Identification of Dss1 as a 12-O-Tetradecanoylphorbol-13-acetate-responsive Gene Expressed in Keratinocyte Progenitor Cells, with Possible Involvement in Early Skin Tumorigenesis*

This study identifies genes expressed early in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin carcinogenesis in genetically initiated Tg·AC v-Ha-rastransgenic 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. Dss1overexpression 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.

This study identifies genes expressed early in 12-Otetradecanoylphorbol-13-acetate (TPA)-induced skin carcinogenesis in genetically initiated Tg⅐AC v-Ha-ras transgenic mice. Keratinocyte progenitor cells from TPA-treated Tg⅐AC 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)(2)(3). The Tg⅐AC 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 -globin gene promoter (4). Tg⅐AC 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. Tg⅐AC mice develop hyperplasia in skin keratinocytes after exposure to tumor promoters such as TPA 1 (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 Tg⅐AC mouse model is a valuable tool to study the early stages of skin carcinogenesis.
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)(13)(14)(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 ␣ 6 bri 10G7 dim . 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 ␣ 6 bri CD71 dim . 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.
Previous studies identified ϳ30 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 ␣ 6 ϩ CD34 ϩ keratinocyte progenitor cells were isolated from hyperplastic skin of TPAtreated animals and their gene expression analyzed using cDNA microarray. This method employs FACS, switching mechanism at the 5Ј end of RNA templates (SMART) cDNA amplification, and mouse cDNA array technology. Eleven TPAresponsive genes were identified in the Tg⅐AC 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.
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 Tg⅐AC 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% CO 2 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 g/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 l of acetone was applied topically to groups of 5 homozygous male Tg⅐AC 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
Atlas TM Mouse 1.2 MicroArray carrying 1176 cDNAs was obtained from Clontech (Palo Alto, CA). The keratinocytes were harvested from the dorsal skin of Tg⅐AC mice (29), and the integrin ␣ 6 ϩ CD34 ϩ keratinocyte progenitor cells were isolated using FACS (18). Total RNAs were extracted from TPA-treated or -untreated keratinocyte progenitor cells using StrataPrep R Total RNA Miniprep Kit (Stratagene, La Jolla, CA). Ten nanograms of total RNA was reverse-transcribed and amplified using the Atlas SMART TM system (Clontech). Five hundred nano-grams of purified SMART cDNA were labeled for 30 min at 50°C with [␣-33 P]dATP (10 Ci/l; Ͼ2500 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 [␣-33 P]dATP-labeled probes (3.7 ϫ 10 6 cpm/ml). The membrane was washed four times in 2ϫ saline sodium citrate (SSC), 1% SDS for 30 min at 68°C, and two times in 0.1ϫ 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).

Vector Constructions
The full-length Dss1 cDNA was amplified by RT-PCR using Tg⅐AC mice skin total RNA. The Dss1 forward and reverse primers were 5Ј-CAC CAT GTC TGA AAA GAA GCA GCC-3Ј and 5Ј-TGA TGT CTC CAT CTT GTA GCC GTG CTT-3Ј, respectively. The PCR amplified Dss1 cDNA was cloned into V5-His-tagged pcDNA3.1D/V5-His-TOPO c 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 pLNCX 2 (Clontech) using the Li-gaFast TM 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 LigaFast TM 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 BigDye TM 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 PLUS TM reagents (Invitrogen) or by infection with virions packaged with ecotropic packaging cells RetroPack TM PT-67 (Clontech). The recombinant pLNCX 2 retroviral vector carries mouse Dss1 gene driven by the cytomegalovirus promoter and neo R 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 g/ml Geneticin (G418) (Invitrogen). Cells were analyzed by Western blot or RT-PCR to confirm the expression of Dss1.

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 [␣-32 P]UTP-labeled Dss1 antisense RNA (1 ϫ 10 6 cpm/ml). The riboprobe was prepared using in vitro Strip-EZ TM T7 RNA transcription kit (Ambion) with EcoRI-linearized T3/T7-U19-Dss1 as a template. Autoradiographs were developed using Amersham Biosciences hyperfilm TM MP at Ϫ80°C. The integrity of total RNA is good, and the ratio of 28 and 18 S ribosomal RNAs is ϳ2:1 in all samples. The signals were quantified using ImageQuant 5.1 software (Molecular Dynamics).

In Situ Hybridization
The in situ hybridization assay was performed as previously described (30). Briefly, the cutaneous tumors were removed from Tg⅐AC mice and fixed overnight in 10% neutral buffered formalin. The tissues were paraffin-embedded, and sections (6 m) 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 2ϫ SSC, then applied with ϳ2 ϫ 10 6 cpm of 35 Slabeled 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-EZ TM T7 or T3 RNA transcription kit (Ambion). Following 40°C overnight hybridization, the tissues were washed in 2ϫ SSC plus 50% formamide at 40°C, then in 2ϫ SSC, 1ϫ SSC, 0.5ϫ SSC, and 0.5ϫ SSC, 30 min each wash at room temperature. To remove unbound probe, the tissues were incubated with 20 l 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 ϫ 10 5 cells/well using LipofectAMINE PLUS TM 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 1ϫ phosphatebuffered saline buffer (150 mM NaCl, 10 mM Na 2 HPO 4 , 10 mM KH 2 PO 4 , pH 7.4) and fixed in 2% paraformaldehyde in 1ϫ 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 63ϫ 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 ϫ 10 6 ) were transiently transfected with 4 g of pcDNA3.1D/Dss1-V5-His in a 10-cm tissue culture plate using LipofectAMINE PLUS TM reagents (Invitrogen). Cells were incubated for 48 h, and 2 ϫ 10 5 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 FIG. 1. a, identification of genes differentially expressed between TPA-treated and -untreated skin integrin ␣ 6 ϩ CD34 ϩ keratinocyte progenitor cells using a microarray analysis. [␣-33 P]dATP-labeled SMART-amplified cDNA probes, prepared from TPA-treated (a) or -untreated (b) Tg⅐AC mouse skin integrin ␣ 6 ϩ CD34 ϩ keratinocyte progenitor cells total RNAs (10 ng), were hybridized to separate Atlas TM Mouse 1.2 MicroArray according to the user manual (Clontech). The cDNAs in Atlas TM 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 Tg⅐AC mouse skin integrin ␣ 6 ϩ CD34 ϩ keratinocyte progenitor cells treated or untreated with TPA were assayed by semiquantitative RT-PCR using the Dss1specific primers and housekeeping gene ␤ 2 microglobulin-specific primers, as described under "Materials and Methods." RT-PCR products were analyzed electrophoretically on 2% agarose gels.

Transformation Assays
Focus-forming Activity-Cells were seeded overnight at a density of 2 ϫ 10 5 cells/well in six-well plates. Cells were transfected with 1 g 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 ϭ 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 ϫ 10 4 ) 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 ϫ 10 4 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% CO 2 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 Tg⅐AC 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. Tg⅐AC mice were treated with TPA, and keratinocyte progenitor cells carrying the cell surface markers, integrin ␣ 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 [␣-33 P]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).
Dss1 Is a TPA-responsive Gene Induced Early in Skin Tumorigenesis-Previous studies demonstrated that chronic topical application of TPA to the skin of Tg⅐AC mice induces epidermal hyperplasia (30). Dss1 expression was analyzed in hyperplastic skin in Tg⅐AC 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 (ϳ2-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 Tg⅐AC 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-V5tagged 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 Tg⅐AC mouse skin tumors, including eight papillomas (2.5 Ϯ 0.4-fold) and three malignant tumors (one spindle cell tumor and two squamous cell carcinomas) (6.2 Ϯ 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).
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 ϳ2-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 ϫ 10 4 in 60-mm soft agar plates to assay for anchorage-independent growth. The colony-formation efficiency increased ϳ5or 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 neo R were inserted into a bicistronic construct using an ecotropic retroviral vector pLNCX 2 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 (Ͼ100 colonies) (pLNCX 2 /Dss1-GR). Eight individual clones (designated as pLNCX 2 and pLNCX 2 /Dss1-C13 -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 pLNCX 2 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 pLNCX 2 -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).
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.

DISCUSSION
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 tech- nique, 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 Tg⅐AC mice. Keratinocyte progenitor cells were isolated using FACS to select cells that express the progenitor cell markers, integrin ␣ 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 doublestrand 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 ϩ CD34 ϩ 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).
A previous report indicates that skin tumorigenesis may be initiated by cellular transformation of KSCs (36). Elevation of ␤-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, Tg⅐AC 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.
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 GFPprotein 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 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, ϫ100.
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. and NIH/3T3 were plated at a density of 1 ϫ 10 3 , 5 ϫ 10 3 , or 1 ϫ 10 4 and then selected, respectively, in 400 g/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 PLUS TM reagents (Invitrogen) and seeded (1 ϫ 10 4 ) into 0.33% soft agar over a 0.5% agar bottom layer. Colony with greater than 8 cells was counted 18 days after seeding.
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 2ϫ 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, anchorageindependent growth assay. One pLNCX 2transduced stable cell clone and eight Dss1-transduced JB6 Cl 41-5a stable cell clones (pLNCX 2 /Dss1-GR and pLNCX 2 / Dss1-C13-C7) were seeded at a density of 1 ϫ 10 4 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.
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 pLNCX 2 /Dss1 retroviral vector, produced by ecotropic packaging cells RetroPack TM 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 Tg⅐AC 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 ϳ25-30%, 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 pLNCX 2 /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 (pLNCX 2 /Dss1-GR) and seven individual stable clones (pLNCX 2 /Dss1-C13-C7) were found to vary, but all were higher than that in control vector only clone (pLNCX 2 ) (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.
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 transactivation domain deletion mutant (TAM67) or AP-1 transrepressing 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 transactivation. 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 TPAinduced 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.