Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention AACR Conference on Molecular Diagnostics - 2008
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Curran, J. A.
Right arrow Articles by Wilson, J. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Curran, J. A.
Right arrow Articles by Wilson, J. B.
[Cancer Research 61, 6730-6738, September 15, 2001]
© 2001 American Association for Cancer Research

Carcinogenesis

Epstein-Barr Virus Encoded Latent Membrane Protein-1 Induces Epithelial Cell Proliferation and Sensitizes Transgenic Mice to Chemical Carcinogenesis1

John A. Curran, Fiona S. Laverty, Donald Campbell, Jennifer Macdiarmid and Joanna B. Wilson2

Robertson Laboratory, Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom [D. C., J. M., J. B. W.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EBV is found to be associated with 100% of poorly or undifferentiated nasopharyngeal carcinomas, a tumor of epithelial origin. The latent membrane protein-1 (LMP1) of EBV, may play a causal role in the development of this disease. The experiments initiated here were designed to examine the activity of LMP1 in vivo in the epidermis of PyLMP1 transgenic mice in relation to its putative role in carcinogenesis. Transgenic positive epidermis showed a 2–3-fold increase in the mitotic index, coupled with an increased level of expression of proliferative cytokeratin markers (K6 and K14) over controls. These results provide direct evidence that LMP1 induces proliferation in otherwise normal epithelial cells in vivo. To assess the role of LMP1 in tumorigenic progression, transgenic mice were treated topically with chemical carcinogens. PyLMP1 mice were highly sensitive to chemical carcinogens, developing significantly more small papillomas at a faster rate than controls. Furthermore, LMP1 could substitute for 12-O-tetradecanoylphorbol-13-acetate treatment in tumor promotion. However, LMP1 inhibited expansion of the benign lesions and did not enhance progression of the lesions to carcinomas or the progression of these to the more malignant spindle cell carcinomas. These data demonstrate that, early in the carcinogenic process, LMP1 acts as a tumor promoter after chemical initiation; but, paradoxically, it may also introduce a hurdle against expansion or progression of a lesion.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EBV is associated with malignancies of B-cells, epithelial cells, and T-cells, including Burkitt’s lymphoma, Hodgkin’s disease, nasal T-cell lymphoma, gastric carcinoma and NPC3 (reviewed in Ref. 1 ). Of these, EBV is most tightly associated with NPC, particularly the poorly differentiated or undifferentiated forms common to high-incidence regions. Monoclonal viral DNA is present in every malignant cell of these tumors, strongly indicative of an etiological role for the virus in this malignancy.

NPC incidence is greatest in Southern China (up to 4/104/year), of intermediate occurrence (3–10/105/year) among native Alaskans and Inuits, indigenous Southeast Asian populations and among certain populations of North Africa and Arabia. Most other populations have a low incidence of NPC (<1/105/year; Ref. 2 ). The demographic pattern of high NPC incidence along with the anthropological relationship between Southeast Asian and Inuit and native Alaskan populations suggests that there might be genetic loci associated with NPC susceptibility, and there is some evidence to support this idea (3) . In addition, environmental and dietary factors may influence the risk of developing the disease. In particular, the high levels of carcinogenic volatile nitrosamines in the salted fish and other preserved foods eaten by people of high incidence groups has been cited as a potential risk factor (4, 5, 6, 7) . As such, environmental/dietary and genetic factors in addition to EBV infection are likely contributors to NPC risk.

Viral gene expression in the malignant epithelial cells of NPC includes the EBER transcripts (8) and consistent expression of EBV nuclear antigen-1 (9) in the absence of the other nuclear antigens. In addition, LMP1 has been detected in approximately two-thirds of NPC specimens tested, whereas LMP1 mRNA has been detected by reverse transcription-PCR assays in the great majority of NPC biopsies, (10, 11, 12) . LMP2 transcripts are also consistently detected in NPC cells (11 , 13) . In addition, the multiply spliced rightward BamHI-A transcripts (BARTs/CSTs) are abundant in NPC cells (14, 15, 16, 17) and capable of encoding several proteins including RPMS1, A73, BARFO and RK-BARFO (18, 19, 20) .

Genetic deletion studies have revealed that LMP1 is one of the latent genes required for in vitro B-cell immortalization by EBV (21 , 22) . Moreover, LMP1 is oncogenic in nonlymphoid cells, first demonstrated by its ability to induce growth transformation in certain immortalized rodent fibroblast cell lines (23, 24, 25) . Importantly, this oncogenic action extends to epithelial cells. Expression of LMP1 in the epidermis of transgenic PyLMP1 mice induces hyperplasia, an early step in the carcinogenic process (26) . Furthermore, in cultured carcinoma cell lines, heterologous expression of LMP1 leads to reduced serum requirements, loss of anchorage dependence, increased invasive capacity, and, in some cases, inhibition of terminal differentiation (27, 28, 29, 30, 31) . Moreover, growth characteristics of NPC tumors have been correlated with LMP1 expression levels. Detectable LMP1 protein is linked with the expression of EGFR and Ki67 in NPC biopsies (32) , and LMP1-positive NPC tumors appear to grow faster and more expansively than LMP1-negative NPC tumors (33) .

In B-cells, LMP1 has been shown to act similarly to a constitutively active CD40 receptor in prolonging cell survival and supporting proliferation (34, 35, 36) . Like CD40, LMP1 binds to tumor necrosis factor receptor-associated factors 1, 2, 3 and 5 (37, 38, 39, 40) . In addition, LMP1 associates with the tumor necrosis factor receptor-associated factor-associated death domain proteins TRADD and RIP (41 , 42) and to JAK3 (43) . Through these interactions, LMP1 causes the activation of NF{kappa}B, JNK, and STAT proteins (43, 44, 45, 46, 47) , which ultimately results in a plethora of changes in cellular gene expression. In epithelial cell lines, LMP1 can induce the expression of CD40 and CD54 (both expressed in NPC cells; Ref. 48 , 49 ), as well as the antiapoptotic gene A20, EGFR, and IL6 (50, 51, 52) . In vivo, transgenic epidermal expression of LMP1 leads to up-regulation of the proliferative cytokeratin marker, K6 (26) .

In transgenic modeling, the accessibility of the mouse skin makes it an ideal tissue in which to study the carcinogenic process. Moreover, the mouse skin model of multistage carcinogenesis has been well characterized with respect to genetic and epigenetic changes (reviewed in Ref. 53 ). Using chemical carcinogens, the stages of initiation, promotion to a benign tumor (papilloma), malignant conversion, and metastatic invasion have been defined. The most commonly used tumor induction protocol involves a single treatment with the mutagen DMBA and then repeated treatments with the tumor promoter TPA. In virtually all DMBA-initiated tumors, activation of the H-ras gene has been identified as an initiating event (54 , 55) , and cyclin D1 has been shown to be a critical target of activated ras in this system (56 , 57) . TPA treatment then promotes the clonal survival and outgrowth of initiated cells to form an overt lesion. The hyperplastic response of the epidermis to TPA is thought to be mediated in part through sustained direct activation of PKC (58) , but, in addition, the enhanced keratinocyte proliferation rate is linked to the accompanying inflammatory skin response (59) . Subsequent loss of p53 contributes to malignant conversion, and deletion of the INK4 locus is associated with transformation to a highly invasive tumor phenotype. Several of these steps have been modeled by engineering transgenic strains of mice with activated oncogenes or deleted tumor suppressor genes (60, 61, 62, 63, 64, 65) .

Transgenic mice expressing LMP1 in the epidermis displayed the phenotype of epidermal hyperplasia with hyperkeratosis that did not progress to tumor formation, demonstrating that LMP1 is insufficient for carcinogenesis (26) . The phenotype of LMP1-expressing mice is markedly similar to that displayed by mice expressing activated H-ras, TGF{alpha}, or (to some extent) v-fos in the epidermis, all of which demonstrate increased epidermal proliferation (61 , 62 , 64 , 66 , 67) . These observations raise a number of questions relating to the action of LMP1. First, does LMP1 induce hyperplasia by increasing epidermal proliferation, inhibiting differentiation, or both? Second, can LMP1 act as a classical initiator and elicit tumors similar to those observed by TPA promotion of H-ras transgenic mice (68) ; or can LMP1 act as a tumor promoter in the manner of v-fos (69) . Alternatively, LMP1 may act as either initiator or promoter, depending upon the context, as with TGF{alpha}, or possibly act at a later stage to promote carcinogenic progression. To address the role of LMP1 in inducing hyperplasia in vivo, the action of LMP1 in the transgenic mouse epidermis was examined. We show that LMP1 acts by inducing epithelial cell proliferation. Furthermore, to assess the synergistic molecular events associated with LMP1 in carcinogenesis, PyLMP1 mice were treated with a classical regime of chemical carcinogens, revealing that LMP1 can act as a tumor promoter.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
DNA and RNA Preparation, Blotting, and Probing.
Genomic DNA was isolated from mouse tail segments as described previously (26) . Transgene status was tested by slot blot or Southern blot using Biodyne nylon membrane (ICN). For Southern analyses, gels were electroblotted in a Hoeffer electroblotting apparatus as described previously (26 , 70) . Total RNA was extracted from tissues using RNAzol B according to the manufacturer’s instructions (Biogenesis Ltd). Total RNA (20 µg) was electrophoresed and electroblotted essentially as described (26) .

All blots were hybridized with [{alpha}-32P]dCTP-labeled, randomly primed DNA probe fragments (Prime-it II kit; Stratagene) and washed under stringent conditions [0.1x SSC-0.1% SDS (w/v) at 68°C] before autoradiography. Northern blot signals were quantified by phosphorimage analysis; and a radiolabeled GAPDH probe was used to normalize for loading, dividing sample-specific phosphorimage values with the GAPDH value of the same sample. Short cDNA fragments primarily consisting of the 3' noncoding region were used as probes for specific murine cytokeratin sequences (K1, K6, K10, and K14 described in Ref. 26 ). The K17 probe was a 3' 255-bp cDNA fragment (71) . For the transgene probe, a 3-kb HindIII-EcoRI LMP1 fragment from the PyLMP1 transgene was used.

BrdUrd Labeling and Immunodetection.
The protocol used is essentially as described (72) . One h before being killed, BrdUrd (50 µg/g body weight) in 0.9% saline was injected i.p. into mice. Dorsal skin, tongue, and tail tissues were collected (as well as tissue for transgene status determination), fixed in 10% neutral buffered formalin (20 mM NaH2PO4; 46 mM Na2HPO4; and 4% formaldehyde, v/v), and embedded in paraffin. Seven-µm sections were cut and subjected to immunohistochemical staining using a murine anti-BrdUrd antibody (Sigma Chemical Co.), then an antimouse IgG biotinylated second antibody, and finally the Vectastain elite ABC complex (which contains avidin and biotinylated horseradish peroxidase, and which allows the avidin: biotinylated horseradish peroxidase to bind to the biotinylated secondary antibody). Positively stained nuclei (brown) were then detected using DAB (3,3'-diaminobenzidine tetrahydrochloride; Sigma Chemical Co.), and negative nuclei were counterstained with hematoxylin (blue). The number of BrdUrd-labeled nuclei in the interfollicular areas, per field of vision (x312.5) were counted for 10 fields of vision/section, and a mean value for the tissue was obtained. Tissues from at least three different mice in each group were counted in this way, where the mean value of these for the tissue type is referred to as the labeling index. All counts were conducted blind, and transgene status determined subsequently.

Immunofluorescent Staining.
Immunofluorescent examination of PyLMP1 transgenic epidermis was conducted essentially as described previously (9) . Seven-µm sections of snap-frozen epidermis were taken and stored at -20°C on gelatin-coated slides. Sections were blocked for 30 min in 1x goat wash (1.3 M NaCl; 70 mM Na2HPO40.2H2O; 30 mM NaH2HPO4.H2O; 0.5% sodium azide, v/v; 10% goat serum, v/v; 2% Triton X-100, v/v; and 0.5% Tween 20, v/v). The primary antibody used was rabbit polyclonal anti-loricrin with secondary FITC-conjugated goat antirabbit IgG (Sigma Chemical Co.). Sections were mounted with Vectashield (Vector Laboratories) and viewed using a Leitz orthoplan microscope at x312.5.

Chemical Carcinogen Administration.
The protocol used is essentially as described (73) . Mice of PyLMP1 line 53 were backcrossed (from C57Bl/6 strain) into the chemical carcinogen-sensitive FVB strain for this study and mice from a minimum of three backcrosses were used (average, 87.5% FVB at backcross 3). Chemical treatment was initiated with mice 8 weeks of age. Hair from the mouse dorsal region was removed by shaving 24 h before the initial chemical administration. Thereafter, the dorsal skin was carefully and regularly shaved. Twenty-five µg DMBA was applied topically to the dorsal skin in 200 µl of acetone. One week later, mice were treated with the tumor promoter TPA, twice weekly, with 200 µl of 5 x 10-5 M TPA for 20 weeks (regime 1) or, alternatively, not treated further (regime 3). TPA treatment for 20 weeks without DMBA initiation was also conducted (regime 2). DMBA treatment followed by only 4 weeks of TPA treatment constituted regime 4. The number of benign and malignant tumors on each mouse was recorded weekly during treatment and beyond. The benign tumors were categorized according to size, and the number of lesions in size categories 2–4 were counted [category 1, <0.2 mm (not counted); category 2, 0.2–0.5 cm; category 3, 0.5–1.0 cm; and category 4, >1.0 cm diameter]. Analysis of the data from each papilloma size category, from the total papilloma count, and from the total malignant carcinoma count was facilitated by use of the Minitab statistical analysis package. Normal distribution of the data were confirmed using Rankits plots, and differences between the two groups were analyzed for significance using the two-sample t test. All mice were of the same background strain, with negative siblings of the transgenic mice used as controls, and housed and fed under the same conditions in a conventional facility. Lesion counts were conducted blind of transgenic status.

Mice were killed if the tumor load became excessive; if any tumor inhibited movement, caused discomfort, or was located at a natural orifice; if any individual tumor exceeded 20 mm in diameter or became ulcerated; or at the termination of the experiment. Decision to remove an animal from study because of these criteria was taken blind of transgenic status. Sections of tumor samples were collected for culture and snap frozen or fixed in 10% neutral buffered formalin (20 mM NaH2PO4; 46 mM Na2HPO4; and 4% formaldehyde, v/v) for paraffin embedding and histopathological analysis.

Development of Carcinoma Cell Lines.
Primary cells were established from malignant carcinomas by removing the tumor into explant medium (DMEM containing 87 µg/ml penicillin, 117 µg/ml streptomycin, and 2.9 µg/ml Fungizone). The tumor was then transferred to warmed culture medium (DMEM containing 20% FCS, v/v; 2% L-glutamine, v/v; 12 µg/ml penicillin; 97 µg/ml streptomycin; and 1.2 µg/ml Fungizone) in which it was diced with a sterile scalpel. The small pieces were allowed to adhere to a tissue culture flask briefly before the careful addition of 2.5 ml of culture medium. The samples were incubated at 37°C in 5% CO2 for 7 days before an additional 2.5 ml of culture medium were added. After an additional 7 days, visible outgrowth of adherent cells from the tumor segments could be observed. The cells were trypsinized, washed, and reseeded in culture medium into 25-cm3 culture flask until confluent. Thereafter, the immortal cultures were maintained in culture medium with 10% FCS.

Western Blotting.
Protein was extracted from cells using RIPPA buffer [150 mM NaCl, 0.5% NP40 detergent, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM DTA, and 20 mM Tris (pH 7.5)] with protease inhibitors (protease inhibitor cocktail, P2714; Sigma Chemical Co.). Forty µg of protein from each extract was electrophoretically separated by SDS-PAGE (8%) minigels, at 180 V for 40 min. Proteins from the gel were electroblotted onto Millipore Immobilon-P membranes at 65 V for 1 h. The membranes were blocked overnight at 4°C in 15 ml of blocking buffer (0.4% casein, 0.1% Tween 20, and 0.02% NaN3 in PBS), then incubated for 1 h at room temperature with a 1:20 dilution of S12 anti-LMP1 monoclonal antibody (in blocking buffer). The membranes were washed three times for 5 min each in PBS-Tween, and incubated for 1 h at room temperature in secondary antibody [1:1000 dilution of alkaline phosphatase conjugated antimouse IgG (Sigma Chemical Co.) in blocking buffer]. Antibody binding was visualized using the CDP-Star detection system (detailed in Ref. 74 ).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transgenic Mouse Lines.
In the experiments described herein, transgenic mice expressing LMP1 in the epidermis and tongue epithelium were used. Transgene expression was directed using the Py early promoter and enhancer in the construct PyLMP1 and demonstrated by Northern and Western blotting (26) . Tissues were taken from mice of two lines (PyLMP1.5 and PyLMP1.53, also referred to as lines 5 and 53 where line 5 was shown to express LMP1 at a higher level than line 53) and to display a more severe phenotype of epidermal hyperplasia with some hyperkeratosis (likened to the human condition of psoriasis), described previously (26) . Line 5 mice suffered from an additional phenotype (lethal in male animals) resulting from a transgenic insertional mutation at a cellular locus (75 , 76) . As such, all additional in vivo studies have been conducted with line 53. Mice are maintained in two strains, C57Bl/6 and the chemical carcinogen-susceptible strain FVB.

Expression Levels of Markers of Differentiation and Proliferation.
Up-regulation of K6 protein and mRNA was previously noted in the skin of transgenic LMP1 pups and adults of both lines (26) . Several postnatal time points were examined revealing that the up-regulation of both RNA and protein was apparent from 1 day postnatal, with maximal RNA levels reached by day 10. Thus, additional Northern analyses were focused around day 10. Northern blots of total RNA samples from skins of transgenic and control siblings of line 5 mice at postnatal days 8 and 10 were probed with sequences for the murine cytokeratin genes K1, K6, K10, K14, and K17 and then with GAPDH to control for loading (Fig. 1)Citation . Signals were quantified by phosphorimage analysis and adjusted against the loading control, and transgene and control samples were compared. Samples from several mice of these ages have been compared, and the range of ratios is presented in summary in Table 1Citation .



View larger version (83K):
[in this window]
[in a new window]
  		Fig. 1. Cytokeratin expression in line PyLMP1.5. Duplicate Northern blots of 20 µg total RNA samples isolated from line 5 transgenic mouse pups (+) and negative sibling controls (-) are shown. Skin samples were taken from pups at ages 8 and 10 days postnatal, as indicated. The blots were probed with sequences for K1, K10, K17, K6, and K14 (as shown), and all signals were normalized against GAPDH expression for the given samples.

 

View this table:
[in this window]
[in a new window]
  		Table 1 Summary of cytokeratin expression in transgenic LMP1 skin: total transcript levels compared with control siblings

 
Transgenic samples showed a consistent 2-fold increase in K14 transcript levels (a basal-layer marker for proliferation) when compared with controls at both ages day 8 and day 10. This is consistent with our earlier observations of an expanded K14 protein expression pattern in skin sections from LMP1 mice 1 week of age (26) . K6, a marker of hyperproliferation, showed increased up-regulation from day 8 to day 10 of 2-fold to 5–10 fold. Murine K17 is less well characterized; however, we have found that it is constitutively expressed in murine skin6 and does not alter with the transgenic hyperplastic state. Surprisingly, suprabasal markers of differentiation, K1 and K10, showed higher levels of expression at day 8 in the transgenic skin samples compared with controls; however this returned to levels equivalent to the controls by day 10. This observation is consistent with the phenotype in that the hyperkeratosis is most marked in the skin of the trunk in mice that are 1 week of age. In addition, loricrin (the final differentiation protein to be expressed), revealed an expanded expression pattern in conjunction with increased acanthosis (thickening of the squamous epithelium) in the transgenic skin at 1 week of age (Fig. 2)Citation .



View larger version (90K):
[in this window]
[in a new window]
  		Fig. 2. Skin sections from mice 1 week of age. Transgenic positive samples from line PyLMP1.53 (B and D) are compared with negative sibling control samples (A and C). A and B have been stained for loricrin expression. C and D have been taken from mice injected with BrdUrd, and incorporation into proliferating cells was visualized by anti-BrdUrd staining. Note evidence of suprabasal cell proliferation in D (arrowhead). Basement membrane is indicated by a narrow arrow.

 
These results confirm and extend our earlier observations that markers of proliferation are up-regulated in LMP1-expressing epidermis, detectable by both RNA and protein analyses. Interestingly, markers of differentiation are either equivalent to controls or also up-regulated in the LMP1 transgenic dorsal skin. Thus, no evidence for inhibition of differentiation by LMP1 was observed by these criteria.

In Vivo Proliferation.
To address directly whether cellular proliferation was affected in the transgenic epidermis in vivo, cellular DNA synthesis was measured by BrdUrd nuclear incorporation in epidermal cells after i.p. injection. Eight transgenic mouse pups of line 53 and eight control siblings were injected at 1 week of age (when the phenotype is most obvious in the dorsal skin). Three transgenic adults, when there is no apparent phenotype in the dorsal skin, and three control siblings were also tested.

Dorsal skin, tongue, and tail tissues were taken from pups, dorsal skin was taken from adults, and several sections of each tissue were analyzed for BrdUrd incorporation (Fig. 2)Citation . Ten fields of view were counted for interfollicular positively stained keratinocytes for each tissue section, and the mean value of these for the section was determined. The section values for each tissue were collated for the groups of mice and the mean for each transgenic or control group calculated as the labeling index. The labeling indices for the sample groups are presented graphically in Fig. 3Citation . In dorsal skin from pups 1 week of age, epidermal cell proliferation is 2-fold higher in the transgenic compared with the control samples. The labeling indices of 22.94 and 11.22, respectively, show a highly significant difference (two-sample t test; 95% confidence limit; P = 0.0035). As expected, proliferation in the adult epidermis is much lower than in pups; nevertheless the 2-fold difference between transgenic and control samples is maintained, with labeling indices of 4.24 and 2.1, respectively, with a very high significant difference (P = 0.0004). Similarly, transgenic animals showed increased proliferation in tail epidermis and tongue epithelium compared with controls (Fig. 3)Citation . For 1-week tail epidermis, transgenic and control labeling indices were 56.0 and 17.13, respectively, showing a significant 3-fold elevation of proliferation in the transgenic samples (P = 0.026). For 1-week tongue epithelium, where the hyperplastic phenotype is less evident, the labeling indices for transgenic and control samples were 26.75 and 17.16, showing a 1.6-fold elevation in the LMP1 transgenics (P = 0.02).



View larger version (41K):
[in this window]
[in a new window]
  		Fig. 3. Proliferation in the epithelium. BrdUrd labeling indices are graphically presented (with SD bars) of the interfollicular regions of dorsal epidermis, tail epidermis, and tongue epithelium samples taken from transgene-positive mice of line PyLMP1.53 (LMP1+) and negative sibling controls (CONTROLS). Results from mice 1 week of age (d7) and adults are shown.

 
In addition to the 2–3-fold elevation in proliferating cells in the LMP1 transgenic interfollicular epidermis compared with controls, it was also noted that the proliferating compartment was less spatially restricted. In control mice, all stained cells indicative of division were in the basal cell layer of the epidermis, as expected. However, in the LMP1 transgenic epidermis, occasional cells in the suprabasal layers stained positive for BrdUrd incorporation as well (Fig. 2)Citation .

Chemical Carcinogen Treatment.
To ascertain whether LMP1 can act to augment chemical carcinogens or whether it can substitute for either an initiator or a promoter, three treatment regimes were initially conducted using PyLMP1.53 transgenic mice and their negative sibling controls. Treatment 1 involved the classical single initiating treatment with DMBA and then 20 weeks of promoting TPA-treatment (twice/week). Thirty-two transgenic mice and 26 control siblings were treated under this regime and monitored for up to 55 weeks from first treatment. An artificial limit was placed on carcinoma load in view of animal welfare (see "Materials and Methods"), and as such, mice were removed from the study as required, from week 17 onwards. Regime 2 involved 20 weeks of TPA treatment (twice/week) in the absence of DMBA initiation. Transgenic mice and control siblings placed on this regime were monitored for 46 weeks. Regime 3 involved a single DMBA treatment. Transgenic and control mice placed on this regime were monitored for 35 weeks. Lesion formation was counted weekly, and the papillomas were categorized by size (see "Materials and Methods"). Conversion of a papillomatous lesion to a carcinoma was recorded, and confirmation of lesion status by histopathology was conducted after the final sample collection.

All mice in regime 1 developed papillomas (Table 2)Citation . The mean numbers of papillomas/mouse/week in size categories 2–4 and the conversion to carcinoma are shown graphically in Fig. 4Citation . Lesions appeared earlier in the transgenic group, at week 5, compared with the control mice, in which lesions first appeared at week 7. Transgenic mice showed a marked increase in the number of small papillomas (size 2, 0.2–0.5 mm) formed compared with controls (Fig. 4)Citation . The difference became significant at week 13 of the study (two-sample t test; 95% confidence; 56 degrees of freedom; P = 0.017 at week 13), showing increasing significance for the remainder of the study (e.g., P = 0.0007 at week 25 and P = 0.0001 at week 26).


View this table:
[in this window]
[in a new window]
  		Table 2 Papilloma incidence

 


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 4. PyLMP1 mice: chemical carcinogen treatment regime 1. Mice were treated with a single application of DMBA and then 20 weeks of TPA application. Mean numbers of total papillomas or carcinomas forming/mouse are shown. Papillomas are recorded into one of three size categories (2, 3, or 4, where category 2 is 0.2–0.5 cm; category 3 is 0.5–1.0 cm; and category 4 is >1.0 cm diameter), and conversion of a lesion to a carcinoma is recorded in the lower right graph. (Note: the size 2 graph has a different Y-axis scale compared with the other three graphs.) PyLMP1 transgenic mice (boxes), n = 32; negative sibling controls (diamonds), n = 26. From 17 weeks on, mice were removed from the study because of lesion load (as described in "Materials and Methods"), at which point the lesion number was recorded as constant from then on for the given mouse. The observed reduction of the size 2-papilloma load posttreatment is in part attributable to conversion to a carcinoma (or larger papilloma) and in part attributable to the regression of some lesions. {square}, LMP1 +ve; {diamondsuit}, controls.

 
A proportion of these papillomas grew to the larger sizes with time in both groups. With papilloma size category 3 (0.5–1.0 cm), little difference in the number of lesions developing was observed between transgenic and control groups (Fig. 4)Citation , supported by the statistical analysis showing no significant difference in this category. However, surprisingly, control mice developed significantly more papillomas in the size 4 category (>1.0 cm) than the transgenic mice. The difference became significant at week 16 of the study (P = 0.013) and continued for the remainder of the study. Combining the opposing sets of data, more lesions in total formed with time in the transgenic group compared with the controls, but this did not show consistent significant difference until after cessation of the carcinogen treatment at week 21.

The mean number of carcinomas formed per mouse (2.69 for transgenic and 2.81 for control siblings) does not show significant difference (P = 0.86); however, carcinoma load was given an artificial limit in view of animal welfare. Animals were removed from the study because of lesion load from week 17 in the transgenic group and from week 20 in the controls. Consequently, conversion rates of papilloma to carcinoma have been calculated at the week 31 values (allowing time for conversion), when ~65% of animals remained under study (in both groups). At week 31, the mean percentage of carcinomas/total lesions was 6% for the transgenic mice and 12% for the controls, without showing significant difference (P = 0.14). This indicates that LMP1 did not enhance conversion of papillomatous lesions to carcinomas, although more small papillomas were induced.

After histopathological analysis, lesions that were scored as carcinomas were revealed to be predominantly of the squamous cell type. Two of 37 carcinomas from the LMP1-positive group were of the more malignant spindle cell type, with 1 of 31 in the control group, indicating that LMP1 does not lead to an increased progression to the more aggressive spindle cell carcinoma.

Cell lines were established in culture from several of the carcinomas that developed in the transgenic and control mice. LMP1 expression in the transgenic carcinoma cell lines was confirmed (Fig. 5)Citation .



View larger version (46K):
[in this window]
[in a new window]
  		Fig. 5. Proteins were separated by SDS-PAGE (8% gel). LMP1 was detected using the S12 monoclonal antiserum with alkaline phosphatase-conjugated antimouse IgG secondary. EBV-positive Raji B-cells (R) and EBV-negative Bjab (B) cells were used as controls. The Mr 63,000 LMP1 of Raji is Mr ~1,900 larger than the transgenic LMP1 (tgLMP1), which has a 33-bp deletion in the repeat region (26) . Protein extracts were derived from a control (PyLMP1-negative) mouse carcinoma cell line (217) and two PyLMP1-positive carcinoma cell lines (204 and 278). A nonspecific band (Mr ~70) is detected in murine carcinoma cell line extracts.

 
These data show that the PyLMP1 mice are highly sensitive to chemical carcinogen treatment and that LMP1 appears to promote lesion formation. However, paradoxically, LMP1 inhibits expansion of the benign lesion. Moreover, LMP1 does not affect the rate of conversion of papillomas to carcinomas or promote the progression of squamous carcinoma to spindle cell type. Therefore, LMP1 seems to augment the action of chemical carcinogens only in the early stages of lesion formation.

Because the PyLMP1 mouse phenotype shows some similarities with the phenotype of mice transgenic for oncogenic H-ras, regime 2 (20 weeks TPA treatment only) was conducted to address the ability of LMP1 to functionally substitute for oncogenic ras. After 46 weeks of monitoring (including the 20-week treatment period), no (0 of 11) LMP1-positive mice developed lesions, whereas one control animal (1 of 17) developed a single papilloma, at week 29 of the study, which did not progress to carcinoma (Table 2)Citation . Therefore, LMP1 is not able to substitute for the action of oncogenic ras in this system.

To investigate whether LMP1 could substitute for TPA-induced tumor promotion, regime 3 (a single DMBA treatment) was conducted. After 35 weeks of monitoring, four (4 of 14) LMP1-positive mice developed papillomas (1, 1, 2, and 5 papillomas, in each case) whereas no controls (0 of 12) developed lesions (Table 2)Citation . This result suggests that LMP1 may be able to act as a weak tumor promoter.

To investigate this further, a new regime was conducted. Weak (or second-stage) promoter activity can be revealed after minimal TPA treatment (77 , 78) . Regime 4 involved a single DMBA treatment and then 4 weeks of TPA treatment (twice/week). Seven transgenic and six control mice were placed on this regime and monitored for up to 70 weeks (although mice were removed from the study, as described above, from week 31). In this treatment regime, control mice remained papilloma free for 52 weeks; subsequently two mice in this group developed size 1 (<0.2 cm) papillomatous lesions (at the end of the study period), one of which regressed after a few weeks (Fig. 6Citation ; Table 2Citation ). In contrast, 7 of 7 LMP1-expressing mice formed papillomas after the minimal TPA treatment, some of which converted to carcinoma. Mice in the transgenic group developed papillomas from week 9 of the study period, all mice having lesions by week 12. Expression of LMP1 in the epidermis is therefore able to substitute for prolonged TPA promotion to induce lesion formation. As such, LMP1 acts as a weak (or second-stage) tumor-promoting agent in this system.



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 6. PyLMP1 mice: chemical carcinogen treatment regime 4. Mice were treated with a single application of DMBA and then 4 weeks of TPA application. Mean numbers of total papillomas forming/mouse are shown. PyLMP1 transgenic mice (boxes), n = 7; negative sibling controls, (diamonds), n = 6. From 31 weeks, mice were removed from the transgenic group because of lesion load (as described in "Materials and Methods"), at which point the lesion number is recorded as constant from then on for the given mouse.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that LMP1 stimulates epithelial cell proliferation in vivo in the transgenic mouse epidermis and tongue epithelium, with a mitotic index 2–3-fold higher than controls. This has been demonstrated with many samples to yield highly statistically significant values. The result is consistent with the observed induction of the proliferative cytokeratin markers, K6 and K14. This is also entirely consistent with the hyperplastic epidermal phenotype in this line of mice, which is visible to the eye soon after birth and intensifies during the first postnatal week. The increase in the mitotic index of the interfollicular keratinocytes in the epidermis was evident when the mice were 1 week of age and was observed at the same increased level in adults. The visible phenotype on the trunk in these mice diminishes by 2 weeks of age (although transgene expression continues), becoming apparent again in more mature mice only in regions with a normally thick epidermis and low hair follicle density (tail, ears, foot pads, and scrotum). The skin has an enormous capacity for homeostatic regulation, which may explain the diminishing adult phenotype despite the continued underlying induction of proliferation by the LMP1 transgene.

A transient induction of the RNA levels of differentiation markers K1 and K10 was observed in the transgenic skin compared with controls at a time when the juvenile phenotype is maximal. This may underlie the hyperkeratotic aspect of the phenotype. LMP1 has been shown to activate JNK, which regulates transcriptional control through the AP-1 complex. Functional AP-1 sites have been shown to activate the transcription of several epithelial differentiation-specific genes, including K1 and involucrin (79, 80, 81, 82) . In addition, both LMP1 expression and CD40 ligation in epithelial cell lines result in activation of NF{kappa}B (52) . Activation of NF{kappa}B in epidermal suprabasal cells is necessary for growth inhibition and differentiation (83) . As such, LMP1 may be able support differentiation in vivo while promoting proliferation.

We have gone on to show that expression of LMP1 in the transgenic mouse epidermis can act in a tumor-promoting fashion, to augment the action of chemical carcinogens. This is consistent with the ability of LMP1 to induce proliferation. TPA treatment of mouse skin similarly induces proliferation and is thought to act in part by binding to PKC and activating it (mimicking diacylglycerol). Activated PKC is proposed in turn to activate AP-1 (among other substrates) by the dephosphorylation of sites on c-Jun, which are inhibitory to DNA binding. This is mediated by PKC phosphorylation and inhibition of glycogen synthase kinase-3ß, a c-Jun kinase (84) . It is tempting to speculate that LMP1 may bring about a similar effect by the activation of the JNK pathway.

Paradoxically, expression of LMP1 in the epidermis appears to inhibit the expansion of the benign papillomas (to the larger size lesions), although more small lesions have formed. Similarly, the numbers of carcinomas developing in each group were equivalent, despite the greater abundance of small papillomas in the transgenic group. These seemingly opposite activities may be explained in the ability of LMP1 to activate pathways involved in proliferation (such as the JNK pathway) and in epithelial differentiation (such as the NF{kappa}B pathway). Whereas LMP1 may provide the proliferative environment which promotes lesion formation, it could simultaneously present a hurdle to lesion expansion by maintaining cell differentiation or senescence. A prediction of this theory would be that the factors (or the loss thereof) which lead to the inhibition of differentiation or senescence could overcome the hurdle and possibly cooperate with LMP1 in lesion expansion or progression.

There are striking similarities between mice expressing TGF{alpha} in the epidermis (62 , 66) and the PyLMP1 mice. In addition to similarities in pup phenotype and the diminution of phenotype in adults, the mice in both series show an increase in BrdUrd mitotic index in the epidermis (64 , 85) . However, TPA treatment (in the absence of initiating DMBA) of transgenic mice overexpressing TGF{alpha} in the basal layer induced papillomas in the transgenic mice (86 , 87) , which is in contrast with our findings with the LMP1 mice. However, the inability of LMP1 to induce benign papillomas upon TPA treatment alone may be a function of expression level or the precise site of expression of the transgene. We are currently investigating whether the tumor-promoting activity of LMP1 may be mediated through the TGF{alpha}/EGFR pathway.

The importance of the inflammatory response in tumor promotion has been revealed using TNF{alpha}-null mice (88) . TPA treatment of TNF{alpha}-null mice does not result in tumor promotion. Again, it is tempting to suggest that LMP1 may be activating inflammatory signaling pathways through interaction with TRAFs and TRADD to induce lesion formation.

In the viral context in vivo, as a latent and lytic protein, LMP1 may promote cellular proliferation to expand the viral pool and support differentiation to facilitate the lytic cycle. In conjunction with other viral proteins and/or cellular oncogenic mutations that inhibit or redirect differentiation, a potential consequence is tumorigenesis.

NPC has a complex etiology with a possible genetic predisposition, potential contribution by dietary carcinogens, and a viral component. In the transgenic system we have analyzed here, the genetic background of the mice is critical in that different strains of mice show markedly different responses to chemical carcinogens. We have concentrated our efforts on mice bred to a background of genetic predisposition to chemical carcinogens. We have studied one viral component, LMP1, and found that it induces epithelial proliferation in vivo and renders the transgenic mice highly sensitive to chemical carcinogens. Minimizing the carcinogen treatment such that no lesions are formed in control mice nevertheless leads to carcinogenesis in the PyLMP1 mice. LMP1 acts early in the carcinogenic process, increasing the frequency of lesion formation, but can also act to inhibit expansion of the benign lesions, and it does not enhance progression of the lesions. This leads to the hypothesis that LMP1 may be an early factor in the genesis of EBV-associated NPC, which would be consistent with the observation that LMP1 is expressed in preinvasive NPC lesions (89) . Moreover, the expression of LMP1 in EBV-infected cells may increase the risk of lesion formation associated with dietary carcinogens (7) . Whether LMP1 continues to be a tumorigenic factor in later stages remains to be elucidated.


   ACKNOWLEDGMENTS
 
We acknowledge Jürgen Schweizer for supplying the murine K17 plasmid and Stuart Yuspa for the anti-loricrin antibody. We thank Willie Thomson for assistance in sectioning skin samples, Uta Boeger-Brown for discussion during the progress of the work, and David Stevenson for critical reading of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The work was supported by grants from the Royal Society and from the Cancer Research Campaign. Back

2 To whom requests for reprints should be addressed, at Institute of Biomedical and Life Sciences, Division of Molecular Genetics, Robertson Building, University of Glasgow, 54 Dumbarton Road, Glasgow, G11 6NU, United Kingdom. Phone: 44-141-330-5108; Fax: 44-141-330-4878; E-mail: Joanna.Wilson{at}bio.gla.ac.uk Back

3 The abbreviations used are: NPC, nasopharyngeal carcinoma; LMP, latent membrane protein; EGFR, epidermal growth factor receptor; JNK, c-Jun NH2-terminal kinase; NF{kappa}B, nuclear factor {kappa}B; DMBA, dimethylbenzanthracene; TPA, 12-O-tetradecanoylphorbol-13-acetate; PKC, protein kinase C; TGF, transforming growth factor; BrdUrd, bromodeoxyuridine; Py, polyomavirus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AP-1, activator protein-1. Back

6 Laverty and Wilson, unpublished observations. Back

Received 9/ 8/00. Accepted 7/10/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Rickinson A. B., Kieff E. Epstein-Barr virus Ed. 3 Fields B. N. Knipe P. M. Howley P. M. eds. . Fields Virology, : 2397-2446, Lippincott-Raven Philadelphia, PA 1996.
  2. IARC Monographs on the evaluation of carcinogenic risks to humans. Epstein-Barr Virus and Kaposi’s Sarcoma Herpesvirus/Human Herpesvirus 8. World Health Organization, 70: 164-194, 1997.
  3. Lu S. J., Day N. E., Degos L., Lepage V., Wang P. C., Chan S. H., Simons M., McKnight B., Easton D., Zeng Y., et al Linkage of a nasopharyngeal carcinoma susceptibility locus to the HLA region. Nature (Lond.), 346: 470-471, 1990.[Medline]
  4. Poirier S., Ohshima H., de-The G., Hubert A., Bourgade M. C., Bartsch H. Volatile nitrosamine levels in common foods from Tunisia, south China and Greenland, high-risk areas for nasopharyngeal carcinoma (NPC). Int. J. Cancer, 39: 293-296, 1987.[Medline]
  5. Yu M. C., Ho J. H., Lai S. H., Henderson B. E. Cantonese-style salted fish as a cause of nasopharyngeal carcinoma: report of a case-control study in Hong Kong. Cancer Res., 46: 956-961, 1986.[Abstract/Free Full Text]
  6. Yu M. C., Huang T. B., Henderson B. E. Diet and nasopharyngeal carcinoma: a case-control study in Guangzhou, China. Int. J. Cancer, 43: 1077-1082, 1989.[Medline]
  7. Zou X. N., Lu S. H., Liu B. Volatile N-nitrosamines and their precursors in Chinese salted fish—a possible etiological factor for NPC in China. Int. J. Cancer, 59: 155-158, 1994.[Medline]
  8. Gilligan K. J., Rajadurai P., Lin J. C., Busson P., Abdel-Hamid M., Prasad U., Tursz T., Raab-Traub N. Expression of the Epstein-Barr virus BamHI A fragment in nasopharyngeal carcinoma: evidence for a viral protein expressed in vivo. J. Virol., 65: 6252-6259, 1991.[Abstract/Free Full Text]
  9. Smith P. R., Griffin B. E. Transcription of the Epstein-Barr virus gene EBNA-1 from different promoters in nasopharyngeal carcinoma and B-lymphoblastoid cells. J. Virol., 66: 706-714, 1992.[Abstract/Free Full Text]
  10. Smith P. R., Griffin B. E. Differential expression of Epstein-Barr viral transcripts for two proteins (TP1 and LMP) in lymphocyte and epithelial cells. Nucleic Acids Res., 19: 2435-2440, 1991.[Abstract/Free Full Text]
  11. Brooks L., Yao Q. Y., Rickinson A. B., Young L. S. Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP1, and LMP2 transcripts. J. Virol., 66: 2689-2697, 1992.[Abstract/Free Full Text]
  12. Chen F., Hu L. F., Ernberg I., Klein G., Winberg G. Coupled transcription of Epstein-Barr virus latent membrane protein (LMP)-1 and LMP-2B genes in nasopharyngeal carcinomas. J. Gen. Virol., 76: 131-138, 1995.[Abstract/Free Full Text]
  13. Busson P., McCoy R., Sadler R., Gilligan K., Tursz T., Raab-Traub N. Consistent transcription of the Epstein-Barr virus LMP2 gene in nasopharyngeal carcinoma. J. Virol., 66: 3257-3262, 1992.[Abstract/Free Full Text]
  14. Hitt M. M., Allday M. J., Hara T., Karran L., Jones M. D., Busson P., Tursz T., Ernberg I., Griffin B. E. EBV gene expression in an NPC-related tumour. EMBO J., 8: 2639-2651, 1989.[Medline]
  15. Gilligan K., Sato H., Rajadurai P., Busson P., Young L., Rickinson A., Tursz T., Raab-Traub N. Novel transcription from the Epstein-Barr virus terminal EcoRI fragment. DIJhet, in a nasopharyngeal carcinoma. J. Virol., 64: 4948-4956, 1990.[Abstract/Free Full Text]
  16. Karran L., Gao Y., Smith P. R., Griffin B. E. Expression of a family of complementary-strand transcripts in Epstein-Barr virus-infected cells. Proc. Natl. Acad. Sci. USA, 89: 8058-8062, 1992.[Abstract/Free Full Text]
  17. Chen H. L., Lung M. M., Sham J. S., Choy D. T., Griffin B. E., Ng M. H. Transcription of BamHI-A region of the EBV genome in NPC tissues and B cells. Virology, 191: 193-201, 1992.[Medline]
  18. Sadler R. H., Raab-Traub N. Structural analyses of the Epstein-Barr virus BamHI A transcripts. J. Virol., 69: 1132-1141, 1995.[Abstract]
  19. Fries K. L., Sculley T. B., Webster-Cyriaque J., Rajadurai P., Sadler R. H., Raab-Traub N. Identification of a novel protein encoded by the BamHI A region of the Epstein-Barr virus. J. Virol., 71: 2765-2771, 1997.[Abstract]
  20. Smith P. R., de Jesus O., Turner D., Hollyoake M., Karstegl C. E., Griffin B. E., Karran L., Wang Y., Hayward S. D., Farrell P. J. Structure and coding content of CST (BART) family RNAs of Epstein-Barr virus. J. Virol., 74: 3082-3092, 2000.[Abstract/Free Full Text]
  21. Kaye K. M., Izumi K. M., Mosialos G., Kieff E. The Epstein-Barr virus LMP1 cytoplasmic carboxy terminus is essential for B-lymphocyte transformation; fibroblast cocultivation complements a critical function within the terminal 155 residues. J. Virol., 69: 675-683, 1995.[Abstract]
  22. Kaye K. M., Devergne O., Harada J. N., Izumi K. M., Yalamanchili R., Kieff E., Mosialos G. Tumor necrosis factor receptor associated factor 2 is a mediator of NF-{kappa}B activation by latent infection membrane protein 1, the Epstein- Barr virus transforming protein. Proc. Natl. Acad. Sci. USA, 93: 11085-11090, 1996.[Abstract/Free Full Text]
  23. Wang D., Liebowitz D., Kieff E. An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell, 43: 831-840, 1985.[Medline]
  24. Baichwal V. R., Sugden B. Transformation of Balb 3T3 cells by the BNLF-1 gene of Epstein-Barr virus. Oncogene, 2: 461-467, 1988.[Medline]
  25. Moorthy R. K., Thorley-Lawson D. A. All three domains of the Epstein-Barr virus-encoded latent membrane protein LMP-1 are required for transformation of rat-1 fibroblasts. J Virol., 67: 1638-1646, 1993.[Abstract/Free Full Text]
  26. Wilson J. B., Weinberg W., Johnson R., Yuspa S., Levine A. J. Expression of the BNLF-1 oncogene of Epstein-Barr virus in the skin of transgenic mice induces hyperplasia and aberrant expression of keratin 6. Cell, 61: 1315-1327, 1990.[Medline]
  27. Fåhraeus R., Rymo L., Rhim J. S., Klein G. Morphological transformation of human keratinocytes expressing the LMP gene of Epstein-Barr virus. Nature (Lond.), 345: 447-449, 1990.[Medline]
  28. Dawson C. W., Rickinson A. B., Young L. S. Epstein-Barr virus latent membrane protein inhibits human epithelial cell differentiation. Nature (Lond.), 344: 777-780, 1990.[Medline]
  29. Fåhraeus R., Chen W., Trivedi P., Klein G., Obrink B. Decreased expression of E-cadherin and increased invasive capacity in EBV-LMP-transfected human epithelial and murine adenocarcinoma cells. Int. J. Cancer, 52: 834-838, 1992.[Medline]
  30. Hu L. F., Chen F., Zheng X., Ernberg I., Cao S. L., Christensson B., Klein G., Winberg G. Clonability and tumorigenicity of human epithelial cells expressing the EBV encoded membrane protein LMP1. Oncogene, 8: 1575-1583, 1993.[Medline]
  31. Nicholson L. J., Hopwood P., Johannessen I., Salisbury J. R., Codd J., Thorley-Lawson D., Crawford D. H. Epstein-Barr virus latent membrane protein does not inhibit differentiation and induces tumorigenicity of human epithelial cells. Oncogene, 15: 275-283, 1997.[Medline]
  32. Zheng X., Hu L., Chen F., Christensson B. Expression of Ki67 antigen, epidermal growth factor receptor and Epstein-Barr virus-encoded latent membrane protein (LMP1) in nasopharyngeal carcinoma. Eur. J. Cancer B Oral Oncol., 30B: 290-295, 1994.
  33. Hu L. F., Chen F., Zhen Q. F., Zhang Y. W., Luo Y., Zheng X., Winberg G., Ernberg I., Klein G. Differences in the growth pattern and clinical course of EBV-LMP1 expressing and non-expressing nasopharyngeal carcinomas. Eur. J. Cancer, 5: 658-660, 1995.
  34. Gires O., Zimber-Strobl U., Gonnella R., Ueffing M., Marschall G., Zeidler R., Pich D., Hammerschmidt W. Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule. EMBO J., 16: 6131-6140, 1997.[Medline]
  35. Zimber-Strobl U., Kempkes B., Marschall G., Zeidler R., Van Kooten C., Banchereau J., Bornkamm G. W., Hammerschmidt W. Epstein-Barr virus latent membrane protein (LMP1) is not sufficient to maintain proliferation of B cells but both it and activated CD40 can prolong their survival. EMBO J., 15: 7070-7078, 1996.[Medline]
  36. Kilger E., Kieser A., Baumann M., Hammerschmidt W. Epstein-Barr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor. EMBO J., 17: 1700-1709, 1998.[Medline]
  37. Mosialos G., Birkenbach M., Yalamanchili R., VanArsdale T., Ware C., Kieff E. The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell, 80: 389-399, 1995.[Medline]
  38. Franken M., Devergne O., Rosenzweig M., Annis B., Kieff E., Wang F. Comparative analysis identifies conserved tumor necrosis factor receptor-associated factor 3 binding sites in the human and simian Epstein-Barr virus oncogene LMP1. J. Virol., 70: 7819-7826, 1996.[Abstract]
  39. Brodeur S. R., Cheng G., Baltimore D., Thorley-Lawson D. A. Localization of the major NF-{kappa}B-activating site and the sole TRAF3 binding site of LMP-1 defines two distinct signaling motifs. J. Biol. Chem., 272: 19777-19784, 1997.[Abstract/Free Full Text]
  40. Sandberg M., Hammerschmidt W., Sugden B. Characterization of LMP-1’s association with TRAF1, TRAF2, and TRAF3. J. Virol., 71: 4649-5466, 1997.[Abstract]
  41. Izumi K. M., Kieff E. D. The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF- {kappa}B. Proc. Natl. Acad. Sci. USA, 94: 12592-12597, 1997.[Abstract/Free Full Text]
  42. Izumi K. M., McFarland E. C., Ting A. T., Riley E. A., Seed B., Kieff E. D. The Epstein-Barr virus oncoprotein latent membrane protein 1 engages the tumor necrosis factor receptor-associated proteins TRADD and receptor-interacting protein (RIP) but does not induce apoptosis or require RIP for NF-{kappa}B activation. Mol. Cell. Biol., 19: 5759-5767, 1999.[Abstract/Free Full Text]
  43. Gires O., Kohlhuber F., Kilger E., Baumann M., Kieser A., Kaiser C., Zeidler R., Scheffer B., Ueffing M., Hammerschmidt W. Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins. EMBO J., 18: 3064-3073, 1999.[Medline]
  44. Huen D. S., Henderson S. A., Croom-Carter D., Rowe M. The Epstein-Barr virus latent membrane protein-1 (LMP1) mediates activation of NF-{kappa}B and cell surface phenotype via two effector regions in its carboxy-terminal cytoplasmic domain. Oncogene, 10: 549-560, 1995.[Medline]
  45. Mitchell T., Sugden B. Stimulation of NF-{kappa}B-mediated transcription by mutant derivatives of the latent membrane protein of Epstein-Barr virus. J. Virol., 69: 2968-2976, 1995.[Abstract]
  46. Kieser A., Kilger E., Gires O., Ueffing M., Kolch W., Hammerschmidt W. Epstein-Barr virus latent membrane protein-1 triggers AP-1 activity via the c-Jun N-Terminal kinase cascade. EMBO J., 16: 6478-6485, 1997.[Medline]
  47. Eliopoulos A. G., Young L. S. Activation of the c-Jun N-terminal kinase (JNK) pathway by the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1). Oncogene, 16: 1731-1742, 1998.[Medline]
  48. Busson P., Braham K., Clausse B., Tursz T. Constitutive expression of HLA class II antigens on EBV positive malignant cells from nasopharyngeal carcinoma: possible involvement in T cell infiltration. Cancer Detect. Prev., 12: 363-368, 1988.[Medline]
  49. Busson P., Zhang Q., Guillon J. M., Gregory C. D., Young L. S., Clausse B., Lipinski M., Rickinson A. B., Tursz T. Elevated expression of ICAM1 (CD54) and minimal expression of LFA3 (CD58) in Epstein-Barr-virus-positive nasopharyngeal carcinoma cells. Int. J. Cancer, 50: 863-867, 1992.[Medline]
  50. Laherty C. D., Hu H. M., Opipari A. W., Wang F., Dixit V. M. The Epstein-Barr virus LMP1 gene product induces A20 zinc finger protein expression by activating NF-{kappa}B. J. Biol. Chem., 267: 24157-24160, 1992.[Abstract/Free Full Text]
  51. Miller W. E., Earp H. S., Raab-Traub N. The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor. J. Virol., 69: 4390-4398, 1995.[Abstract]
  52. Eliopoulos A. G., Stack M., Dawson C. W., Kaye K. M., Hodgkin L., Sihota S., Rowe M., Young L. S. Epstein-Barr virus-encoded LMP1, and CD40 mediate IL-6 production in epithelial cells via an NF-{kappa}B pathway involving TNF receptor-associated factors. Oncogene, 14: 2899-28916, 1997.[Medline]
  53. Akhurst R. J., Balmain A. Genetic events and the role of TGFß in epithelial tumour progression. J. Pathol., 187: 82-90, 1999.[Medline]
  54. Bizub D., Wood A. W., Skalka A. M. Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons. Proc. Natl. Acad. Sci. USA, 83: 6048-6052, 1986.[Abstract/Free Full Text]
  55. Quintanilla M., Brown K., Ramsden M., Balmain A. Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis. Nature (Lond.), 322: 78-80, 1986.[Medline]
  56. Robles A. I., Rodriguez-Puebla M. L., Glick A. B., Trempus C., Hansen L., Sicinski P., Tennant R. W., Weinberg R. A., Yuspa S. H., Conti C. J. Reduced skin tumor development in cyclin D1-deficient mice highlights the oncogenic ras pathway in vivo. Genes Dev., 12: 2469-2474, 1998.[Abstract/Free Full Text]
  57. Rodriguez-Puebla M. L., LaCava M., Conti C. J. Cyclin D1 overexpression in mouse epidermis increases cyclin-dependent kinase activity and cell proliferation in vivo but does not affect skin tumor development. Cell Growth Differ., 10: 467-472, 1999.[Abstract/Free Full Text]
  58. Zhang G., Kazanietz M. G., Blumberg P. M., Hurley J. H. Crystal structure of the cys2 activator-binding domain of protein kinase C {delta} in complex with phorbol ester. Cell, 81: 917-924, 1995.[Medline]
  59. Schlatterer K., Krauter G., Schlatterer B., Hecker E., Chandra P. Mechanistically different acting tumor promoters induce consistent qualitative and quantitative changes in protein expression patterns of murine epidermis. Anticancer Res., 19: 385-395, 1999.[Medline]
  60. Bailleul B., Surani M. A., White S., Barton S. C., Brown K., Blessing M., Jorcano J., Balmain A. Skin hyperkeratosis and papilloma formation in transgenic mice expressing a ras oncogene from a suprabasal keratin promoter. Cell, 62: 697-708, 1990.[Medline]
  61. Greenhalgh D. A., Rothnagel J. A., Quintanilla M. I., Orengo C. C., Gagne T. A., Bundman D. S., Longley M. A., Roop D. R. Induction of epidermal hyperplasia, hyperkeratosis, and papillomas in transgenic mice by a targeted v-Ha-ras oncogene. Mol Carcinog., 7: 99-110, 1993.[Medline]
  62. Dominey A. M., Wang X. J., King L. E., Jr., Nanney L. B., Gagne T. A., Sellheyer K., Bundman D. S., Longley M. A., Rothnagel J. A., Greenhalgh D. A., et al Targeted overexpression of transforming growth factor-{alpha} in the epidermis of transgenic mice elicits hyperplasia, hyperkeratosis, and spontaneous, squamous papillomas. Cell Growth Differ., 4: 1071-1082, 1993.[Abstract]
  63. Jhappan C., Takayama H., Dickson R. B., Merlino G. Transgenic mice provide genetic evidence that transforming growth factor-{alpha} promotes skin tumorigenesis via H-ras-dependent and H-ras-independent pathways. Cell Growth Differ., 5: 385-394, 1994.[Abstract]
  64. Greenhalgh D. A., Wang X. J., Donehower L. A., Roop D. R. Paradoxical tumor inhibitory effect of p53 loss in transgenic mice expressing epidermal-targeted v-rasHa, v-fos, or human transforming growth factor-{alpha}. Cancer Res., 56: 4413-4423, 1996.[Abstract/Free Full Text]
  65. Brown K., Strathdee D., Bryson S., Lambie W., Balmain A. The malignant capacity of skin tumours induced by expression of a mutant H-ras transgene depends on the cell type targeted. Curr. Biol., 8: 516-524, 1998.[Medline]
  66. Vassar R., Fuchs E. Transgenic mice provide new insights into the role of TGF-{alpha} during epidermal development and differentiation. Genes Dev., 5: 714-727, 1991.[Abstract/Free Full Text]
  67. Greenhalgh D. A., Rothnagel J. A., Wang X. J., Quintanilla M. I., Orengo C. C., Gagne T. A., Bundman D. S., Longley M. A., Fisher C., Roop D. R. Hyperplasia, hyperkeratosis and benign tumor production in transgenic mice by a targeted v-fos oncogene suggest a role for fos in epidermal differentiation and neoplasia. Oncogene, 8: 2145-2157, 1993.[Medline]
  68. Leder A., Kuo A., Cardiff R. D., Sinn E., Leder P. v-Ha-ras transgene abrogates the initiation step in mouse skin tumorigenesis: effects of phorbol esters and retinoic acid. Proc. Natl. Acad. Sci. USA, 87: 9178-9182, 1990.[Abstract/Free Full Text]
  69. Greenhalgh D. A., Wang X. J., Eckhardt J. N., Roop D. R. 12-O-tetradecanoylphorbol-13-acetate promotion of transgenic mice expressing epidermal-targeted v-fos induces rasHA-activated papillomas and carcinomas without p53 mutation: association of v-fos expression with promotion and tumor autonomy. Cell Growth Differ., 6: 579-586, 1995.[Abstract]
  70. Wilson J. B., Drotar M. E. Considerations in generating transgenic mice: DNA, RNA and protein extractions from tissues-rapid and effective blotting Wilson J. B. May G. H. W. eds. . Epstein-Barr Virus Protocols. Methods in Molecular Biology Series, Vol. 174: 361-377, Humana Press Totowa, New Jersey 2001.
  71. Knapp B., Rentrop M., Schweizer J., Winter H. Three cDNA sequences of mouse type I keratins. Cellular localization of the mRNAs in normal and hyperproliferative tissues. J. Biol. Chem., 262: 938-945, 1987.[Abstract/Free Full Text]
  72. Curran J. In vivo assay of cellular proliferation Wilson J. B. May G. H. W. eds. . Epstein-Barr Virus Protocols. Methods in Molecular Biology Series, Vol. 174: 379-389, Humana Press Totowa, New Jersey 2001.
  73. Curran J. Topical chemical carcinogen treatment in mice Wilson J. B. May G. H. W. eds. . Epstein-Barr Virus Protocols. Methods in Molecular Biology Series, Vol. 174: 391-399, Humana Press Totowa, New Jersey 2001.
  74. Rowe M., Jones M. Detection of EBV latent proteins by Western blotting Wilson J. B. May G. H. W. eds. . Epstein-Barr Virus Protocols. Methods in Molecular Biology Series, Vol. 174: 229-242, Humana Press Totowa, New Jersey 2001.
  75. Wilson J. B., Ferguson M. W. J., Jenkins N. A., Lock L. F., Copeland N. G., Levine A. J. Transgenic mouse model of X-linked cleft palate. Cell Growth Diff., 4: 67-76, 1993.[Abstract]
  76. Laverty H. G., Wilson J. B. Murine CASK is disrupted in a sex-linked cleft palate mouse mutant. Genomics, 53: 29-41, 1998.[Medline]
  77. Hennings H., Boutwell R. K. Studies on the mechanism of skin tumor promotion. Cancer Res., 30: 312-320, 1970.[Abstract/Free Full Text]
  78. Chiba M., Aldaz C. M., Conti C. J., Klein-Szanto A. J. Metastatic potential of mouse skin carcinomas produced by different protocols of chemical carcinogenesis. Invasion Metastasis, 11: 288-296, 1991.[Medline]
  79. Lu B., Rothnagel J. A., Longley M. A., Tsai S. Y., Roop D. R. Differentiation-specific expression of human keratin 1 is mediated by a composite AP-1/steroid hormone element. J. Biol. Chem., 269: 7443-7449, 1994.[Abstract/Free Full Text]
  80. Welter J. F., Crish J. F., Agarwal C., Eckert R. L. Fos-related antigen (Fra-1), junB, and junD activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity[published erratum appears in J. Biol. Chem., 271: 11034, 1996]. J. Biol. Chem., 270: 12614-12622, 1995.[Abstract/Free Full Text]
  81. Rossi A., Jang S. I., Ceci R., Steinert P. M., Markova N. G. Effect of AP1 transcription factors on the regulation of transcription in normal human epidermal keratinocytes. J. Investig. Dermatol., 110: 34-40, 1998.[Medline]
  82. Efimova T., LaCelle P., Welter J. F., Eckert R. L. Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway. J. Biol. Chem., 273: 24387-24395, 1998.[Abstract/Free Full Text]
  83. Seitz C. S., Lin Q., Deng H., Khavari P. A. Alterations in NF-{kappa}B function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-{kappa}B. Proc. Natl. Acad. Sci. USA, 95: 2307-2312, 1998.[Abstract/Free Full Text]
  84. Goode N., Hughes K., Woodgett J. R., Parker P. J. Differential regulation of glycogen synthase kinase-3ß by protein kinase C isotypes. J. Biol. Chem., 267: 16878-16882, 1992.[Abstract/Free Full Text]
  85. Wang X. J., Greenhalgh D. A., Lu X. R., Bickenbach J. R., Roop D. R. TGF{alpha} and v-fos cooperation in transgenic mouse epidermis induces aberrant keratinocyte differentiation and stable, autonomous papillomas. Oncogene, 10: 279-289, 1995.[Medline]
  86. Vassar R., Hutton M. E., Fuchs E. Transgenic overexpression of transforming growth factor-{alpha} bypasses the need for c-Ha-ras mutations in mouse skin tumorigenesis. Mol. Cell. Biol., 12: 4643-4653, 1992.[Abstract/Free Full Text]
  87. Wang X. J., Greenhalgh D. A., Eckhardt J. N., Rothnagel J. A., Roop D. R. Epidermal expression of transforming growth factor-{alpha} in transgenic mice: induction of spontaneous and 12-O-tetradecanoylphorbol-13-acetate-induced papillomas via a mechanism independent of Ha-ras activation or overexpression. Mol. Carcinog., 10: 15-22, 1994.[Medline]
  88. Moore R. J., Owens D. M., Stamp G., Arnott C., Burke F., East N., Holdsworth H., Turner L., Rollins B., Pasparakis M., Kollias G., Balkwill F. Mice deficient in tumor necrosis factor-{alpha} are resistant to skin carcinogenesis. Nat. Med., 5: 828-831, 1999.