. Control cells were harvested from animals not treated with TPA.
The cDNA was prepared from 10 ng of RNase-free DNase 1-treated
total RNA from keratinocyte progenitor cells by two independent
methods: reverse transcription and PCR-based SMART amplification. The
cDNA was labeled using Klenow-mediated incorporation of
[
-33P]dATP. The hybridization signals were quantified
densitometrically using ImageQuant 5.1 software. The genes were
characterized if their expression changed 2-fold or more in TPA-treated
cells. Eleven genes were identified by gene expression profiling for which expression was up- or down-regulated by TPA (Fig.
1A). Nine genes
were up-regulated and two genes were down-regulated by TPA (see Table
I). Dss1 was induced 3.5-fold,
and it was selected for further study. Dss1 expression was
also verified in TPA-treated and untreated keratinocyte progenitor
cells by semiquantitative RT-PCR. As shown in Fig. 1B, the
result was consistent with the microarray experiment (i.e.
2-3-fold increase in Dss1 expression in cells exposed to
TPA).
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Fig. 1.
a, identification of genes
differentially expressed between TPA-treated and -untreated skin
integrin 6+ CD34+ keratinocyte
progenitor cells using a microarray analysis.
[
-33P]dATP-labeled SMART-amplified cDNA probes, prepared from TPA-treated
(a) or -untreated (b) Tg·AC mouse skin integrin
6+ CD34+ keratinocyte progenitor
cells total RNAs (10 ng), were hybridized to separate
AtlasTM Mouse 1.2 MicroArray according to the user manual
(Clontech). The cDNAs in AtlasTM
Mouse 1.2 MicroArray containing 1176 genes are printed in single spots.
Results were quantitated using ImageQuant 5.1 software. The remarkably
similar array results were obtained in three independent experiments,
one of which was shown. Dss1 gene is indicated by a
circle. b, RT-PCR. Ten nanograms of total RNAs
from Tg·AC mouse skin integrin
6+
CD34+ keratinocyte progenitor cells treated or untreated
with TPA were assayed by semiquantitative RT-PCR using the
Dss1-specific primers and housekeeping gene
2
microglobulin-specific primers, as described under "Materials and
Methods." RT-PCR products were analyzed electrophoretically on 2%
agarose gels.
Differentially expressed genes in TPA-treated keratinocyte progenitor
cells
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Fig. 2.
A, overexpression of Dss1
mRNA in early TPA-induced hyperplastic skin tissues. The multiple
doses of TPA treatment of Tg·AC mouse skins were extracted for total
RNAs using TRIzol reagent (Invitrogen). Isolated RNA (8 µg) was
separated electrophoretically on a 1% agarose gel containing glyoxal,
transferred to the nylon membrane, and cross-linked by UV Stratalinker.
The membrane was probed with [ -32P]UTP-labeled
Dss1 (top panel) or GAPDH
(lower panel) antisense RNA (1 × 106 cpm/ml). Autoradiographs were developed with Amersham
Biosciences hyperfilmTM MP at
80 °C. RNA quantitation
was determined by ImageQuant 5.1 software. The arrows
indicate that the sizes in 0.5 and 1.4 kb are Dss1 and
GAPDH, respectively. GAPDH was served as an
internal control and attested to the equivalent amounts of total RNA
loaded in each lane. B, TPA-induced transcriptional
activation of Dss1 was in a dose- and
time-dependent manner. TPA-susceptible JB6 Cl 41-5a
epidermal cells were either treated for 18 h with indicated TPA
concentrations (a) or exposed to 10 ng/ml TPA at indicated
time points (b). Cells were harvested and total RNA was
extracted using TRIzol reagent (Invitrogen). Eight micrograms of total
RNAs were loaded in Northern blot analysis.
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Fig. 3.
Dss1 is expressed in a variety of
Tg·AC mouse tissues. Tissue samples of the
brain (B), heart (H), small intestine
(SI), kidney (K), liver (Li), lung
(Lu), ovary (O), spleen (Sp), stomach
(St), and skin (Sk) were homogenized and
extracted for total RNA using TRIzol reagent (Invitrogen). Mouse tissue
total RNA (8 µg) Northern blot membrane was probed with
[ -32P]UTP-labeled Dss1 (top
panel) or GAPDH (lower
panel) antisense RNA.
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Fig. 4.
Subcellular localization of Dss1.
A, Western blot analysis. Twenty micrograms of the
whole-cell lysates from JB6 Cl 41-5a cells transiently expressing EGFP
alone (M) or EGFP-Dss1 fusion protein (D) were
subjected to a 10% SDS-polyacrylamide gel. The membrane was probed
with an anti-EGFP rabbit polyclonal antibody (top
panel) or anti- -tubulin mouse monoclonal antibody
(lower panel). B, fluorescence
microscopies. JB6 Cl 41-5a cells (2 × 105) were
plated on eight-well culture slides (Falcon) and transiently
transfected with pEGFP-C3/Dss1 plasmid DNA using LipofectAMINE
PLUSTM reagents (Invitrogen). Cells were incubated at
37 °C for 48 h, fixed in 2% paraformaldehyde, and mounted with
the Prolong antifade medium (Molecular Probes). Fluorescence
microscopies reveal the localization of Dss1 that is indicated by
arrows in nucleus (N) and cytoplasm
(C). Panels I and IV
represent JB6 Cl 41-5a cells expressing EGFP-Dss1 fusion protein,
whereas panels II and V are cells in
panels I and IV, respectively, stained
with DAPI. Panel III is an overlap of
panels I and II, whereas
panel VI is a merge of panels
IV and V. Original magnification, ×630.
C, immunocytochemical staining. JB6 Cl 41-5a cells were
transiently transfected for 48 h with pcDNA3.1D/Dss1-V5-His
plasmid DNA carrying V5-tagged Dss1 gene by LipofectAMINE
PLUSTM reagents (Invitrogen). Dss1-transduced cells were
plated on eight-well culture slides and fixed with methanol, probed
with normal mouse IgG or anti-V5 tag mouse monoclonal antibody, and
stained with the M.O.M. Immunodetection Kit (Vector). Light
microscopies reveal the localization of V5-Dss1 that is indicated by
arrows in nucleus and cytoplasm. I represents
Dss1-transduced JB6 Cl 41-5a cells labeled with normal mouse IgG
(negative control), whereas II is the Dss1-transduced JB6 Cl
41-5a cells probed with anti-V5 tag mouse monoclonal antibody. Original
magnification, ×400.
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Fig. 5.
Elevated expression of Dss1
in neoplasms from Tg·AC mice.
A, Northern blot analysis. Total RNAs were prepared
from eight Tg·AC mice normal skin tissues (NS1 NS8) and
tumors, including eight papillomas (P1
P8) and three
malignancies (M1
M3) with one spindle cell tumor
(M1) and two squamous cell carcinomas (M2 and
M3). Normal skin tissues and tumors were homogenized and
extracted for total RNAs using TRIzol reagent (Invitrogen). Total RNA
(8 µg) Northern blot membrane was probed with
[
-32P]UTP-labeled Dss1 (top
panel) or GAPDH (lower
panel) antisense RNA. B, in situ
hybridization. To detect expression of the Dss1 message,
in situ hybridization assay was performed on sections of
TPA-induced skin tumors, including papilloma (I,
II, and III) and squamous cell carcinoma
(IV, V, and VI), using Dss1
sense (III and VI) and antisense (II
and V) riboprobes. The silver grains indicate the signals in
probe hybridization, and slides were counterstained with hematoxylin
(I and IV). The photographs were taken under
light field (I and IV) and dark field
(II, III, V, and VI)
conditions. Original magnification, ×100.
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Fig. 6.
Functional properties of ectopic
Dss1 expression were evaluated by transfection with
pcDNA3.1D/Dss1-V5-His plasmid DNA. A, Western blot
analysis. The whole-cell lysates (60 µg) from parental
(P), mock-transfected (M), or
Dss1-transfected (D) cell lines including COS-1,
JB6 Cl 30-7b, JB6 Cl 41-5a, Rat-1, and NIH/3T3 were separated
electrophoretically on 12% SDS-polyacrylamide gels. The proteins were
transferred to polyvinylidene difluoride membrane and probed with an
anti-V5 tag mouse monoclonal antibody (top panel)
or anti- -tubulin mouse monoclonal antibody (lower panel).
B, Dss1 overexpression confers focus-forming
activity. JB6 Cl 41-5a, JB6 Cl 30-7b, and NIH/3T3 cells were
transfected with mock or Dss1 expression construct for
48 h. Mock-transfected or Dss1-transfected cell lines
including JB6 Cl 41-5a, JB6 Cl 30-7b, and NIH/3T3 were plated at a
density of 1 × 103, 5 × 103, or
1 × 104 and then selected, respectively, in 400 µg/ml G418 for 14-21 days. Foci were fixed, stained, and counted.
C, elevated Dss1 expression enhances
transformation efficiency in epidermal cell lines. JB6 Cl 41-5a, JB6 Cl
30-7b, and NIH/3T3 cells were stably transfected with mock or
pcDNA3.1D/Dss1-V5-His plasmid DNA by LipofectAMINE
PLUSTM reagents (Invitrogen) and seeded (1 × 104) into 0.33% soft agar over a 0.5% agar bottom layer.
Colony with greater than 8 cells was counted 18 days after
seeding.
-C7) were isolated that stably expressed
Dss1 or the vector control. The stable
Dss1-transduced clones produced a transcript of 443 bp
detected by RT-PCR using a pair of pLNCX2 forward
(2882-2906) and reverse (3057-3032) sequencing/PCR primers (Fig.
7A), indicating that
Dss1 was successfully integrated and expressed. These clones expressed Dss1 mRNA at a variable level, but all stable
clones expressed more Dss1 than control cells
(1.4-3.2-fold; Fig. 7B). The vector
pLNCX2-transduced cells with a band of 176 bp (Fig. 7A, lane 4) were served as a negative
control and showed a low level expression of endogenous Dss1 (Fig.
7B, lane 2).
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Fig. 7.
Constitutive expression of Dss1
remarkably enhances neoplastic transformation in
Dss1-transduced JB6 Cl 41-5a epidermal cell stable
clones. A, detection of the integrated Dss1
gene in JB6 Cl 41-5a Dss1 stable clones. RT-PCR was employed to detect
the presence of Dss1 gene with a pair of PCR primers located
on pLNCX2 retroviral vector. The forward (2882-2906) and
reverse primers (3057-3032) are 5'-AGC TGG TTT AGT GAA CCG TCA GAT
C-3' and 5'-ACC TAC AGG TGG GGT CTT TCA TTC CC-3', respectively. Only
the Dss1-transduced clones, JB6 Cl
41-5a/pLNCX2/Dss1-GR, -C1 -C7, and positive control
retroviral vector pLNCX2/Dss1 showed a 443-bp band
(top panel). The vector
pLNCX2-transduced cells with a band of 176 bp and water
blank served as negative controls.
-Actin with a band of 540 bp
served as an internal control (lower panel).
B, a significant increase of Dss1 transcript in
JB6 Cl 41-5a Dss1 stable clones. Northern blot analysis in
Dss1-transduced cell clones, JB6 Cl
41-5a/pLNCX2/Dss1-GR, -C1
-C7, and positive control
plasmid pcDNA3.1D/Dss1-V5-His showed a band of 0.5 kb in size. The
vector pLNCX2-transduced stable cell clone, in
lane 2, showed a low level expression of
endogenous Dss1 and served as a negative control. C,
Dss1 stable clones demonstrate growth advantage in monolayer
culture. Growth curves were generated for JB6 Cl 41-5a cells stably
expressing mock or Dss1, as described under "Materials and
Methods." Cells were counted in triplicate every other day for 8 days. D, anchorage-independent growth assay. One
pLNCX2-transduced stable cell clone and eight
Dss1-transduced JB6 Cl 41-5a stable cell clones
(pLNCX2/Dss1-GR and pLNCX2/Dss1-C1
-C7) were
seeded at a density of 1 × 104 in a 0.33% soft agar
over a 0.5% agar bottom layer. Colony with greater than 8 cells was
counted in triplicate at 18 days.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6 and CD34. This novel approach was highly effective and identified in vivo
TPA-inducible effector genes that might lead to neoplastic
transformation in skin. Eleven differentially expressed genes were
identified (Fig. 1 and Table I); nine are up-regulated genes, such as
those for galectin-7, nucleoside diphosphate kinase B (NDP kinase B),
cytoskeletal epidermal keratin 14 (CK14), Dss1, DNA double-strand break
repair RAD21 homolog, transcription termination factor 1 (TTF1),
thymosin
4, calpactin I light chain, and 40 S ribosomal protein SA,
and two are down-regulated genes like apolipoprotein E precursor and type 1 cytoskeletal keratin 15 (CK15). Dss1 is one of the
most interesting identified genes and was selected for further
characterization in this study. Here, our data have demonstrated that
TPA was able to induce a high level of Dss1 expression in
integrin
6+ CD34+ keratinocyte
progenitor cells (Fig. 1) and in early hyperplastic skins in
vivo (Fig. 2A), and in JB6 Cl 41-5a preneoplastic
epidermal cells in vitro (Fig. 2B). In addition,
Dss1 is persistently overexpressed in TPA-induced skin
tumors including papillomas and malignant tumors (spindle cell tumor
and squamous cell carcinoma) (Fig. 5). Furthermore, constitutive
expression of Dss1 could promote cell proliferation (Fig.
7C) and enhance the ability of preneoplastic epidermal
cells, JB6 Cl 30-7b and JB6 Cl 41-5a, to grow in soft agar (Figs.
6C and 7D).
-catenin levels
enhances proliferative potential of keratinocytes, increases stem cell
self-renewal, and decreases stem cell differentiation (37). In
addition, activation of the Wnt signaling pathway stimulates carcinogenesis in epithelial cells (38). Trempus et al. in
our laboratory showed that integrin
6 and CD34 were
useful markers for hemopoietic stem and progenitor cells that are
expressed in keratinocytes of the hair follicle bulge. Cells expressing
integrin
6 and CD34 are quiescent and highly clonogenic
progenitor cells (18). In this study, Tg·AC mouse was topically
applied with multiple doses of TPA and dorsal skins were digested with
trypsin and type IV collagenase. The candidate keratinocyte progenitor cells were isolated and enriched with anti-integrin
6
and anti-CD34 antibodies by FACS. Our results showed that the
expression level of Dss1 was elevated in TPA-treated
keratinocyte progenitor cells (Fig. 1) and is associated with the
promotion stage of skin carcinogenesis in mice (Fig. 2A). In
addition, Dss1 expression increases in a time- and
dose-dependent manner (Fig. 2B) and occurs
consistently in TPA-induced skin tumors, eight papillomas and three
malignant tumors (one spindle cell tumor and two squamous cell
carcinomas), with malignant tumors having the highest level of
Dss1 (Fig. 5). These results indicate that Dss1
is a TPA-responsive gene that may be a useful marker for early skin tumorigenesis.
-C7) were found to vary, but all
were higher than that in control vector only clone (pLNCX2)
(Fig. 7B). The magnitude of enhancement of transformation was proportional to the transcription levels of Dss1
mRNA. Taken together, in addition to promoting cell proliferation,
Dss1 strongly provided a crucial role in cellular transformation.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Anton Jetten (Laboratory of Pulmonary Pathobiology, NIEHS, National Institutes of Health, Research Triangle Park, NC) and Cindy Afshari (Laboratory of Molecular Carcinogenesis, NIEHS) for critically reading the manuscript and giving us invaluable advice. We also thank John Otstot (Laboratory of Molecular Carcinogenesis, DNA Sequencing Core, NIEHS) for confirming the DNA sequence, Robert Wine (Laboratory of Neurotoxicology, NIEHS) for manipulating fluorescence microscope, Drs. Dong-Seok Lee (Genome Research Center, Korea Research Institute of Bioscience and Biotechnology, Taejon, South Korea) and Alexandra Heinloth (National Center for Toxicogenomics, NIEHS) for technical suggestions and comments, and Dr. Wen K. Yang (Institute of Biomedical Sciences, Academia Sinica, Taiwan, Republic of China) for communicating results prior to publication.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: National Center for Toxicogenomics, NIEHS, National Institutes of Health, Bldg. 101, Rm. F-149, M. D. F1-05, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-316-4660; Fax: 919-541-1460; E-mail: wei2@niehs.nih.gov.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M206328200
2 S.-J. Wei, C. S. Trempus, R. E. Cannon, C. D. Bortner, and R. W. Tennant, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; Dss1, Deleted in split hand/split foot 1; FACS, fluorescence-activated cell sorting; KSC, keratinocyte stem cell; SHFM1, split hand/split foot malformation; SMART, switching mechanism at the 5' end of RNA templates; TA, transit amplifying cell; RT, reverse transcription; MEM, minimal essential medium; FBS, fetal bovine serum; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; DAPI, 4,6-diamidino-2-phenylindole.
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