Transgenic expression of survivin in keratinocytes counteracts UVB-induced apoptosis and cooperates with loss of p53 (original) (raw)
Keratin 14-survivin transgenic mice. The survivin transgene was constructed using a pGEM2-based plasmid containing 2.1-kb upstream elements of the keratin-14 (K14) gene and 2.2 kb of the human growth hormone (HGH) gene and polyadenylation sequence (Figure 1a). This expression vector was kindly provided by William Philbrick (Yale University School of Medicine, New Haven, Connecticut, USA) with permission of Elaine Fuchs (University of Chicago, Chicago, Illinois, USA) and has been described previously (30, 31). A cDNA encoding the 140 amino acids and stop codon of mouse survivin (32) was cloned into the _Bam_HI site between the K14 and HGH sequences (Figure 1a), and the internal and 3′ flanking _Eco_RI sites in the survivin gene were simultaneously disrupted by site-directed mutagenesis using the respective oligonucleotides 5′-CCGTCAGTGAgTTCTTGAAAC-3′ and 5′-CTTGATATCGAgTTCCTGCAGC-3′ and the GeneEditor system (Promega Corp., Madison, Wisconsin, USA) according to the manufacturer’s instructions. Loss of these _Eco_RI sites and the integrity of the survivin cDNA and flanking sequences were confirmed by restriction mapping and DNA sequencing. Following purification by ion-exchange chromatography (QIAGEN Inc., Valencia, California, USA), the K14/survivin/HGH fusion product was released with _Eco_RI, separated on a 0.9% agarose gel, and electroeluted into 12,000 molecular weight cutoff dialysis tubing. The fragment was further purified on an Elutip-d column (Schleicher & Schuell Inc., Keene, New Hampshire, USA), according to the manufacturer’s instructions, and dialyzed against injection buffer (10 mM Tris, 0.1 mM EDTA, pH 7.4) by floating the sample on 0.05-μm VMWP 01300 filters (Millipore Corp., Bedford, Massachusetts, USA). Finally, the fragment was diluted in injection buffer to 5 ng/μl, sterile-filtered (0.45 μm), and microinjected into B6C3F2 embryos that were implanted into B6C3F1 pseudopregnant females.
Construction and characterization of K14-survivin transgenic mice. (a) The 0.5-kb mouse survivin cDNA, including the stop codon, was cloned into a _BamH_I site flanked by 2.1 kb of K14 promoter sequences and 2.2 kb of HGH and polyadenylation sequences, and the expression cassette was released from the vector by _EcoR_I digest. Arrows indicate the approximate location of PCR primers used for genotyping and RT-PCR. (b) RNA isolated from epidermal cells of K14-survivin transgenic (Tg+) and nontransgenic (Tg–) mice was incubated in the presence or absence of reverse transcriptase (RT) and amplified by PCR with primers complementary to K14 and HGH (left) or mouse keratin 2E (right). Molecular-weight (MW) markers are in the far left lanes, and arrows designate predicted products. (c) Lysates (50 μg) prepared from epidermal cells of K14-survivin (Tg+) and nontransgenic (Tg–) mice or aliquots (5 ng) of recombinant survivin (rSurv) were electrophoresed, transferred to nylon membranes, and blotted with Ab’s to survivin or β-actin, as indicated. Molecular-weight markers are shown on the left.
Of the five mice (out of 26 potential founders) that carried the transgene by tail DNA genotyping, one developed hydrocephalus at 3 weeks of age and was euthanized and two did not transmit the transgene to offspring in Mendelian fashion. One founder (no. 19) of the two remaining mice yielded offspring with detectable transgene expression, and these were propagated by repeated mating with C57BL/6NCR mice (National Cancer Institute, Bethesda, Maryland, USA). Litters from the second and third backcrosses were used for these studies.
Genotyping. Potential founders and littermates were screened for the transgene by PCR. Genomic DNA was prepared by incubation of 2 mm of tail in 0.3 ml digestion buffer (50 mM NaCl, 50 mM Tris, pH 8, 100 mM EDTA, 1% SDS) containing 133 μg/ml proteinase K (Sigma Chemical Co., St. Louis, Missouri, USA) at 56°C for 4 hours, followed by addition of RNase A (100 μg/ml, Roche Molecular Biochemicals, Indianapolis, Indiana, USA) and further incubation at 37°C for 1 hour. Following repeated extraction with phenol/chloroform/isoamyl alcohol (25:24:1; Roche Molecular Biochemicals), genomic DNA was precipitated with isopropanol, washed in 70% ethanol, and resuspended in 10 mM Tris, 1 mM EDTA, pH 8. The transgene was amplified in 25 μl with 0.6 U Taq polymerase (Roche Molecular Biochemicals) from 0.1 μg genomic DNA using 200 μM 2′-dinucleoside 5′-triphosphates (Roche Molecular Biochemicals) and primers (10 pmol of each) corresponding to the flanking K14 (5′-TAGCCTGTGGGTGATGAAAG-3′) and HGH (5′-CTGAGATTGGCCAAATACTGG-3′) sequences. PCR reactions were carried out as follows: 2 minutes of denaturation at 94°C; 30 cycles of 94°C for 30 seconds, 56°C for 1 minute, and 72°C for 1 minute; and a 7-minute extension at 72°C. Five microliters was sufficient to visualize the 0.75-kb product on ethidium bromide–stained 1% agarose gels.
Preparation of keratinocytes. Epidermal keratinocytes were isolated from 7- to 9-week-old mice as described elsewhere (33), with modifications. Hair was immediately removed from killed mice using Oster A5 clippers and a #40 blade (VWR Scientific, Bridgeport, New Jersey, USA), followed by a Braun 3612 shaver (Braun GmbH, Kronberg, Germany). Shaved skin was excised, residual subcutaneous tissues were scraped away, and skin was cut into 0.5-cm strips. After floating dermal side down in trypsin buffer (148 mM NaCl, 5.4 mM KCl, 5.5 mM glucose, pH 7.6) containing 2.3 mg/ml trypsin (T-8003; Sigma Chemical Co.) at 37°C for 1 hour, the epidermis was peeled off and keratinocytes were dissociated in trypsin buffer containing 12,000 U per milliliter DNase I (Calbiochem-Novabiochem Corp., La Jolla, California, USA) by vigorous agitation at 37°C for 7 minutes and then placed on ice. Cells were diluted 1:2 in ice-cold complete medium consisting of Dulbecco’s minimal Eagle’s medium (GIBCO-BRL; Life Technologies Inc., Grand Island, New York, USA) containing 10% FCS (Gemini Bio-Products, Woodland, California, USA), 2 mM L-glutamine (BioWhittaker Inc., Walkersville, Maryland, USA), 100 U/ml penicillin (BioWhittaker Inc.), 100 μg/ml streptomycin (BioWhittaker Inc.), and 1,200 U/ml DNase I and filtered through autoclaved Nitex nylon mesh (Safer America Inc., Depew, New York, USA). Cells were pelleted and suspended in complete medium, centrifuged over Histopaque-1083 (Sigma Chemical Co.) to remove dead cells, and then washed in PBS, pH 7.4. For Western blot analysis, keratinocytes were not subjected to gradient centrifugation.
RT-PCR and Western blot analysis. Total RNA was purified from epidermal cells using TriReagent (Molecular Research Center Inc., Cincinnati, Ohio, USA). RT-PCR was performed with 0.5 μg RNA and the Superscript Preamplification System (GIBCO-BRL; Life Technologies Inc.) according to the manufacturer’s instructions. Oligo-dT was used to prime reverse transcription, and the K14 and HGH primers noted above were used for PCR. Primers 5′-TTGCCAGATCTCCTGCAGGTC-3′ and 5′-CCACCTCCAAATCGGCCTCCG-3′, corresponding to the mouse keratin-2E gene, were used as a positive control for nontransgenic animals. Reverse transcriptase was not added to some reactions as a negative control. In another series of experiments, epidermal cell lysates isolated from K14-survivin transgenic animals or control littermates were subjected to Western blot analysis using a rabbit polyclonal Ab raised against full-length recombinant human survivin (currently available from NOVUS Biologicals Inc., Littleton, Colorado, USA), which cross-reacts with mouse survivin, followed by chemiluminescence, as described (34).
Immunohistochemistry and immunofluorescence microscopy. Freshly excised whole skin samples from K14-survivin transgenic mice or control normal littermates were embedded in OCT compound and frozen. Five-micrometer tissue sections were cut, fixed in ice-cold acetone for 10 minutes, air dried for 15 minutes, and rehydrated in Tris-buffered saline (TBS), pH 7.0, for 10 minutes. Slides were then placed over moistened paper towels in a covered tray. The sections were blocked with 0.5% normal goat serum (Vector Laboratories, Burlingame, California, USA) in TBS, pH 7.0, for 5 minutes. For immunocytochemistry, a polyclonal Ab raised against the survivin sequence A3-I19 and used in previous studies on mouse tissues (20) was applied at a concentration of 5 μg/ml in TBS, pH 7.0, containing 1% BSA and incubated overnight at 4°C. Binding of the primary Ab was visualized using a goat anti-rabbit polyclonal Ab (1:100) and Vectastain Elite ABC and 3-amino-9-ethyl carbazole (AEC) peroxidase substrate kits (Vector Laboratories) according to the manufacturer’s instructions. In control experiments, the survivin Ab was replaced with a rabbit polyclonal Ab to enhanced green flourescent protein (EGFP). At the end of the incubation, slides were washed in water, counterstained for 5 seconds with Harris hematoxylin-2 (Sigma Chemical Co.), and washed again in water, prior to mounting and coverslipping. In another series of experiments, paraffin sections were cut from shaved formalin-fixed skin of 8-week-old K14-survivin transgenic mice or control normal littermates and treated as described (34), except without an antigen-retrieval step. Skin sections were incubated with 2 μg/ml nonimmune rabbit IgG (Sigma Chemical Co.) or rabbit Ab’s to mouse keratin-1 or keratin-14 (MK1, MK14; Covance Research Products Inc., Richmond, California, USA) for 1 hour at 22°C. Staining was visualized using a Histomouse-SP kit (Zymed Laboratories Inc., South San Francisco, California, USA) with AEC as the chromophore. For subcellular localization of transgenic survivin, keratinocytes from K14-survivin transgenic mice and control littermates were isolated as described above and incubated overnight in complete medium to adhere to 13 mm no. 1 optical glass coverslips (ProSciTech, Thuringowa Central, Queensland, Australia). Cells were fixed at –20°C in methanol for 5 minutes followed by acetone for 5 seconds and then air dried. Analysis for survivin expression by fluorescence microscopy was performed using the polyclonal rabbit Ab to full-length survivin (NOVUS Biologicals Inc.). Binding of the primary Ab was visualized using Texas red–conjugated goat anti-rabbit IgG (Molecular Probes Inc., Eugene, Oregon, USA), and cell nuclei were detected by simultaneous labeling with Hoechst 33342. Coverslips were analyzed on a Zeiss Axiophot microscope (Carl Zeiss GmbH, Jena, Germany) equipped with ×63/NA planopochromatic lens, and image files were collected and assembled in Photoshop 5.0.
UVB-induced apoptosis in vivo. Mice 7–9 weeks of age were shaved to expose two 1 × 1.5-cm patches of dorsal skin and 24 hours later irradiated unrestrained in open cages at a dose of approximately 600 J/m2 (at a rate of 2.5 J/m2 per second) using a bank of three sun lamps (FS20T12-UVB; National Biological Corp., Twinsburg, Ohio, USA). These bulbs emit wavelengths between 250 and 420 nm (72.6% UVB, 27.4% UVA, 0.01% UVC), with peak emission at 313 nm, according to the manufacturer. A filter (Kodacel TA422; Eastman Kodak Co., Rochester, New York) was placed over the cages to block residual UVC. Dosimetry was determined using a newly calibrated UVB-500C meter (National Biological Corp.). Animals were killed 24 hours after irradiation, and the exposed patches of skin were excised and fixed in 10% buffered formalin. Skin was then cut into 1- to 2-mm strips, which were stacked on top of each other for embedding in paraffin and routine staining with hematoxylin and eosin. This technique allowed for histologic analysis of 6–7 linear centimeter of skin on a single slide. Apoptotic “sunburn” cells were identified within the epidermis by the presence of eosinophilic cytoplasm and condensed, fragmented, or absent nuclei, as described (11). TUNEL staining was carried out on unstained sections using the ApopTag kit (Intergen Co., Purchase, New York, USA), as described previously (34).
Keratinocyte proliferation and hyperplasia in vivo. Mice were irradiated with 600 J/m2 UVB, and 72 hours later were injected intraperitoneally with 5-bromo-2′-deoxyuridine (BrdU, 50 mg/kg; Sigma Chemical Co.) in PBS, pH 7.4. After 2 hours, mice were killed, and both irradiated and unirradiated (unshaven) skin was excised and processed as above. In separate experiments, mice were shaved and 0.2 ml acetone or acetone containing 6.8 nmol PMA (Sigma Chemical Co.) was applied every 3 days for a total of four treatments. Forty-eight hours after the last application, mice were killed and skin was excised and processed. Tissue sections were stained for BrdU using a kit (Zymed Laboratories Inc.), as described previously (35). In sections stained with hematoxylin and eosin, epidermal thickness was measured from the top of the granular layer to the basement membrane at up to 20 sites in multiple strips of skin.
UVB-induced apoptosis in vitro. Freshly isolated keratinocytes were resuspended in PBS, pH 7.4, gently spread as a thin film (50–100 μl) onto the center of 35 × 10–mm polystyrene dishes (Falcon no. 353001), and irradiated with UVB. Complete medium was then carefully added (2 ml per dish) from the side so as not to disrupt the loosely adherent keratinocytes, and dishes were placed in a humidified incubator (37°C, 5% CO2). In some experiments, the pan-caspase inhibitor ZVAD-fmk (40 μM; Enzyme Systems Products Inc., Livermore, California, USA) was included in the medium. In other experiments, medium containing recombinant mouse IFN-γ (Calbiochem-Novabiochem Corp.) and hamster anti-mouse Fas Ab (clone Jo2, PharMingen, San Diego, California, USA) was added to unirradiated cells. After 20–24 hours, the adherent cells were washed, fixed, stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co.), and assessed for apoptotic morphology by fluorescence microscopy, as described previously (34).
Keratin 14-survivin p53+/– mice. K14-survivin transgenic mice were crossed with p53-deficient (p53–/–) mice on identical background strain (Taconic Farms, Germantown, New York, USA) to generate transgenic and nontransgenic p53+/– animals. Littermates were genotyped for the presence of the survivin transgene by PCR as discussed above. No gross or histologic differences were noted between transgenic and nontransgenic p53+/– animals observed from birth to 4 months.
Statistics. Data derived from multiple animals or determinations were subjected to an unpaired t test using Welch’s correction (Prism; Graphpad Software for Science Inc., San Diego, California, USA). P values less than 0.05 were considered statistically significant.