JOURNAL OF VIROLOGY, May 2004, p. 4797–4805 Vol. 78, No. 90022-538X/04/$08.00�0 DOI: 10.1128/JVI.78.9.4797–4805.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Increased Incidence of Squamous Cell Carcinomas in Mastomysnatalensis Papillomavirus E6 Transgenic Mice during

Two-Stage Skin CarcinogenesisIris Helfrich,* Min Chen,† Rainer Schmidt, Gerhard Furstenberger,

Annette Kopp-Schneider, David Trick, Hermann-Josef Grone,Harald zur Hausen, and Frank Rosl

Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany

Received 7 August 2003/Accepted 23 December 2003

Papillomaviruses cause certain forms of human cancers, most notably carcinomas of the uterine cervix. Incontrast to the well-established involvement of papillomavirus infection in the etiology of cervical carcinomasand in carcinomas of a rare hereditary condition, epidermodysplasia verruciformis, a causative role for cu-taneous human papillomavirus types in the development of nonmelanoma skin cancer has not been proven. Inorder to better understand the functions of individual genes of cutaneous papillomavirus types, we generatedtransgenic mice carrying oncogene E6 of the Mastomys natalensis papillomavirus (MnPV), which causes kerato-acanthomas of the skin in its natural host. In the present study, we demonstrate that under conditions ofexperimental two-stage skin carcinogenesis, fast-paced squamous cell carcinomas develop in nearly 100% ofMnPV E6 transgenic mice in comparison to 10% in their nontransgenic littermates (log rank test; P < 0.0001).Therefore, we conclude that the MnPV E6 transgene favors the malignant progression of chemically inducedtumors. Whereas an activating H-ras mutation is a consistent feature in benign and malignant tumors inwild-type mice, the majority of papillomas and keratoacanthomas and all squamous cell carcinomas obtainedin MnPV E6 transgenic mice contain nonmutated ras alleles. These results indicate that the development ofsquamous cell carcinomas in MnPV E6 transgenic mice does not depend on an activated H-ras oncogene.

Papillomaviruses are widely known to play a causative role inthe development of tumors in humans. Certain genotypescause mostly benign epidermal tumors in humans and animals.Another subgroup of genotypes, the so-called “high-risk” ano-genital human papillomavirus (HPV) types, are causally in-volved in the development of malignant tumors, such as car-cinoma of the cervix (17, 47, 49). The carcinogenic potentialwas first detected in 1935, when Rous and Bernard showed thatinoculation into domestic rabbits of the filterable extracts fromwarts of cottontail rabbits was able to induce skin carcinomas(37). A direct involvement of cutaneous HPV types in thedevelopment of squamous cell carcinomas, one of the mostfrequent malignancies in humans of Caucasian origin, is sus-pected but still not proven (7, 34). The earliest hints of aninvolvement of specific HPV types in human skin cancer orig-inated from studies of patients suffering from the very rarehereditary disease epidermodysplasia verruciformis (EV) (32,21). Only a limited number of HPV types, commonly referredto as EV-HPV types, such as HPV5, -8, and -14, are frequentlydetected in squamous cell carcinomas that develop in �30% ofcases within multiple flat warts in sun-exposed areas of the skinin such patients (25). More recent data suggested a role forEV-related papillomaviruses in the origin of skin cancer inimmunosuppressed patients (39) and those with renal trans-

plants (28). Thus, specific high-risk cutaneous HPV types com-parable to carcinogenic anogenital HPV types have not beenidentified (48, 49).

The tumorigenic potential of some cutaneous animal papil-lomaviruses has been demonstrated (for a review, see refer-ence 10), including that of Mastomys natalensis papillomavirus(MnPV) (29), a rodent papillomavirus known to cause kera-toacanthomas in its natural host, M. natalensis (1). Reinacheret al. (36) described spontaneous keratoacanthomas in M. na-talensis and observed MnPV particles in keratinized cells of thestratum granulosum and the stratum corneum. Infection ofyoung M. natalensis with MnPV particles resulted in benignskin tumors in 40% of the infected animals (36). During theirlife spans, the animals remained persistently infected, and thenumber of viral genomes in their skins steadily increased. Atthe age of 1 year, skin tumors developed spontaneously (1).

In the case of high-risk anogenital HPV types, the E6 and E7genes cooperate in the transformation of human cells andinduce carcinomas in transgenic mice (2, 3, 15, 16, 19, 24). Inorder to assess the role of the E6 protein of a cutaneouspapillomavirus type in vivo, we generated transgenic mice ex-pressing the MnPV E6 gene in the skin under the control ofthe human cytokeratin-14 promoter. MnPV E6 transgenic miceshowed an increased incidence of squamous cell carcinomaswhen the classical mouse skin two-stage carcinogenesis proto-col (6, 13) was applied. These results demonstrated a cooper-ating function of the MnPV E6 protein with chemical carcin-ogens.

The ras family oncogenes have been detected with highfrequency in both human (26) and animal (4, 31) cancers. The

* Corresponding author. Mailing address: ForschungsschwerpunktAngewandte Tumorvirologie, Abteilung Virale Transformationsmech-anismen, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld280, D-69120 Heidelberg, Germany. Phone: 496221/424919. Fax:496221/424902. E-mail: i.helfrich@dkfz.de.

† Present address: Texas Heart Institute, Houston, TX 77303.

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mouse model system for skin tumorigenesis has been invalu-able in dissecting tumor development into distinct stages, i.e.,initiation, promotion, and progression (17, 46). Molecularanalyses of the stages of chemical skin carcinogenesis haveindicated that the H-ras gene is a major target for a mutationevent that takes place at the time of initiation (4, 5). MutantH-ras alleles are found in a high proportion of premalignanttumors, indicating that the H-ras mutation is an early event (4).Furthermore, the specific mutations observed depend on thenature of the carcinogen used as an initiator (5, 35), suggestinga direct interaction between the carcinogen and the H-rasgene. Treatment with chemical carcinogens, specifically with7,12-dimethylbenz[a]anthracene (DMBA), mainly resulted inmutations and the frequent accumulation of tumor cells har-boring a nucleotide transversion at position 2 of H-ras codon61 (35). Also, mutations in K-ras have been reported after theuse of DMBA–12-O-tetradecanoylphorbol-13-acetate (TPA)during a classical two-stage mouse carcinogenesis in H-ras nullmutant mice (22). Therefore, we examined the mutation ratesof H-ras and K-ras oncogenes for potential codon 12, 13, and61 mutations in tumors obtained from MnPV E6 transgenicmice and nontransgenic littermates exposed to the two-stageskin carcinogenesis protocol. In both mouse lines, no muta-tions were found for K-ras in codons 12, 13, and 61. Tumorsobtained from nontransgenic animals revealed H-ras muta-tions at position 2 in codon 61 in 70%, in contrast to 24%, ofMnPV E6 transgenic mice. Most notably, no mutated H-rasgene was found in any malignant tumors expressing high levelsof MnPV E6. The same was true for a cell line established froma squamous cell carcinoma of the MnPV E6 transgenic-mouseline after chemical carcinogenesis.

MATERIALS AND METHODS

Generation of transgenic mice. Mice were purchased from Charles River Wiga(Sulzfeld, Germany). We generated hemizygous MnPV E6 transgenic mice in aninbred FVB/N genetic background by injection of vector-free DNA into a pro-nucleus of fertilized oocytes. Transgenic animals were identified by PCR (30cycles of 94°C for 60 s, 55°C for 60 s, and 72°C for 120 s) with DNAs obtainedfrom tail biopsies (44) using primers ATV11 and ATV12, specific for the lacZreporter gene, together with primers Mgp1 and Mgp2 for the endogenous myo-genin gene as a control. The MnPV E6 transgene was assembled in pBluescriptKS (Stratagene, La Jolla, Calif.) by inserting a 2-kb XhoI fragment carrying thepromoter of human cytokeratin-14 and a 650-bp BamHI-EcoRI fragment con-taining the rabbit �-globin intron from plasmid pHR2 (44) (a generous gift of S.Werner, ETH Zurich, Zurich, Switzerland), a 440-bp HindIII fragment with thecomplete open reading frame of the MnPV E6 gene obtained by PCR from aMnPV genomic construct (42) (kindly provided by E. Amtmann, DeutschesKrebsforschungszentrum, Heidelberg, Germany), a 3.9-kb SpeI-XbaI fragmentfrom plasmid pIRESlacZ containing the encephalomyocarditis virus internalribosome entry site (IRES) element (provided by G. Sczakiel, University ofLubeck, Lubeck, Germany), and the lacZ gene including a simian virus 40polyadenylation signal from plasmid BGZ40 (45). For injection, the 7-kb MnPVE6 transgene construct was excised from the vector by cutting with the restrictionendonucleases SalI and NotI.

Analysis of transgene expression. Transgene expression from the bicistronicMnPV E6 transgene was assessed by visualizing �-galactosidase reporter enzymeactivities with X-Gal (5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside) onwhole embryos or on 5- to 10-�m-thick cryostat sections of adult mouse skin orskin tumor tissue (20). Expression of the E6 transgene was monitored by reversetranscriptase (RT) PCR performed on total RNA from primary keratinocytecultures isolated from 2- to 3-day-old mice. Briefly, the skin of each animal wasfloated on 0.25% trypsin in phosphate-buffered saline (PBS) at 4°C overnight.The epidermis was peeled off, minced, and stirred for 25 min in KeratinocyteSFM (Life Technologies, Karlsruhe, Germany) supplemented with 0.05 mMCaCl2. After filtration of the cell suspension through nylon nets, the cells were

pelleted for 15 min at 1,200 � g, washed with PBS, and seeded onto fibronectin-and collagen-coated plates in Keratinocyte SFM with 0.05 mM CaCl2, 50 �g ofamphotericin B (Fungizone)/ml, and 20 �g of gentamicin/ml. The cells weremaintained at 37°C in a humidified atmosphere with 5% CO2. Dead and differ-entiated cells were washed off with PBS the next day. Total RNA was preparedfrom primary keratinocytes or from M. natalensis skin tumor tissue using Trizol(Life Technologies) according to the manufacturer’s instructions. Samples weretreated with DNase I and reverse transcribed with SuperScript II (Life Technol-ogies) using the primer ATV175 or T22. After the removal of RNA by RNase H(Roche, Mannheim, Germany), PCRs for E6 were performed with Taq polymer-ase and ATV176 and ATV177 primers for 30 cycles (94°C for 30 s, 50°C for 45 s,and 72°C for 60 s). Control reactions for �-actin employed PCRs with primersATV49 and ATV50 for 27 cycles (94°C for 30 s, 55°C for 45 s, and 72°C for 60 s).Control PCRs were routinely carried out using samples without prior reversetranscription. The PCR products were visualized on ethidium bromide-stainedagarose gels.

Two-stage mouse skin carcinogenesis. Induction of tumor development wasperformed on 6-week-old female MnPV E6 transgenic mice of the stronglyE6-expressing line HP22-42 and their nontransgenic littermates. The dorsal skinsof the mice were shaved 1 week prior to topical application of a single dose of theinitiator DMBA (0.1 �mol dissolved in 100 �l of acetone) or acetone only (18).Starting 1 week later, the dorsal skins were treated twice weekly with the tumorpromoter TPA (5 nmol in 100 �l of acetone) or acetone alone for 20 weeks. SixtyMnPV E6 transgenic and 56 nontransgenic mice were treated with DMBA-TPA.Controls receiving acetone or acetone-TPA consisted of 10 MnPV E6 transgenicanimals and 10 nontransgenic littermates per treatment. DMBA-acetone con-trols had 37 animals per group. The tumor incidence and number were recordedindividually for each mouse once per week. All of the animals were observeduntil week 36. Mice that developed tumors with a diameter of �1 cm weresacrificed. Tumors were scored by morphological appearance as papillomas orkeratoacanthomas/squamous cell carcinomas at the time of collection. Papillo-mas and all other tumors were reexamined histologically on hematoxylin-eosin-stained paraffin sections for detailed diagnoses. A part of each tumor tissue wasfrozen in liquid nitrogen for further analyses.

Analysis of tumor data. The average number of papillomas per animal wascalculated by dividing the total papilloma count by the total number of survivinganimals. The papilloma incidence was monitored over time as the percentage ofpapilloma-bearing animals in a group. The distribution functions describingcarcinoma and keratoacanthoma incidences were based on individual animalobservations and were determined by using the Kaplan-Meier estimator. Differ-ences in carcinoma and keratoacanthoma occurrence times between MnPV E6transgenic and wild-type mice were evaluated using the log rank test (13).

Detection of H-ras mutation at position 2 in codon 61. The presence of theactivating A-to-T transversion of the second nucleotide of codon 61 of the H-rascoding region generates a diagnostic XbaI restriction enzyme recognition site,which was identified through PCR-based RNA and DNA analyses after isolationfrom tumor tissue. Following RNA isolation using Trizol (Life Technologies),the samples were treated with DNase I and reverse transcribed with SuperScriptII using primer ATV192. After treatment with RNase H, PCRs for H-ras wereperformed with Taq polymerase and ATV190 and ATV192 primers for 35 cycles(94°C for 30 s, 65°C for 30 s, and 72°C for 30 s). Control reactions for �-actinusing primers ATV49 and ATV50 and reactions without prior reverse transcrip-tion were made to exclude amplification of genomic DNA. For DNA isolation,the tumors were digested with 0.5 mg of proteinase K/ml in lysis buffer (100 mMNaCl, 10 mM Tris [pH 7.5], 1 mM EDTA, 1% sodium dodecyl sulfate) at 55°Covernight. After phenol-chloroform extraction and ethanol precipitation, theresulting DNA was subjected to 30 cycles of PCR (94°C for 30 s, 60°C for 30 s,and 72°C for 30 s) to amplify the respective H-ras fragment using ATV190 andATV192 primers. PCR products from RNA and DNA were gel purified anddigested with XbaI. The resulting fragments were separated by electrophoresison 5% MetaPhor agarose gels (Biozym, Hessisch Oldendorf, Germany).

Sequencing analysis of Ras gene mutation. Isolated RNAs from papillomas,keratoacanthomas, and squamous cell carcinomas of MnPV E6 transgenic miceand wild-type controls were used as templates for RT-PCR. PCRs for H-ras usingATV193 and ATV194 primers for 35 cycles (94°C for 30 s, 64°C for 30 s, and72°C for 30 s) and K-ras using ATV195 and ATV196 primers for 35 cycles (94°Cfor 30 s, 62°C for 30 s, and 72°C for 30 s) were performed. PCR products fromRNA were gel purified using a Qiaex II gel extraction kit (Qiagen). Nucleotidesequences containing codons 12, 13, and 61 of the H-ras or K-ras gene weredetermined by dye terminator cycle sequencing (Applied Biosystems) using theprimers listed below.

Southern blot analysis. RNAs isolated from papillomas, keratoacanthomas,and squamous cell carcinomas of MnPV E6 transgenic mice were used as tem-

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plates for RT-PCR. PCRs for MnPV E6 and �-actin were performed usingprimers ATV176, ATV177, ATV49, and ATV50 in one reaction for 29 cycles(94°C for 30 s, 50°C for 45 s, and 72°C for 60s) and separated on 1.5% agarosegels in the presence of ethidium bromide. The amplified PCR products weretransferred to GeneScreen Plus membranes (DuPont NEN). For hybridization,2.5 ng of transgenic MnPV E6 construct and 2.5 ng of linearized pHF�A1 usingBamHI were labeled with [32P]dCTP by random priming using the HexaLabelPlus DNA-labeling kit (MBI Fermentas). The filters were washed in 2� SSC (1�SSC is 0.15 M NaCl plus 0.015 M sodium citrate) including 0.1% sodium dodecylsulfate at 68°C and exposed by phosphorimager (Amersham Pharmacia Biotech).The counts were quantified using ImageQuant (Amersham Pharmacia Biotech).

PCR primers. The DNAs were synthesized in an Applied Biosystems synthe-sizer using phosphoramitide chemistry and further purified by high-performanceliquid chromatography.

The following primers were used: ATV11 (5�-GCGACTTCCAGTTCAACATC-3�), ATV12 (5�-GATGAGTTTGGACAAACCAC-3�), ATV49 (5�-ACCCACACTGTGCCCATCTACGA-3�), ATV50 (5�CTTGCTGATCCACATCTGCT-GGA-3�), ATV175 (5�-AGACGGCAATATGGTG-3�), ATV176 (5�TGCACATTCTGCTCGAGGTT-3�),; ATV177 (5�-CCCTCTTTCTGCACACTCTA-3�), ATV190 (5�-CTGGAGGCGTGGGAAAGAGTG-3�), ATV192 (5�-CTGTACTGATGGATGTCCTCGAAG-3�), ATV193 (5�GCAGCCGCTGTAAAAGCTAT-3�), ATV194 (5�-GTCCTCGAAGGACTTGGTGT-3�), ATV195 (5�GGCCTGCTGAA-AATGACTGAG-3�), ATV196 (5�-CTAACTCCTGAGCCTGTTTCGTG-3�), Mgp1 (5�-CCAAGTTGGGTCAAAAGCC-3�), and Mgp2 (5�-CTCTCTGCTTTAAGGAGTCAG-3�).

RESULTS AND DISCUSSION

Phenotype of K14MnPV E6 transgenic mice. In order toanalyze the role of a cutaneous papillomavirus type in skintumor formation, we generated transgenic mouse lines carry-ing the MnPV E6 gene under the control of the human cyto-keratin-14 promoter. This directs transgene expression to thebasal layer of the skin (Fig. 1A) (43). The mRNA transcribedfrom the transgene was designed as a bicistronic message. TheMnPV E6 gene constituted the first open reading frame, andthe lacZ reporter constituted the second open reading frame,preceded by an encephalomyocarditis virus IRES element forefficient translation of the lacZ reporter. This permits easy andfast detection of transgene expression by monitoring �-galac-tosidase activities in transgenic animals. Initial analysis ofMnPV E6 transgenic mouse lines hemizygous for the trans-gene and derived from different founders was performed bywhole-mount staining of 15.5-day-old embryos with the chro-mogenic �-galactosidase substrate X-Gal. This revealed thepresence of �-galactosidase in developing hair follicles andepidermis, a pattern expected for the activity of the cytokera-tin-14 promoter at this stage (Fig. 1B and C) (9). Variations inthe strength of transgene expression in 15.5-day-old embryosamong eight expressing MnPV E6 transgenic lines corre-sponded to �-galactosidase reporter activities observed in cry-ostat sections of the respective adult skins (Fig. 1B to E).�-Galactosidase activities were detected throughout the epi-dermal layers of adult skin in the strongly expressing lines,reflecting the production of large amounts of this enzyme andits stability throughout differentiation processes of the mouseepidermis (Fig. 1D). �-Galactosidase activity in skin sections ofweakly expressing MnPV E6 transgenic lines was typicallypatchy and confined to the basal layer of the epidermis (Fig.1E). Expression of the MnPV E6 gene was analyzed by RT-PCR. Reproducibly, the RT-PCR signals for E6 were strongestin those lines with the highest �-galactosidase activities. Thispoints to equal MnPV E6 expression levels and �-galactosidase

reporter activities, as expected from the translation of thebicistronic E6 IRES-lacZ transcript (Fig. 1F).

Mice from different MnPV E6 transgenic lines displayed notransgene-dependent phenotype and did not develop sponta-neous tumors by the age of 2.5 years. The strong E6-expressinglines HP22-42, HP21-2, and HP21-13 were used for furtheranalyses. For a complete phenotyping of the MnPV E6 trans-genic animals, hematoxylin and eosin-stained paraffin sectionsfrom the skin of different regions of the body (back, tail, andear) and also from the tongue, salivary glands, esophagus,thyroid gland, heart, liver, lung, spleen, small intestine, thymus,adrenal glands, and pancreas of 10-day- and 3-, 6-, 12-, 24-, and36-month-old MnPV E6 transgenic mice and wild-type con-trols were prepared (three animals per time point). We foundthat MnPV E6 transgenic mice were histologically indistin-guishable from their nontransgenic littermates. They showedno increase in proliferation, as was also demonstrated by im-munohistochemical staining using Ki-67 antibody (data notshown). Thus, the MnPV E6 transgene expression per se didnot perturb normal skin differentiation and tissue homeostasis.This is in contrast to results obtained with HPV16 E6 trans-genic mice (41), where two out of five transgenic lines showeda consistent thickening of the epidermis due to hyperprolifera-tion of the basal keratinocytes. A small percentage of theseHPV16 E6 transgenic mice developed spontaneous, mostlymalignant skin tumors by the age of 1 year (41). The differ-ences between HPV16 E6 and transgenic MnPV E6 lines maybe indicative of distinct biological properties of MnPV E6 as“skin-type” papillomavirus in comparison to anogenital high-risk-type HPV16 E6.

MnPV E6 increases the yield of squamous cell carcinomasafter treatment with chemical carcinogens and primarily en-hances malignant progression. High-risk papillomavirus E6protein cooperates with a second viral oncoprotein, E7, duringtumor formation and immortalization or transformation ofprimary human cells (16, 30, 40). The expression of the E6protein of HPV16 alone was found to affect malignant tumorprogression in chemically induced skin tumors (40). We choseto test MnPV E6 transgenic mice of the strongly positive lineHP22-42 in a two-stage mouse skin carcinogenesis assay usingDMBA as an initiator and TPA as a tumor promoter. Thistreatment results in the formation of papillomas, keratoacan-thomas, and squamous cell carcinomas almost exclusively as aconsequence of the clonal selection and expansion by TPA ofkeratinocytes carrying a DMBA-induced mutation in the H-rasgene (8, 35).

In MnPV E6 transgenic mice and wild-type littermates,treatment with DMBA and TPA predominantly produced pap-illomas. Less frequent tumor types were diagnosed as squa-mous cell carcinomas and keratoacanthomas (Fig. 2). The in-cidence of papillomas and the papilloma number per survivinganimal were monitored weekly over a period of 36 weeks afterDMBA initiation. The mean papilloma number per survivinganimal was associated with large variability (variation coeffi-cient, 80 to 120% in both groups). No significant differenceswere found between the numbers and incidences of papillomasfor transgenic and wild-type mice (Fig. 3A and B). MnPV E6transgenic mice, however, developed 27 squamous cell carci-nomas, while only 2 squamous cell carcinomas grew in thenontransgenic animals. Most squamous cell carcinomas ap-

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peared after cessation of TPA treatment between weeks 21 and36. By using the Kaplan-Meier estimator, taking into accountanimals that had to be sacrificed prior to the end of the ob-servation time due to large carcinoma size, the percentages ofanimals with carcinomas were calculated at the end of thetreatment to exceed 90% among MnPV E6 transgenic micewhile reaching �10% among the nontransgenic control mice(Fig. 3C). The difference in carcinoma incidence evaluatedover the whole study period was highly significant (log ranktest; P � 0.0001).

A statistically significant difference (P 0.02) was also ob-served for the incidence of keratoacanthomas: MnPV E6transgenic mice developed keratoacanthomas earlier thantheir nontransgenic littermates (Fig. 3D). No tumors devel-oped in groups of mice treated with acetone only, DMBA-acetone, or acetone-TPA. This result suggests that the MnPVE6 transgene acts neither as an initiator nor as a tumor pro-moter of mouse skin carcinogenesis.

In a second MnPV E6 transgenic mouse line, HP21-2, weobtained essentially the same results by using the same proto-

FIG. 1. Transgene expression in MnPV E6 transgenic mice. (A) Schematic representation of the transgene construct. K14, promoter of thehuman cytokeratin-14 gene; �-glo, 5� untranslated region of the rabbit �-globin gene with intron; E6, coding region of the MnPV E6 gene; IRES,IRES element of encephalomyocarditis virus; LacZ, Escherichia coli lacZ gene encoding the reporter enzyme �-galactosidase; pA, simian virus 40polyadenylation signal. (B and C) Reporter gene expression monitored by �-galactosidase activities in 15-day old transgenic mouse embryos(lateral views). Transgene expression in embryos from the lines with the strongest expression, such as HP22-42 (B), was seen in the epidermis anddeveloping hair follicles all over the body, whereas in embryos of weakly expressing lines, such as HP22-12 (C), transgene expression was restrictedto the epidermis of the extremities, face, ears, and prominent early-developing hair follicles, such as the whiskers. (D and E) In the skin of adulttransgenic mice, expression of the transgene was confined to the epidermis and hair follicles. The scale bar in panel E corresponds to 50 �m (Dand E) and 0.9 mm (B and C). Nontransgenic animals showed no �-galactosidase activities on day 15 of development or in adult skin (not shown).(F) E6 expression in primary keratinocytes from MnPV E6 transgenic lines analyzed by RT-PCR. Lanes: wt, wild-type control; Mn tumor, positivecontrol with RNA from MnPV-infected tumor tissue from M. natalensis; HP22-42, HP21-2, HP21-13, HP22-3, HP22-21, HP22-12, HP21-23, andHP21-24, RT-PCR products with RNAs from different MnPV E6 transgenic lines. Top, PCR products for the amplified MnPV E6 gene; bottom,amplified fragments of the �-actin control. No PCR products were obtained with samples without RT.

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FIG. 2. Histological analysis of skin tumors from MnPV E6 transgenic mice developed during chemical carcinogenesis. All tumors were paraffinembedded, cut into 2-�m-thick sections, and stained with hematoxylin-eosin. (A) Papilloma developed during week 12 of chemical-carcinogenesistreatment. The papillomas showed hyperplasia of the squamous cell layer (left arrow), a typical hyperkeratinized layer (middle arrow), and anintact basal membrane (lower arrow) (magnification, �50). (B) Squamous cell carcinoma developed during week 10 of treatment (overview). Thearrow shows the infiltration of the carcinoma into the underlying dermis (magnification, �25). (C) Enlargement of boxed portion of panel Bshowing typical carcinoma-like areas, including cells with enhanced mitosis (upper arrow). Infiltrations from carcinoma into the muscle layer areclearly visible (middle and lower arrow). (D) Keratoacanthoma developed during week 12 of treatment (overview) (magnification, �25) showinga horn-filled crater and clear demarcation of the muscle layer (lower arrow). (E) Enlargement of boxed area in panel D showing dysplastic epithelia(upper arrow), keratin pearls (middle arrow), and inflammation areas (lower arrow), typical characteristics of keratoacanthomas (magnification,�25).

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col (data not shown). Thus, our data point to an involvementof the MnPV E6 protein in the malignant progression of chem-ically induced skin neoplasias (41).

Given that the MnPV E6 protein is involved in malignantexpression, we reasoned that the expression level of the trans-gene may be increased in squamous cell carcinomas carryingnormal ras alleles. To scrutinize this assumption, RNAs frombenign and malignant tumors of MnPV E6 transgenic micewere isolated for RT-PCR using specific primers for MnPV E6and �-actin (Fig. 4B). As depicted in Fig. 4C, all squamous cellcarcinomas lacking an H-ras mutation expressed at least athreefold-higher steady-state level of E6 (as calculated byphosphorimager analysis) than benign tumors, using �-actin asan internal reference. In addition, we also examined a cell lineestablished from a DMBA-TPA-induced squamous cell carci-noma of the MnPV E6 transgenic line HP22-42 for the rele-vant mutation of H-ras (Fig. 4, HP22-42 scc cell line). In agree-ment with the tumor data, these cells also expressed high levelsof MnPV E6 and contained nonmutated ras alleles.

The molecular mechanism by which E6 enhances malignantprogression remains unknown. The ability of E6 proteins fromhigh-risk anogenital HPV types to interfere with p53-regulatedpathways and to cause genomic instability may be involved inthis process. It has indeed been reported that the absence ofp53 enhances the malignant progression of DMBA-TPA-in-duced tumors (23). Moreover, high-risk HPV E6 protein isknown to neutralize p53 by binding to p53 and the ubiquitinligase E6-AP, thereby recruiting the latter to p53 and trigger-ing its proteasome-mediated degradation (38). Interactionwith E6-AP and an efficient degradation of p53 has not yetbeen shown for E6 proteins of cutaneous HPV types (12).Further studies are required to test whether MnPV E6 caninduce p53 degradation or whether it interferes with p53-reg-ulated processes.

H-ras gene is not activated in chemically induced skin tu-mors with high expression of MnPV E6. The majority of tu-mors generated according to the two-stage carcinogenesis pro-tocol are known to harbor DMBA-induced activating ras

FIG. 3. Time course of tumor formation in mice treated initially with 100 nmol of DMBA, followed by treatment with 5 nmol of TPA asdescribed in the text. The number of tumors was examined weekly to the end of the observation time. MnPV E6 transgenic mice (solid lines)developed similar average numbers of papillomas per mouse (A), with time courses (B) similar to those of nontransgenic (wild-type) controls(shaded lines) during the 21 weeks of initiation and promotion. After the end of tumor promotion, the average number of papillomas decreasedin both groups due to spontaneous regression of papillomas (not shown). MnPV E6 transgenic mice showed a highly significant increase in thepercentage of carcinoma-bearing animals compared to wild-type controls throughout the time of the experiment (C), as well as an earlierappearance of keratoacanthomas (D). No tumors were found in mice with mock treatments.

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mutations (14, 22, 33, 35). The most frequent mutation re-sulted in a CAA-to-CTA transversion of codon 61 in the H-rasgene (11). In addition, an activated H-ras was found to coop-erate with HPV18 E6 in malignant cell transformation in tissueculture experiments (1). To investigate whether an activatingmutation of H-ras is involved in tumor development in MnPVE6 transgenic animals, we PCR amplified the relevant part ofthe H-ras gene after RNA and DNA isolation and digested theproduct with XbaI, allowing the detection of activating muta-tions at position 2 in codon 61 by virtue of an XbaI restrictionfragment length polymorphism (24). Seventy-one tumors, in-cluding papillomas, keratoacanthomas, and squamous cell car-cinomas, derived from MnPV E6 transgenic mice and 45 tu-mors from control mice were screened for mutations by thismethod. We found mutations of H-ras at the second position ofcodon 61 in 24% of MnPV E6 transgenic tumors, in contrast to70% in tumors obtained from nontransgenic mice. Figure 5Ashows a panel of tumors received from nontransgenic andMnPV E6 transgenic mice (papillomas, keratoacanthomas,and squamous cell carcinomas), revealing in some tumors amutated H-ras allele in a heterozygous state. To determinewhich allele was transcribed, RT-PCR was performed. Theratio of the transcription efficiencies of the two H-ras allelescorresponds to that obtained in DNA analysis (compare Fig.5A and B).

Additionally, we evaluated the tumors for the presence ofother activating mutations of the H-ras and K-ras genes, espe-cially codons 12, 13, and 61, by PCR amplification and directsequencing of PCR products (Table 1). In all tumors of MnPVE6 transgenic mice and wild-type controls, no mutations werefound in codons 12, 13, and 61 of K-ras. Also, no mutation incodons 12 and 13 of H-ras was detectable, in accordance withprevious results using DMBA (22, 35). Thirteen of 34 E6transgenic papillomas contained an A-to-T transversion at the

second position of codon 61, in comparison to 24 out of 34 inthe nontransgenic littermates, where single CAA-to-CTT andCAA-to-wAG transversions were also found (w indicates thatboth nucleotides A and T were detected at the same position).A lower mutation rate was also recognized for transgenickeratoacanthomas. Four of 14 MnPV E6 transgenic kerato-acanthomas harbor the A-to-T mutation in the middle base of

FIG. 4. H-ras activation in comparison to MnPV E6 expressionlevel. Tumors induced during two-stage carcinogenic treatment weretested for H-ras activation. All MnPV E6 transgenic squamous cellcarcinomas containing high levels of E6 had no mutations in H-ras.(A) Selection of PCR products for amplified H-ras gene in squamouscell carcinomas (scc), keratoacanthomas (kerato.), and papillomas(pap.) derived from MnPV E6 transgenic (tg) mice. The HP22-42 scccell line was established from a squamous cell carcinoma developedduring tumorigenic treatment. No mutations in H-ras were detected inmalignant MnPV E6 transgenic tumors or the HP22-42 cell line.(B) For quantification of the MnPV E6 expression level, PCR wasperformed using specific MnPV E6 and �-actin primers for reference.(C) Hybridization of signals (shown in panel B) using Southern blot-ting technique followed by phosphorimager quantification.

FIG. 5. Activation of H-ras in skin tumors developed in lineHP22-42 of MnPV E6 transgenic mice during chemical carcinogenesis.The presence of the activating A-to-T transversion of the second nu-cleotide of codon 61 of the H-ras coding region generates a diagnosticXbaI restriction enzyme recognition site. Tumors were analyzed usingrestriction fragment length polymorphism. The following cell lineswere used for control: SN161, derived from squamous cell carcinoma(a gift from A. Balmain, University of California—San Francisco);MK2 immortalized BALB mouse keratinocytes (a generous gift fromS. Aaronson, National Institutes of Health); and 3P2, derived from asquamous cell carcinoma induced in NMRI mice by chemical carcino-genesis using DMBA-aplysiatoxin (R. Schmidt, unpublished data).(A) PCR products for amplified H-ras gene in tumors (papillomas[pap.], keratoacanthomas [kerato.], and squamous cell carcinomas[scc]) derived from wild-type (wt) controls and MnPV E6 transgenic(tg) mice and control cell lines. (B) RT-PCR products for H-ras geneamplified after RNA isolation in the same tumors and cell lines as forpanel A.

TABLE 1. ras gene mutations in skin tumors

Genotype HistologyaMutant

gene andcodon

MutationbNo. of mutanttumors/totalno. tested

Wild-type Papilloma 0/34H-ras 13 0/34H-ras 61 CAA 3 CwA 24/34H-ras 61 CAA 3 CTT 1/34H-ras 61 CAA 3 wAG 1/34

Keratoacanthoma H-ras 12 0/9H-ras 13 0/9H-ras 61 CAA 3 CwA 4/9

SCC H-ras 12 0/2H-ras 13 0/2H-ras 61 CAA 3 CwA 1/2

E6 transgenic Papilloma H-ras 12 0/34H-ras 13 0/34H-ras 61 CAA 3 CwA 13/34

Keratoacanthoma H-ras 12 0/14H-ras 13 0/14H-ras 61 CAA 3 CwA 4/14

SCC H-ras 12 0/23H-ras 13 0/23H-ras 61 CAA 3 CwA 0/23

a SCC, squamous cell carcinoma.b w indicates that both nucleotides, A and T, were detected at the same

position. No mutations were found in codons 12, 13, and 61 of K-ras.

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codon 61, in contrast to 4 of 9 tumors in the wild-type controls.Of particular interest is the mutation incidence in malignanttumors. One of the two carcinomas obtained in wild-typemice harbored an A-to-T transversion at position 2 of codon61. In contrast, no mutations were detectable in 23 testedsquamous cell carcinomas of the MnPV E6 transgenic mouseline.

The majority of papillomas and keratoacanthomas and allsquamous cell carcinomas in MnPV E6 transgenic animalscarry normal ras alleles. Nevertheless, their formation criticallydepended on initiation by DMBA, which induces an activatingras mutation in wild-type skin. This may indicate that in MnPVE6 transgenic mice, the development of ras-mutated papillo-mas is impaired in the presence of the transgene protein. Also,keratinocytes initiated by DMBA-induced genetic alterations,other than ras mutations, appear to be preferentially selectedand expanded to papillomas with normal ras alleles, which inaddition exhibit an accelerated transgene-dependent malig-nant progression. The wild-type papillomas show the knownlow progression rate. This observation may point to an addi-tional function of the MnPV E6 protein in tumor promotion ofDMBA-initiated keratinocytes with normal ras alleles.

ACKNOWLEDGMENTS

We thank I. Moll for help with histological preparations, K. Wayssfor tumor material from M. natalensis, A. Balmain (University ofCalifornia—San Francisco) for providing SN161 cells, S. Aaronson(National Institutes of Health, Bethesda, Md.) for MK2 cells, A. Hun-ziker for sequencing the tumor samples, H. Poepperl for generation ofthe MnPV E6 transgenic mice, and B. Goedecke and G. Schulze forexcellent animal care. M. Chen was supervised by H. Poepperl.

This work was supported by the Bundesministerium fur Gesundheit,Bonn, Germany.

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