Suprabasal expression of the human papillomavirus type 16 oncoproteins in mouse epidermis alters expression of cell cycle regulatory proteins

James F. Crish1, Frederic Bone1, Sivaprakasam Balasubramanian1, Tarif M. Zaim4, Thomas Wagner6, Jeung Yun6, Ellen A. Rorke7 and Richard L. Eckert1,2,3,4,5,8

1 Department of Physiology and Biophysics,
2 Department of Biochemistry,
3 Department of Reproductive Biology,
4 Department of Dermatology,
5 Department of Oncology and
7 Department of Environmental Health Sciences, Case Western Reserve University School of Medicine, 2109 Adelbert Road, Cleveland, OH 44106-4970 and
6 Edison Biotechnology Center, Athens, OH, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human papillomavirus (HPV) survives by reactivating DNA replication in post-mitotic cells. In the present study, we describe a mouse model of HPV-dependent disease. In these mice, DNA synthesis is activated in suprabasal keratinocytes, leading to acanthosis, parakeratosis and enhanced desquamation. The full-length E6/E7 transcript and two alternately spliced products are produced and in most lines the predominant product is E6*. In the present study, we examine the effects of E6/E7 on cell cycle regulatory protein expression. E6/E7 expression in mouse epidermis is correlated with increased levels of the p53, p21, p27, cdk2, cdk4, cdk6, cyclin D1 and cyclin E regulatory proteins. Hyperproliferation is also observed in the buccal mucosa and the tongue epithelia of E6/E7 mice, and p53 levels are markedly increased in these epithelia. These results suggest that the major changes in cell cycle regulatory protein expression are in response to the presence of E7 and that E6 has a lesser impact.

Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; HPVs, human papillomaviruses.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human papillomaviruses (HPVs) are double-stranded DNA tumor viruses that induce hyperproliferative lesions in cutaneous and mucosal epithelia. Various HPV types have been implicated as causative agents in the genesis of epithelial diseases in laryngeal, oral, epidermal and cervical epithelia (1,2). HPVs encode two proteins, E6 and E7, that are important for cell immortalization (3). E6 and E7 interact with the tumor suppressor proteins p53 and pRb, respectively. E6 facilitates degradation of p53 through association with an accessory protein, E6-AP, a component of the ubiquitin proteolytic degradation pathway (46). p53 normally acts to increase the levels of p21 (7), a cyclin kinase inhibitor (8,9). A reduced p53 level results in less p21 expression with an accompanying loss of cell cycle checkpoint regulation. E7 binds to pRb, and the related pocket proteins p107 and p130, leading to functional inactivation of these proliferation regulators (10,11). In quiescent cells, pRb is complexed to the transcription factor E2F (12,13). Approaching S phase of the cell cycle, pRb becomes hyperphosphorylated, causing release of E2F, which then activates expression of growth-associated genes. E7 bypasses this pRb-dependent control by binding to pRb and causing the non-physiological release of active E2F (12).

Recent studies have significantly advanced our understanding of the effects of E6 and E7 on the keratinocyte phenotype and on the level of cell cycle regulatory proteins. In normal stratifying surface epithelia, keratinocytes migrate out from the basal layer towards the body surface. The suprabasal cells exit the cell cycle and undergo a process of biochemical and structural remodeling that results in specific changes in gene expression and alteration of cell morphology (14). The morphological changes include loss of cell organelles, including the nucleus, and assembly of the cornified envelope (14,15). The ultimate fate of these cells is desquamation from the body surface. Biochemical changes include the differentiation-dependent expression of markers of keratinocyte differentiation. For example, the involucrin and transglutaminase genes, and the keratin K1 and K10 genes are turned on when cells differentiate, while the {alpha}4/ß6 integrin, K5 and K14 genes are turned off (reviewed in refs 16,17). In HPV-infected tissue, this differentiation process is altered and the keratinocytes do not completely differentiate.

In cultured keratinocytes, E6 (18) and E7 (1923) have been reported to regulate differentially the level and/or activity of cell cycle regulatory proteins. These studies implicate the E7 protein as being primarily responsible for these phenotypic changes and the loss of ability to exit the cell cycle (2426). Moreover, these studies suggest that the HPV promoter activates transcription in the differentiated epithelial layers (26). Some of these changes have been confirmed in studies of human tumor tissues (27); however, the effects of E6/E7 on cell cycle regulatory protein level have not been examined using an animal model of HPV-dependent disease. We have targeted HPV16 E6/E7 expression to the epidermal suprabasal layers using a promoter that mimics the pattern of HPV transcription. We use these animals to study the effect of E6/E7 reading frame expression on cell cycle regulatory protein expression.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibodies
Mouse monoclonal antibodies specific for cyclin D1 (sc-246), p27/Kip1 (sc-1641), p53 (sc-099), pRb (sc-102) and p21/Waf1 (sc-6246), rabbit polyclonal antibodies specific for cyclin E (sc-481), cyclin-dependent kinase (cdk) 2 (sc-163) and cdk 6 (sc-7181), and a goat polyclonal antibody for cdk 4 (sc-601) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at dilutions specified by the manufacturer.

Preparation of buccal, tongue and epidermal cell lysates
Mice were killed by cervical dislocation, the dorsal skin was removed, and the epidermis was separated from the dermis by scrapping. The epidermis was homogenized in ice-cold lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5% NP-40, 1% Triton X-100, 1 mM PMSF, 10 aprotinin and 10 µg/ml leupeptin, pH 7.4) and placed on ice for 30 min. The lysate was centrifuged at 15 000 g for 15 min at 4°C and the supernatant (total cell lysate) was used immediately or stored at –80°C. Buccal epithelium (25 mg) and tongue epithelium (100 mg) were harvested by scraping (buccal) or slicing (tongue) and homogenized in 50 or 200 µl of lysis buffer, respectively. The samples were held on ice for 30 min, clarified by centrifugation for 10 min at 15 000 g and stored at –80°C.

Immunoblot analysis
For immunoblot analysis, cell lysates (25 µg protein) were electrophoresed on denaturing 6–10% polyacrylamide gels (28), and the separated proteins were transferred to nitrocellulose membranes. To block non-specific binding, membranes were incubated in 10 mM Tris–HCl pH 7.2 containing 100 mM NaCl, 0.1% Tween-20 and 5% non-fat dry milk. The blot was incubated with the appropriate primary antibody for 1h at 25°C or for 12h at 40°C. Antibody binding was detected by incubation with the appropriate horseradish peroxidase-conjugated anti-mouse IgG (Calbiochem, La Jolla, CA) or anti-rabbit or anti-goat IgG (Santa Cruz Biotechnology) at 1:5000 dilution for 1 h at 25°C. The blots were washed and antibody binding was visualized by chemiluminescent detection methods. Relative band intensity was compared by densitometry.

Construction of hINV-E6/E7-SV40 transgene and production of transgenic mice
The complete HPV16 genome cloned at the BamHI site in pUC8, kindly provided by Dr Harold zur Hausen (29), was digested with KpnI/EcoRI to release a 1330 bp fragment containing the E6/E7 coding region. This fragment was transferred to KpnI/EcoRI-restricted pUC19 to yield pUC19HPV16 (E7453/K880)1. This plasmid was digested with EcoRI/PpuMI to release the HPV16 upstream regulatory region, which was replaced with a double-stranded DNA linker designed to reconstruct the E6 transcription start site beginning at HPV16 nucleotide 79 (29). The resulting plasmid, pUC19HPV16 (79/K880)1, was digested with BamHI/KpnI and the E6/E7 insert was transferred to BamHI/KpnI-restricted pKSM13(-). The pKSM13(-)HPV16 E6/E71 plasmid was digested with XbaI/KpnI and the E6/E7 reading frame fragment was ligated with BamHI/XbaI-restricted pUC19 and a BamHI/KpnI fragment from pECE (30) containing the SV40 transcription terminator. The resulting plasmid, pUC19HPV16 E6/E7-SV401, contains an E6/E7-SV40 cassette as a HindIII/BamHI fragment. pINV-E6/E7-SV40 contains the involucrin promoter linked to the E6/E7 reading frames and an SV40 terminator. This plasmid was produced by replacing the luciferase reporter gene in pINV-2473 (31,32) with the E6/E7-SV40 cassette using HindIII/BamHI. The hINV-E6/E7-SV40 transgene was released by digestion with NheI/BamHI and purified. The purified fragment was injected into one-cell embryos derived from B6SJL (C57BLxSJL) mice. The presence of the transgene in newborn mice was assessed by Southern blotting of tail DNA (33).

Histological methods
For histological analysis, tissue was collected and fixed immediately in 10% formalin in phosphate-buffered saline. The samples were dehydrated in ethanol, transferred to xylene and embedded in paraffin. Four-micrometer sections were prepared and stained with hematoxylin and eosin prior to examination.

BrdU labeling
5-Bromo-2'-deoxyuridine (BrdU) labeling was performed by injecting animals intraperitoneally (100 µg/g body weight) with 5 mg/ml of BrdU (Sigma) in 10 mM Tris–0.9% saline–1 mM EDTA pH 8. After 60 min, the animals were killed. Tissues obtained by biopsy were fixed, processed, embedded in paraffin and sectioned. Sections (5 µm) were deparaffinized, rehydrated and immersed in 2 N HCl for 1 h, followed by a tap water rinse and equilibration in phosphate-buffered saline. The sections were blocked with normal goat serum and then incubated with anti-BrdU antibody (Br-3, Cal-Tag) diluted 1:50 for 12 h at 4°C in phosphate-buffered saline. Antibody binding was detected using a peroxidase–avidin–biotin complex (Vector Elite) and 3,3'-diaminobenzidine (Sigma, St Louis, MO).

Detection of HPV RNA expression
HPV16 transcript production was detected by RT–PCR using the primers and PCR conditions described by Falcinelli et al. (34). Mouse epidermis was shaved and the excised skin was placed in Hank's balanced salt solution containing 10 mg/ml dispase (35). After 4 h at 4°C, the epidermis was removed by scraping and frozen in liquid nitrogen. After epidermal pulverization, the total RNA was isolated using the single-step method (36). Total RNA (1 µg) was reverse transcribed in a 20 µl reaction containing 50 mM Tris–HCl pH 7.6, 60 mM KCl, 1 mM dithiothreitol, 0.5 µg oligo-dT primer, 8 mM ddNTPs, 40 U RNasin, 10 mM MgCl2 and 25 U AMV reverse transcriptase (Boehringer Mannheim, Germany). After 1 h at 42°C, the reaction was boiled for 2 min and diluted in 100 µl of sterile H2O. A 5 µl aliquot was then used as a PCR template using HPV16-specific primers (34). These primers produce bands of 395, 213 and 95 bp for the full-length, E6* and E6** products. The PCR reactions contained 1 µM each primer, 0.8 mM dNTPs, 60 mM Tris–HCl pH 8.5, 3.5 mM MgCl2 and 1.25 U Taq polymerase (Boehringer Mannheim) in a 50 µl reaction. The reaction was pre-incubated with 275 ng Taq Start antibody for 15 min at 25°C (Clontech). The PCR cycling was 2 min at 94°C, 2.5 min at 55°C and 2.5 min at 72°C for 35 cycles. The first and last cycles, respectively, included a 5 min denaturation at 94°C and a 4.5 min extension at 72°C. PCR products were detected by electrophoresing a 10 µl aliquot on a 2% agarose gel for Southern blot analysis.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
E6/E7 expression produces acanthosis in stratifying epithelia
HPV16 E6/E7 expression was targeted to stratified epithelia in mice using the vector shown in Figure 1Go. Eleven independent HPV16-positive mouse lines were developed. These animals displayed visible epidermal phenotypes including hair loss, and epidermal scaling. The severity of these phenotypes varies among the lines from most to least severe as follows: 22, 54 > 12, 15 > 62, 64, 71 > 56, 49, 33, 30. Virtually 100% of the animals display acanthosis, parakeratosis and hyperproliferation in epidermis and in other stratifying surface epithelia. The E6/E7 animal epidermis is much thicker (10–15 cell layers) compared with the epidermis of non-transgenic littermates (Figure 2Go). Figure 3Go shows that numerous suprabasal cells incorporate BrdU in the transgenic animals, indicating cell division in the suprabasal layers. Although not shown in these sections, BrdU-positive cells were also detected in the basal layer. Basal BrdU incorporation was also observed in normal littermates (not shown).



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Fig. 1. Structure of involucrin-E6/E7 targeting vector. hINV-E6/E7 was constructed as outlined in Materials and methods. It contains the human involucrin gene promoter (33,50) driving transcription of the HPV16 E6/E7 reading frames. Transcription is terminated by the SV40 transcriptional terminator. (B) Nucleotide sequence surrounding the fusion point. The upstream box contains sequence derived from the involucrin promoter, unboxed sequences are derived from linker and the downstream box show sequences derived from HPV16. The arrow indicates the transcriptional start position of the viral P97 promoter.

 


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Fig. 2. Histological phenotype of the E6/E7 mouse epidermis. Epidermis from line 22 E6/E7 and control mice was sectioned, fixed and stained with hematoxylin–eosin. The extent of the epidemis is indicated (Epi) and the arrows point to the cells of the epidermal basal layer.

 


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Fig. 3. Suprabasal BrdU uptake in E6/E7 mice. Twelve-week-old (line 22) E6/E7 mice were injected with BrdU and after 60 min the animals were killed. Biopsy samples were fixed, embedded and sectioned. The sections were then immunostained using a BrdU-specific antibody. The BrdU-positive cell nuclei are stained brown. The arrows indicate the epidermal basal layer.

 
Epidermal expression of HPV16 E6/E7 reading frames
The E6/E7 coding region of HPV16 is known to be alternately spliced within the E6 reading frame (3741). We assayed for HPV transcript production by RT–PCR. Figure 4Go compares HPV transcript production in the epidermis of several E6/E7 mouse lines. Most lines produce the E6* transcript and lower levels of transcripts encoding full-length E6 and E6** (i.e. 71, 15, 22, 30 and 64). However, the predominant transcript in lines 12 and 56 is full-length E6. HPV16 transcripts from CaSki cells, shown as a control, encode mostly E6* (41).



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Fig. 4. Presence of alternately spliced E6/E7 transcripts. Epidermis was isolated from representative adult mice from E6/E7 lines 12, 15, 22, 30, 56, 64 and 71. Total RNA was prepared from the epidermis and analyzed for the presence of HPV transcripts by RT–PCR. Three primer-dependent bands were detected at 395, 213 and 95 bp, corresponding, respectively, to the full-length E6/E7 transcript and the E6* and E6** spliced transcripts (34). RNA prepared from CaSki cells, an HPV-positive human cervical cell line that transcribes varying amounts of each transcript, was used as a control.

 
Expression of tumor suppressor genes
E6 and E7 are thought to interfere with the function of p53 and pRb, respectively (11,42). We therefore measured the level of p53 and pRb in epidermal extracts. As shown in Figure 5Go, p53 levels are markedly increased in E6/E7 mouse epidermis. Repeated attempts, using multiple antibodies, were made to measure pRb levels, but pRb was not detected in epidermis from normal or E6/E7 mice.



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Fig. 5. Detection of p53 expression in epidermis. Epidermis, including both basal and differentiated layers, was isolated from adult E6/E7 mice (line 22) for the preparation of keratinocyte total cell extracts. Extract samples were layered onto denaturing polyacrylamide gels, and blotted to nitrocellulose. Layering was normalized based on total protein concentration. p53 was detected using specific antibodies. Antibody binding was visualized using ECL detection reagents.

 
Cell cycle regulatory proteins
Cyclin-dependent kinase activity is regulated by cyclins and cyclin-dependent kinase inhibitors. To assess changes in cell cycle proteins, we measured the level of cyclin-dependent kinases. cdk2, cdk4 and cdk6 levels were slightly increased in E6/E7 mouse epidermis (Figure 6Go). As shown in Figure 6Go, cyclin D1, which activates cdk2 and cdk4 (12), is markedly increased in E6/E7 mouse epidermis. In addition to increased cyclin D1 levels, p21 and p27, cyclin-dependent kinase inhibitors, are also elevated (Figure 6Go).



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Fig. 6. Detection of cell cycle regulatory proteins. Full-thickness epidermis was isolated from adult E6/E7 mice (line 22) for the preparation of keratinocyte total cell extracts. Extract samples were layered onto denaturing polyacrylamide gels, and blotted to nitrocellulose. Layering was normalized based on total protein concentration. Cyclin-dependent kinases (cdk6, cdk4, cdk2), cyclin-dependent kinase inhibitors (p27, p21) and cyclin D1 and cyclin E were detected using specific antibodies. Antibody binding was visualized using ECL detection reagents. This experiment is representative of three separate repeats.

 
E6/E7 effects on cervical and oral cavity epithelium
We also examined the effects of E6/E7 reading frame expression in other epithelia. Acanthosis and hyperproliferation were evident in the tracheal, buccal mucosa, tongue and the ectocervical epithelium. In the cervix, this was associated with the formation of fingers that project into the underlying stroma (not shown). In the buccal epithelium (Figure 7Go), HPV16 E6/E7 reading frame expression resulted in marked hyperproliferation. We were able to collect quantities of oral buccal and tongue mucosa to permit monitoring of p53 and p21 levels. As shown in Figure 8Go, p53 levels are markedly increased in E6/E7 mouse buccal and tongue mucosa. p21 was not detected in either buccal or tongue epithelial extracts.



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Fig. 7. Oral epithelial morphology. Oral buccal mucosa from E6/E7 mice (line 22) was sectioned, fixed and stained with hematoxylin–eosin.

 


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Fig. 8. p53 and p21 levels in oral and tongue epithelia. Tongue and buccal epithelial tissue, including basal and suprabasal layers, from E6/E7 line 22 was isolated and extracts containing equivalent quantities of protein were layered onto denaturing polyacrylamide gels, and blotted to nitrocellulose. p53 and p21 were detected using specific antibodies.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Suprabasal expression of HPV16 E6/E7 in stratifying epithelia
HPV consists of an 8 kb circular double-stranded DNA genome that encodes a small number of genes expressed early in the viral life cycle, and at least two genes expressed late in the life cycle (1,2). After infection, the virus exists as an episome, but, in the case of the cancer-causing viral forms, the DNA subsequently becomes integrated into the host cell genome. The majority of cervical tumors contain integrated HPV DNA (1,2) and express RNA encoding the E6 and E7 reading frames. Because of the absence of HPV-dependent disease models in animals, an effort has been made to produce HPV-positive mice using transgenic methods. Several gene regulatory units have been used to target HPV reading frame expression to epidermis and cervix, the major HPV-susceptible tissues, including promoters for keratin 14 (4345), keratin 1 (46) and keratin 6 (47). None of these promoters closely mimic the pattern of HPV expression in human tissues. Each of these promoters, including the modified K1 promoter (46), drive expression in the basal proliferative layer.

Previous studies show that HPV DNA replication and RNA expression are differentiation dependent (i.e. restricted to the upper spinous and granular layers in epidermis) (48,49). This suggests that viral survival depends upon the ability of the virus to reactivate DNA replication in cells that have previously ceased dividing. Based on this evidence, we have developed a mouse model in which HPV16 E6/E7 expression is targeted to the epidermal upper spinous and granular layers (48,49). This was achieved by regulating HPV gene expression using the human involucrin promoter (33,50). Involucrin gene expression is confined to the upper spinous and granular layers in stratifying epithelia (33,5052), a pattern essentially identical to that of HPV.

HPV effects on keratinocyte differentiation/proliferation and tumor formation
Eleven separate animal lines were derived, each displaying a similar phenotype. All animals display extensive epidermal hyperproliferation, acanthosis and parakeratosis. Hyperproliferation is also observed in other epithelia, including the esophageal, vaginal, oral and cervical epithelia, and the increased proliferation is associated with dramatically increased desquamation. BrdU incorporation is observed in the suprabasal epidermal layers and basal layers, and visual inspection reveals mitosis in many suprabasal cells. This pattern is reminiscent of that observed in human HPV-positive epithelia (48,49). DNA synthesis is also present in the suprabasal layers when HPV18 E7-expressing keratinocytes are grown in in vitro raft cultures (26).

Tumor formation is the ultimate manifestation of HPV-dependent disease. In humans, this process can have a very long latency period and tumor development is thought to require alterations in additional genes (53,54). We did not observe epithelial papilloma formation in any of these animals, regardless of age (maximum 1.5 years). This is in contrast to some mouse HPV models where papillomas are observed in older animals (46). It is possible that the absence of tumor formation is related to the fact that the E6/E7 reading frames are expressed in the suprabasal location. The absence of tumor formation is reminiscent of the disease process in human cervix, where hyperproliferation is present in HPV-positive lesions, but tumors are not present early in the disease process (53). It is also consistent with observations indicating that HPV can immortalize cells in culture, but that the cells do not grow in soft agar or form tumors in nude mice (55,56). Tumor formation of cells in vitro is enhanced by secondary oncogene activation (56,57), and in studies in progress we have shown that carcinogen treatment can induce papilloma formation.

HPV16 transcript synthesis
In human tumors and in HPV-positive cultured human epidermal or cervical keratinocytes, E6/E7 mRNA is produced as a full-length transcript, and as two spliced forms, E6* and E6** (58). The predominant transcript is generally E6* (5961), and although all three forms are able to produce E7 product (62), it has been suggested that E6* is most important for efficient E7 expression (63). Each of the seven hINV-E6/E7 mouse lines displays similar epidermal hyperproliferation. In most lines E6* is the major transcript, but in several lines the full-length form is most abundant. In addition, the transcript level varies. Based on an analysis of E6/E7 transcript level and splice variant and phenotype, we conclude that neither the specific splice variant present nor the level of RNA produced appears to influence phenotype severity. In human disease, the association of particular E6/E7 spliced products with certain stages of disease remains controversial (59,64). Our results suggest that an equally severe phenotype can be observed whether E6* or the full-length transcript is the major product.

Alteration of cell cycle control
Epidermal histology indicates that cell proliferation is not appropriately regulated in hINV-E6/E7 mice. To investigate the molecular basis for this dysfunction, we measured the level of several cell cycle regulatory proteins. p53 regulates progression through the cell cycle via effects on cell cycle regulators, including p21 (7). p53 increases p21 levels via a transcriptional mechanism (7). p21, in turn, inhibits cyclin-dependent kinase activity. In E6-positive cells, the E6 oncoprotein enhances p53 turnover (6,65) thus reducing p53 levels and resulting in enhanced cell proliferation. Contrary to expectation, immunological analysis of hINV-E6/E7 mouse epidermis indicates that p53 and p21 levels are markedly increased. This suggests that E6 is either not being produced at significant levels or is not functional. The fact that p21 levels are elevated suggests that the p53 is functional, since p21 is a transcriptional target of p53. The finding of elevated p53 levels is not without precedence. A similar p53/p21-positive phenotype has been reported in E7-expressing cultured human keratinocytes (20). In addition, cyclin-dependent kinase 2 (cdk2) activity is also increased in these cells (21). Taken together, these results suggest that E7, and not E6, is determining the phenotype in our mouse model, a hypothesis that is consistent with E7 functioning as the major HPV immortalizing gene (25,6668).

In addition to elevated p53 level, cyclin D1 and cyclin E levels are markedly increased. The cyclin D1 level is known to be increased in tumors, and this increase is correlated with increased cell proliferation (69,70). However, the molecular events responsible for this increase are not known. It will be important in future experiments to determine whether the cyclin D1 level is increased due to transcriptional mechanisms or to amplification. Cyclin-dependent kinase inhibitors, p21 and p27, are also increased, although the increase is not as substantial as the cyclin D1 increase. Cyclin E levels are also markedly increased, suggesting that events in late G1/S are also being stimulated. It is thus possible, overall, that the increase in cell proliferation is being driven by increased cyclin levels.

To determine the state of cell cycle proteins in other epithelia, we monitored p53 and p21 levels in oral epithelia. Our studies indicate that p53 levels are elevated in the mouse E6/E7-positive oral epithelia. This is again consistent with the absence of a role for p53 in determining the biochemical phenotype. However, in contrast to epidermis, we could not detect p21 in oral tissues. These results suggest that the regulatory mechanisms differ in different epithelia, and future studies will be necessary to identify the underlying mechanisms. This result is consistent with the observation that p53 is detected in HPV-positive oral lesions (69).

In summary, the involucrin promoter can be used to target HPV oncoprotein expression in a manner that is similar to that observed in human HPV infections. Targeting E6/E7 expression to the suprabasal epidermis results in a significant alteration in cell cycle regulatory protein expression, cell morphology and DNA content that suggests that the phenotype is predominantly determined by the HPV E7 protein. Additional studies will be required to identify the mechanism of regulation.


    Acknowledgments
 
This work was supported by a Focused Giving Award from Johnson & Johnson and by a grant from the American Institute for Cancer Research (R.L.E.), and utilized the facilities of the Skin Diseases Research Center of Northeast Ohio (NIH, AR39750).


    Notes
 
8 To whom correspondence should be addressed at: Department of Physiology and Biophysics Rm E532, Case Western Reserve University School of Medicine, 2109 Adelbert Road, Cleveland, OH 44106-4970, USA Email: rle2{at}po.cwru.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 20, 1999; revised December 28, 1999; accepted December 30, 1999.