Indirect immunofluorescence.

<p>Kera5, MaFi132, 308 and NIH 3T3 cells were grown on cover slips, fixed with acetone, stained with keratin 14 or vimentin specific antibodies and detected with AlexaFluor488 or AlexaFluor594-conjugated secondary antibodies, respectively. Murine keratinocytes (308 cells) and fibroblasts (NIH 3T3) were used as controls. Green fluorescence indicates keratin 14 expression only in Kera5 and 308 cells, whereas red fluorescence shows vimentin expression only in MaFi132 and NIH 3T3 (original magnification: 200x).</p

Establishment of an Immortalized Skin Keratinocyte Cell Line Derived from the Animal Model <i>Mastomys coucha</i>

<div><p>In the present report we describe the establishment of a spontaneous immortalized skin keratinocyte cell line derived from the skin of the multimammate rodent <i>Mastomys coucha</i>. These animals are used in preclinical studies for a variety of human diseases such as infections with nematodes, bacteria and papillomaviruses, especially regarding cutaneous manifestations such as non-melanoma skin cancer. Here we characterize the cells in terms of their origin and cytogenetic features. Searching for genomic signatures, a spontaneous mutation in the splicing donor sequence of <i>Trp53</i> (G to A transition at the first position of intron 7) could be detected. This point mutation leads to alternative splicing and to a premature stop codon, resulting in a truncated and, in turn, undetectable form of p53, probably contributing to the process of immortalization. <i>Mastomys coucha</i>-derived skin keratinocytes can be used as an <i>in vitro</i> system to investigate molecular and immunological aspects of infectious agent interactions with their host cells.</p></div

Sequencing of the <i>Mastomys</i> p53 gene (<i>Trp53</i>) and partial alignment with mouse and rat sequences.

<p><b>(A)</b> Sequencing of <i>Trp53</i> in Kera5 (passages 8 and 103) shows a G>A transition at the first position of intron 7 (underlined). Freshly isolated primary keratinocytes (Kera5, p0) as well as five individual <i>Mastomys</i> samples do not harbor this mutation and are similar to murine and rat sequences at this position. Numbers refer to the sequences of murine <i>Trp53</i> and rat <i>Tp53</i>. For intron 7, only 5´-start and 3´-ends are shown. Exon 7: green, exon 8: grey, insertion: orange, frames: splicing signals; “a” indicates the original splicing donor, “b” alternative splicing donor signal in intron 7, “c”: splicing acceptor. <b>(B)</b> Sequencing chromatograms of <i>Trp53</i> reveal the G>A transition in a subpopulation of Kera5 cells at p8 which is not present at p0. A switch of the major peak from G to A from p8 to p103 occurs, suggesting the outgrowth of a single cell colony. At p146 only the A peak is left, revealing homozygosity. The arrows indicate the position of the mutation.</p

Calcium-induced differentiation of Kera5.

<p>Kera5 cells at passage 139 were grown on cover slips. After 24 h, the dKSFM (<0.1 mM Ca²⁺) was additionally supplemented with 0.35 mM, 0.7 mM or 1.05 mM Ca²⁺ to induce differentiation. After additional 24 h incubation, the cells were fixed with acetone, stained with an involucrin antibody and detected with an AlexaFluor488 secondary antibody, respectively. Green fluorescence indicates elevated involucrin expression in a dose-dependent manner (original magnification: 200x).</p

Cell morphology.

<p><b>(A)</b><i>Mastomys coucha</i> keratinocytes at passage 6. <b>(B)</b> Kera5 at passage 175, showing a typical cobblestone phenotype and an increased cell size (arrowheads mark ongoing mitoses; magnification: 200x).</p

p53 cDNA sequence and transcriptional analyses.

<p><b>(A)</b> The relevant nucleotides of the mutated p53 cDNA are shown. At passage 13, an insertion of 19 nt is detectable in the cDNA of p53 (exon 7: green, exon 8: grey, insertion: orange). <b>(B)</b> Comparison of translated wildtype and mutant sequences derived from the cDNA. The insertion leads to a premature stop codon (*) that results in a truncated form of p53. <b>(C)</b> Semi-quantitative RT-PCR of <i>Trp53</i> at different passages. <i>GAPDH</i> was used as reference gene. <b>(D)</b> Semi-quantitative RT-PCR to detect the insertion within the p53 cDNA, resulting in a slower-migrating PCR product at higher passages (p27-p105). <i>GAPDH</i> was used as reference gene. <b>(E)</b> Western blotting. Left panel: lack of p53 expression in Kera5. Kera5 (p155) were exposed to UVB (40 or 250 mJ/cm²) or treated with adriamycin (1.0 μg/ml) and harvested as indicated. 50 μg protein/lane were loaded. To control the specificity of the p53 antibody, MaFi132 were transiently transfected with pPK-CMV-E3 containing the cDNA for wildtype (p53wt) or the truncated (p53trunc)) form of p53. Since only 25 μg of protein were loaded for transfected MaFi132, a longer exposure time was chosen for actin. Right panel: NIH 3T3 cells were used as reference for stabilization of p53 after cells damage with UV or Adriamycin, respectively. NIH 3T3 cells were exposed to UVB (20 or 100 mJ/cm²) or treated with Adriamycin (0.75 μg/ml) and harvested as indicated. 50 μg protein/lane were loaded. After low dose UVB, p53 stabilization occurs slowly, whereas after high dose UVB the stabilization is fast, but transient. Adriamycin permanently stabilizes p53 already after 7h. Actin was used as loading control.</p

Study design and tumor development.

<p><b>A)</b><i>Mastomys coucha</i> as a model for cutaneous papillomavirus infection. In the study, naturally MnPV-infected animals (MnPV⁺) as well as virus-free control animals (MnPV^-) were irradiated three times per week with UVB. The starting dose of 150 mJ/cm² was increased weekly by 50 mJ/cm² until the desired final dose was reached (450, 600 or 800 mJ/cm², respectively). Black arrows indicate an increase of the dose, gray arrows the subsequent application of this dose. The irradiation was continued until the animals were sacrificed or died. <b>B)</b> Kaplan-Meier curves demonstrating the percentage of irradiated virus-infected (MnPV⁺, UV⁺), virus-free (MnPV^-, UV⁺) and unirradiated virus-infected (MnPV⁺, UV^-) tumor-bearing animals. <b>C)</b> Two examples of spontaneous skin lesions arising in naturally infected animals. <b>D)</b> Examples of UV-induced keratinizing SCCs (KSCC) with similarities to human keratoacanthomas. <b>E)</b> Examples of UV-induced non-keratinizing SCCs (nKSCC) (C, D and E: scale bars: 10 mm). <b>F)</b> Number of KSCCs and nKSCCs in correlation with the final UV doses. Note that KSCCs preferentially appeared at the lowest dose, nKSCCs preferentially at higher doses (Mean ± SEM; animal numbers: see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006723#ppat.1006723.t001" target="_blank">Table 1</a>; av: average number of tumors).</p

Schematic overview of the mechanism suggested for UV-induced NMSC development in <i>Mastomys coucha</i>.

<p><b>A)</b> MnPV infects basal epithelial cells of the skin of young animals via small injuries. <b>B)</b> MnPV genome is amplified in stratified skin layers (pink and red nuclei) and new virions are released. <b>C)</b> UVB irradiation of the skin. <b>D)</b> UVB-irradiated skin is hyperproliferative, favoring viral replication and virion formation. UVB-induced photoproducts, e.g. in <i>Trp53</i>, occur in keratinocytes (altered nuclei). In uninfected cells, damages are repaired. In infected cells, MnPV-E6/E7 reduce chromosomal stability and inhibit DNA repair. Mutations can accumulate and altered cells become neoplastic. <b>E)</b> Neoplastic squamous cells (light blue) start forming a well-differentiated keratinizing SCC, still representing a permissive system that allows viral replication and formation of virions. <b>F)</b> When neoplastic squamous cells accumulate further mutations (dark blue), a spindle cell phenotype is acquired, forming a poorly differentiated SCC that may become ulcerated. MnPV cannot replicate in dedifferentiated cells and the viral DNA is subsequently lost.</p

MnPV interferes with DNA damage repair.

<p><b>A)</b> Repair kinetics of CPDs in MnPV E6/E7-positive and -negative <i>Mastomys</i> keratinocytes (Mean ± SD; n = 2, measurements were performed in quadruplicates). <b>B)</b> Immunofluorescence staining of γH2AX foci in keratinocytes stably expressing MnPV E6/E7. Cells were irradiated with UVB and further incubated prior to detection and quantification of γH2AX foci (Ctrl: unirradiated, UV: irradiated; Red: γH2AX, blue: nuclei; scale bars: 50 μm). <b>C)</b> Quantification of γH2AX foci (Mean ± SEM; n≥242; 1way-ANOVA, *p<0.05, **p<0.01, ***p<0.001). <b>D)</b> Co-detection of CPDs and γH2AX in MnPV^+/- skin harvested 24h after UV irradiation. Arrows point towards positive cells (Viral loads: animal 3: 13.68 ± 1.66 copies/cell, animal 4: 147.42 ± 14.62 copies/cell; Scale bars: 100 μm). <b>E)</b> Co-detection of CPDs and γH2AX in a KSCC harvested 24h after UV irradiation (Viral load: 611.88 ± 18.75 copies/cell; scale bars: 100 μm).</p

Molecular analyses of tumor-bearing animals.

<p><b>A)</b> Viral load in tissue samples from UV-irradiated and control animals from the MnPV-infected colony analyzed by qPCR and normalized to a plasmid standard. Samples were grouped according to their origin as indicated (ctrl skin: skin from unirradiated animals; ui skin/UV skin: unirradiated or UV-irradiated skin from irradiated animals; KSCC/nKSCC: UV-induced SCCs; non-UV tumor: tumors from non-UV sites of irradiated animals and spontaneous tumors from unirradiated animals). UV^+/- indicates whether the animal was UV-exposed or not (Kruskal-Wallis test, *p<0.05, ***p<0.001, ^nsp>0.05). <b>B)</b> Southern blot analysis of unirradiated and UV-irradiated skins, a KSCC and a non-UV tumor. DNA was digested with ApaI (no cleavage site in MnPV), XbaI (one site) or XhoI (two sites) as indicated (Form I: supercoiled; Form II: relaxed circular; Form III: linear form of MnPV). <b>C)</b> Semi-quantitative RT-PCR for the most abundant MnPV <i>E1^E4</i> transcript in non-UV tumors and UV-induced SCCs or the control <i>GAPDH</i>. <b>D)</b> Semi-quantitative RT-PCR for MnPV <i>E6</i>, <i>E7</i> and <i>L1</i> transcripts in non-UV tumors and UV-induced SCCs or the control <i>GAPDH</i>.</p