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[Cancer Research 65, 8826-8835, October 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Epstein-Barr Virus Latent Membrane Protein 1 (CAO) Up-regulates VEGF and TGF{alpha} Concomitant with Hyperlasia, with Subsequent Up-regulation of p16 and MMP9

David Stevenson, Chrystalla Charalambous and Joanna B. Wilson

Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom

Requests for reprints: Joanna B. Wilson, Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, 54 Dumbarton Road, Glasgow, United Kingdom. Phone: 44-141-330-5108; Fax: 44-141-330-4878; E-mail: joanna.wilson{at}bio.gla.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
EBV latent membrane protein 1 (LMP1) is an oncoprotein frequently expressed in nasopharyngeal carcinoma. We have generated transgenic mice expressing the nasopharyngeal carcinoma–derived CAO strain of LMP1 and LMP1 of the B95-8 strain, using the viral ED-L2 promoter for epithelial expression. LMP1CAO and LMP1B95-8 induce transforming growth factor {alpha} expression and epidermal hyperplasia. However, levels of total epidermal growth factor receptor (EGFR) decline with the appearance of phosphorylated EGFR products, suggesting that the negative feedback loop upon EGFR expression is intact or that there is faster turnover at these early stages of carcinogenesis. In the L2LMP1CAO mice, increased levels of vascular endothelial growth factor are also seen at an early stage in the skin. As the phenotype worsens, with increasing hyperplasia and vascularization leading to keratoacanthoma, p16INK4a and matrix metalloproteinase 9 expression is induced. The lesions can progress spontaneously to carcinoma. Carcinoma cell lines developed from these mice show high levels of total and phosphorylated EGFR. These data show that the induction of signaling through EGFR by LMP1 is an early event in carcinogenesis and that any inhibition upon EGFR expression is lifted during progression. Furthermore, expression of LMP1 is not sufficient to inhibit induction of p16INK4a in response to abnormal proliferation. These data are consistent with the cooperative effects seen between LMP1 and loss of the INK4a locus in transgenic mice and with the frequency of loss of this locus in EBV-associated nasopharyngeal carcinoma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EBV is a human herpesvirus that is associated with the pathogenesis of a number of malignancies of lymphoid and epithelial origin, including Burkitt's lymphoma, nasal T-cell lymphomas, AIDS-associated and posttransplant lymphomas, Hodgkin's disease, nasopharyngeal carcinoma, and gastric carcinoma. Nasopharyngeal carcinoma is the malignancy most strongly associated with EBV (1). The disease occurs at high incidence in southern China, among Southeast Asian ethnic Chinese, and among the indigenous populations of Greenland and Alaska. In addition to EBV therefore, there are most likely environmental and/or genetic susceptibilities factorial in the development of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma cells exhibit a type II pattern of latency (1) in which expression is restricted to latent membrane proteins 1, 2A, and 2B (LMP1, LMP2A, and LMP2B); EBV nuclear antigen 1 (EBNA-1); EBV nonpolyadenylated RNAs; and EBV BamHIA rightward-oriented transcripts.

Of these gene products, a prime candidate for involvement in the development of nasopharyngeal carcinoma is LMP1. LMP1 is an oncogene that can transform rodent fibroblasts in vitro (2) and is required for B-cell immortalization by EBV (3). LMP1 is a trans-membrane protein that interacts with signaling molecules involved in the pathways of the tumor necrosis factor receptor family and results in activation of nuclear factor-{kappa}B (NF-{kappa}B; ref. 4), activator protein 1 (AP-1; ref. 5), p38 mitogen-activated protein kinase (6), and Janus-activated kinase/signal transducers and activators of transcription pathways (7).

Viruses harboring variant forms of LMP1 have been identified in nasopharyngeal carcinoma tissues (8, 9). One particular variant, LMP1CAO, has a number of differences relative to the primary amino acid sequence of the prototype LMP1B95-8 (derived from the B95-8 strain of EBV; ref. 10). These include point mutations and amplification of the internal repeat as well as significant deletions. Of interest is a 10-amino-acid (30 bp) deletion in LMP1CAO relative to LMP1B95-8 that lies between the COOH-terminal activating regions 1 and 2, which are domains of LMP1 required for factor binding and signaling (11). Whereas there is evidence suggesting a role for this deletion in disease progression through increased oncogenicity (12), differences in the signaling activity and phenotypic effects of LMP1CAO and LMP1B95-8, particularly enhanced signaling through NF-{kappa}B, altered AP-1 signaling, and lower turnover of the former, map to the NH2 terminus and transmembrane domains of LMP1 (13–16). Moreover, an analysis of the LMP1 sequence in EBV isolates revealed no association of the 30-bp deletion with nasopharyngeal carcinoma or HD over and above the incidence in the wider local populations (17, 18). Nevertheless, LMP1CAO is less immunogenic than LMP1B95-8 (19). Further analyses of the sequence and immunogenicity of nasopharyngeal carcinoma–associated LMP1 variants has suggested that there is selection in tumorigenesis against immunogenic forms of LMP1, such as the B95-8 strain (9, 20).

To model the role of LMP1 in epithelial tumorigenesis, we have generated a series of transgenic mice expressing LMP1 in murine epithelia. Expression of LMP1B95-8 in the murine epidermis under the control of the polyomavirus promoter/enhancer induced a phenotype of epidermal hyperplasia without progression to tumor in both C57Bl/6 and FVB strains of mice (21). LMP1 induced epithelial cell proliferation and augmented the action of 12-O-tetradecanoylphorbol-13-acetate in tumor promotion but inhibited expansion of the benign lesions (22). The inhibitory effects of LMP1 on the growth of these carcinogen-induced papillomas was found to be mediated by a product of the INK4a locus (p16INK4a and/or murine p19ARF), because the inhibition was absent in INK4a-null mice (23). Interestingly, the LMP1-induced lesion growth inhibition was partly alleviated in heterozygous INK4a-null mice, showing that loss of this locus is not completely recessive as had been previously thought.

Whereas LMP1B95-8 has also been shown to induce growth inhibition in SCC12F cells, LMP1CAO did not elicit this effect (13) nor is it as effective in inducing the expression of several cellular genes including CD40, CD54, CD44, IL-6, and IL-8 (13, 16). To explore the tumorigenic potential of LMP1CAO in vivo, we have generated a further series of transgenic mice aimed at expressing LMP1CAO in murine epithelia. LMP1CAO like LMP1B95-8 induces epidermal hyperplasia through proliferation; however, unlike LMP1B95-8, the phenotype of the LMP1CAO mice can progress to benign lesions and even carcinoma. As a result of our observations, we propose that a primary consequence of LMP1 expression in the setting of an intact epithelium is the up-regulation of transforming growth factor {alpha} (TGF-{alpha}) and activation of epidermal growth factor receptor (EGFR). LMP1B95-8 and LMP1CAO lead to this up-regulation, whereas progression of the LMP1CAO phenotype leads to further changes including up-regulation of matrix metalloproteinase 9 (MMP9) and p16INK4a.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids and generation of transgenic lines. The EBV.B95-8 ED-L2 promoter sequences from plasmid pL2 (3-kb ScaI-XhoI; ref. 24) were ligated to LMP1CAO (4.2-kb NcoI-ScaI; ref. 10) to generate pL2LMP1CAO (Fig. 1A). Similarly, the L2 promoter was linked to LMP1B95-8 sequences (21). A 3.6-kb BamHI linear fragment containing L2LMP1CAO and a 3.7-kb BamHI-EcoRI linear fragment containing L2LMP1B95-8 were used for microinjection of zygote pronuclei.



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  		Figure 1. A, diagram of the linear L2LMP1 transgenes used in the generation of lines described herein. Expression is driven by the EBV ED-L2 promoter (shaded) derived from the 3' end of LMP1B95-8. The LMP1 genes comprise three protein-coding exons (black) and two introns (white). B-G, phenotype of animals in the L2LMP1CAO series. B, spontaneous papillomas on the dorsal skin (shaved) of an animal of line 117 (FVB strain). C, cervical lymph nodes derived from transgenic mouse of line 117 (three above) and transgene-negative sibling control (three below). D, runted transgenic pup of line 104 (right) alongside a transgene-negative sibling (left). E, normal tail of wild-type animal. F, "ring-tail" phenotype of transgenic animal of line 117. G, advancing ear phenotype (stages I-V) of line 117 transgenic mice compared with a transgene-negative (WT) sibling of the stage I transgenic mouse (shown). By ~8 weeks of age, most mice of line 117 show ears of stage II phenotype. The phenotype progresses through stages III and IV from 3 to 8 months old.

 
Transgenic animals were generated by the microinjection of linearized DNA into B6D2.F2 zygotes as previously described (21). Tail genomic DNA was screened by Southern blotting following BglII digestion as detailed (25). Blots were probed with radiolabeled transgene linear fragments. Founder animals were bred into the C57Bl/6 strain of mice to produce stable lines and subsequently bred into the FVB strain. Mice were maintained under conventional housing conditions. All procedures have been conducted under UK Home Office license and the research has complied with Home Office and institutional guidelines and policies. In compliance with these guidelines, most animals displaying the ear phenotype at stage V (Fig. 1G; usually <1-year-old for line 117) were removed from study, precluding a thorough analysis of further phenotypes that could develop with age.

Separation of epidermis from dermis. Skin separation of the epidermis from dermis was carried out using dispase as described (26). Essentially, the skin sample was floated dermis side-down on a solution of 0.5% dispase in PBS at 4°C overnight. Subsequently, the dermis was removed from the epidermis with forceps. The separate epidermal and dermal samples were snap frozen in liquid nitrogen.

Western blotting. Proteins were extracted from tissues by polytron disruption of snap-frozen samples using radioimmunoprecipitation assay buffer [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 5 mmol/L EDTA (pH 8.0), 1.5% NP40, 0.1% SDS, 0.5% deoxycholic acid, with protease and phosphatase inhibitors: 2% aprotinin, 5 mmol/L phenylmethylsulfonyl fluoride, 1x Sigma cocktail, 0.5 mmol/L ß-glycerophosphate, 0.5 mmol/L sodium pyrophosphate, 0.5 mmol/L sodium fluoride, 0.5 mmol/L sodium molybdate, 125 ng/mL leupeptin] and incubated for 10 minutes on ice. For LMP1 detection, proteins were extracted from tissues by homogenization in urea extraction buffer [8 mol/L urea, 5% 2-mercaptoethanol, 25 mmol/L Tris-HCl (pH 9.5)] at 55°C overnight with shaking. Following centrifugation at 16,000 x g for 10 minutes, protein concentration of the supernatant was assessed using Bio-Rad protein assay reagent. Protein extract (40-100 µg) was mixed with sample loading buffer [to a final concentration of 62 mmol/L Tris-HCl (pH 6.8), 10% glycerol, 5% 2-mercaptoethanol, 2% SDS, 0.002% bromophenol blue], heated at 95°C for 5 minutes, separated by SDS-PAGE, and electroblotted onto Immobilon P membrane. Blot probing and washing were carried out as described (21, 23). The primary antibodies used for detection of LMP1 were murine ascites S12 (ref. 27; 1:500 dilution), rat anti-CAO cocktail and rat Ig6 (ref. 28; 1:100 dilution) in blocking buffer [TBST: 20 mmol/L Tris-HCl (pH 7.6), 140 mmol/L NaCl, 0.1% Tween 20, with 5% nonfat milk powder]. The secondary antibody was goat-anti-mouse or anti-rat (as appropriate) IgG-horseradish peroxidase conjugate (1:4,000 dilution) using an ECL+ kit (Amersham Laboratories, Amersham, United Kingdom) for detection. Extracts of EBV-positive (IB4 or Raji) or EBV-negative (Ramos or BJAB) human B cells, or a B-cell line derived from a transgenic lymphoma EµLMP1.39 x EµEBNA1.59 (cell line 3959.48) were used as controls for LMP1 expression.

Antibodies used against cellular proteins include anti-total EGFR, anti-phospho-EGFR (Tyr845, Tyr1068; Cell Signaling Technology, Beverly, MA), anti-TGF-{alpha}, anti-p16, anti-MMP9 (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-vascular endothelial growth factor (VEGF, Oncogene Research Products, Boston, MA), all at a dilution of 1:1,000 in PBST (PBS, 0.1% Tween 20) with 5% nonfat milk powder. Secondary alkaline-phosphatase-conjugated antibodies directed against IgG of relevant species (Santa Cruz Biotechnology) were used (1:4,000 dilution).

Immunohistochemistry and pathology. Tissue slices (10 µm, formalin fixed, and paraffin embedded) were stained with H&E, or with proliferating cell nuclear antigen (PCNA) polyclonal rabbit antiserum (Santa Cruz Biotechnology; 1:200 dilution) followed by biotin-conjugated anti-rabbit IgG (Sigma, Poole, United Kingdom). Detection was done using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of transgenic lines and description of phenotype and pathology. Seven transgene-positive founder animals were generated using the L2LMP1CAO construct and identified as animals 99, 104, 105, 106, 107, 112, and 117. Founder 105 was subsequently shown to harbor two independent transgene insertions (one Y chromosome linked, the other autosomal), which segregated into distinct lineages (identified as lines 105A and 105B). Two transgene-positive founder animals (101X and 101Y) were generated using the L2LMP1B95-8 construct.

Many of the founders and derived lines displayed a common phenotype (with varying severity and detail between the different lines), of hyperplasia of the skin, giving a thickened, wrinkly appearance similar to that seen for PyLMP1B95-8-expressing lines (21). When manifest in the tail, this led to the development of constriction rings, referred to here as a "ring-tail" phenotype (Fig. 1F).

L2LMP1B95-8 founder 101Y died at birth, whereas founder 101X survived for 5 days displaying a severe skin phenotype. L2LMP1CAO founders showed a gradation in phenotype severity (Table 1). Founder 99 had a phenotype of scurfy skin and ring-tail and died on day 11. Similarly, founder 107 displayed the ring-tail phenotype and was runted relative to transgene-negative siblings and died on day 9. Founder 104 showed a severe skin and tail phenotype but survived to breeding age, although it remained stunted in its' growth (as described for PyLMP1 line 5; ref. 21). Transgene-positive pups of this line were more severely affected than the founder (indicative of founder mosaicism; Fig. 1D), dying at an early age and thus preventing further propagation of the line. Founder 112 showed a ring-tail phenotype in adulthood but failed to transmit the transgene to offspring.


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  		Table 1. Summary of phenotypes of L2LMP1 mice

 
No juvenile phenotype was noted in founders 105, 106, or 117, but these animals and their offspring (with the exception of animals of line 105A) developed skin phenotypes on the tail and/or ears in adulthood. The ears of lines 117 and 105B display an age-related progressive phenotype (Fig. 1G), showing hyperplasia and becoming visibly hypervascularized and thicker with age. Histopathologic analysis of the ears of lines 117 and 105B show that both dermis and epidermis are expanded in transgenic relative to negative control animals (Fig. 2). Nucleated cells can be seen in the periphery of the corneal layer and most likely represent aberrantly differentiated epithelial cells. A significant increase in the vasculature of the ear is also evident. Additional effects associated with hyperplastic animal skin, such as melanin incontinence, rete pegs, and ulceration, have also been noted, as well as bacterial infection (possibly resulting from scratching). This ulcerative dermatitis gives the appearance of trauma with secondary infection (stage III onwards). As the lesion progresses, the tissue becomes degenerative. Despite this, severe hyperplasia continues, and in several cases, keratoacanthomas (inverted papillomas) form (Fig. 1G, stage IV). In some advanced instances (~5%), squamous cell carcinoma has been noted at stage V. The ear phenotype seems significantly earlier and progresses faster and further in mice of line 117 relative to 105B, and this becomes even more pronounced on breeding into an FVB background. Mice of both lines 105B and 117 have been bred into the FVB strain. At backcross generation 2, ~40% of the mice of line 117 exhibited spontaneous papilloma formation on the dorsal skin (Fig. 1B), not seen in the C57Bl/6 strain background. In rare cases, these dorsal lesions of 117 transgenic mice have been seen to progress to carcinoma. A papillomatous lesion is also infrequently noted on the dorsal skin of mice of line 105B. In addition to the skin phenotype, mice of line 117 develop hyperplastic cervical lymph nodes often containing one or more pseudocysts, invariably appearing in the right cervical lymph node first. By 8 months of age, all transgenic mice of line 117 show at least one expanded cervical lymph node, whereas negative siblings do not (Fig. 2C). No other phenotypes have been noted in these lines (including tissues of nasopharynx, tongue, esophagus, and forestomach) up to the end of study point when mice show the stage V ear phenotype.



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  		Figure 2. H&E staining of ear sections from (A) a line 105B transgenic mouse (stage II/III) and (B) a negative sibling. A, abnormal nucleated cells at the corneal layer and multiple blood vessels (arrows). Immunohistochemical detection of PCNA in ear sections from (C) a line 105B transgenic mouse, (D) a negative sibling to the same magnification as (C), (E) a line 117 transgenic mouse at lower magnification (stage III/IV), and (F) a negative sibling to the same magnification as (E).

 
Mice of line 106 show a mild ring-tail phenotype only. Mice of line 105A show no skin phenotype, but some display a phenotype of weight loss in the older adult, the cause of which is not clear. Histopathology has revealed that this phenotype may be associated with an infiltration of lymphocytes in the esophageal and stomach tissues, but that the epithelium is unremarkable.

All transgenic founders and the mice of the lines derived using the L2 promoter show a phenotype indicative of transgene expression. None of the transgenic lines were phenotype free. As such, the L2 promoter is unusually effective in driving transgene expression, regardless of insertion site. The prominent phenotype of L2LMP1 mice is that of epidermal hyperplasia, noted at differing sites and with a gradation in severity (Table 1). The most severely affected died young or could not be established into lines. The moderately affected in the L2LMP1CAO series nevertheless go on to develop keratoacanthomas and papillomas, which can progress to squamous cell carcinoma.

Increased cell proliferation in the ear epithelium of L2LMP1CAO mice. Tissue sections from ears of transgene-positive and transgene-negative mice of lines 105B and 117 were probed with anti-PCNA antibodies to detect proliferating cells. Greater numbers of PCNA-positive cells indicative of increased proliferation were evident in the epithelium of transgene-positive animals (Fig. 2C-F).

Expression of the transgene. Using standard protein extraction protocols and Western blotting, expression of LMP1CAO was readily detected in the skin of an adult animal of the severely affected line 104 using the S12 antibody that recognizes both LMP1CAO (66 kDa) and LMP1B95-8 (63 kDa; data not shown). However, expression of LMP1 could not be detected by this method from the phenotypic tissues of L2LMP1CAO lines 117 and 105B nor PyLMP1 line 53. Nevertheless, transcription of the transgene was confirmed by reverse transcription-PCR (RT-PCR) from ear tissue of both lines 117 and 105B (data not shown).

Because the tissues primarily affected are highly keratinized, it was postulated that LMP1 might be localized with the keratins and equally difficult to extract. Therefore, a urea extraction protocol, used to extract the hard keratins from hair and skin (29), was adapted. A comprehensive analysis of tissues from mice of the lines 105A, 105B, and 117 by Western blotting was conducted in this manner. Three LMP1-specific antisera have been used to sequentially probe each blot, S12 (27), anti-CAO cocktail (specific to LMP1CAO), and Ig6 (28), to overcome the problem that each (or the anti-mouse/rat secondaries) detects significant but different murine proteins particularly in epithelia (see transgene-negative samples; Fig. 3). In this manner, LMP1CAO expression has been detected in all of the phenotypic tissues and several tissues that do not display a phenotype (Fig. 3 and summarized in Table 2). Expression of the 66-kDa LMP1CAO in the ear tissue of lines 117 and 105B is clear, whereas the nonphenotypic ears of line 105A showed no expression. Detection of a major 50-kDa product (evident in transgenic samples and not in transgene-negative controls) specifically reacting with all three antisera is seen in tissues from line 117, to a lesser extent in 105B and little in 105A. Whereas this product would be consistent in size with lytic LMP1 (lyLMP1) translated from an ED-L1A promoted transcript, the LMP1CAO sequences are polymorphic at the lyLMP1 ATG start codon (being ATT) and cannot therefore express lyLMP1, although the possibility that other internally initiated products are expressed cannot be ruled out. Further smaller LMP1-specific bands (of ~40, 35, 25, and 20 kDa) were detected in transgenic tissues of line 117 that could be breakdown or turnover products (30). Expression of the 66-kDa LMP1CAO product was detected in line 117 most strongly in ear tissue (with expression at all phenotypic stages) and the hyperplastic lymph nodes, but several other tissues showed expression (Fig. 3; Table 2). The smaller LMP1-specific products could also be detected in some tissues (such as lung and heart) without the 66-kDa product evident. Liver, salivary gland, spleen, and brain showed no detectable expression in all three lines. For mice of line 105B, expression of the 66-kDa LMP1CAO product was detected most strongly in ear tissue. Tissues of line 105B mice showed a similar pattern of LMP1 expression to line 117 but at lower level (data not shown; summarized in Table 2). Tissues of mice from line 105A showed a similar pattern of expression of the 66-kDa LMP1CAO with a notable exception, no expression was detected in ear or dorsal skin tissue. This is entirely consistent with the phenotype in that mice of line 105A do not develop any phenotype in the ear or dorsal skin. In conclusion, LMP1 expression driven by the L2 promoter induces predominantly epithelial phenotypes, the degree of which is consistent with the level of expression detected.



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  		Figure 3. Detection of transgene expression using anti-LMP1 antibody S12, by Western blotting of 100 µg of protein extracts of tissues as indicated, derived from a LMP1CAO-positive transgenic mouse (+) compared with a negative sibling (–) from lines 117 (top) and 105A (bottom). Abbreviations: npr, nasopharyngeal region; oesoph., oesophagus; forestom, forestomach. The EBV-positive human B-cell line Raji expressing a 63-kDa LMP1 (arrow) and Ramos (Ram, EBV negative) are used as controls. The 66-kDa LMP1CAO expressed in some transgenic tissues is indicated. This was confirmed by reprobing the blots with different anti-LMP1 antisera as described. Note that in 117 tissues, several smaller LMP1-related products (triangles, at ~50, 35, 25, and 20 kDa) are detected specifically in the transgenic tissues and not the control sibling samples. Protein marker sizes are indicated in kDa.

 

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  		Table 2. Summary of LMP1CAO expression patterns in transgenic mice

 
Epithelial expression of LMP1 leads to up-regulation of transforming growth factor-{alpha} and activation of epidermal growth factor receptor. Both in PyLMP1B95-8 mice developed previously (21) and in the L2LMP1 mice described here, LMP1 induces epidermal hyperplasia resulting from increased proliferation. The phenotypes of these mice bear a striking resemblance to the phenotypes described for transgenic TGF-{alpha}-expressing mice. L2LMP1CAO mice develop severe hyperplasia and thickening of the epidermis and dermis of the ears and the mice develop papillomas at other sites as do HK14.TGF-{alpha} mice with expression of the transgene in the basal layer of the epidermis (31). PyLMP1 mice develop a hyperplastic, hyperkeratotic phenotype as pups, which is less apparent in adults in lower expressing lines, a phenotype exactly as described for HK1.TGF-{alpha} mice with suprabasal epidermal expression (32). This observation led to the hypothesis that LMP1 might mediate its oncogenic action in vivo, at least in part via TGF-{alpha} and its receptor EGFR. Protein extracts from ears of progressing phenotypic stage from both lines 105B and 117 were examined for TGF-{alpha} expression. Whereas TGF-{alpha} expression could not be detected in transgene-negative sibling controls, expression was evident in the transgenic tissues at all stages and in mice of both lines (Fig. 4). Similarly, examination of skin samples separated into the epidermal and dermal layers from mice of line PyLMP1B95-8.53 shows that LMP1 leads to up-regulation of TGF-{alpha} in the epidermis (Fig. 5A). Therefore, expression of both LMP1CAO and PyLMP1B95-8 in the skin leads to the up-regulation of TGF-{alpha}.



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  		Figure 4. Western blotting of ear protein extracts of transgene-negative animals (–) compared with transgene-positive animals of lines 105B and 117. Phenotypic stage of the ears (st1-st5). Protein/track (40 µg) was separated by SDS-PAGE and following transfer, blots were probed with antisera directed against TGF-{alpha}, VEGF, p16INK4a, and MMP9 as indicated. Specific bands are identified (arrows).

 


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  		Figure 5. Western blotting of 100 µg of protein extract/track, separated by SDS-PAGE. A-C, tissue extracts from transgene-negative 5-day-old pups (3–, 4–, and tg–) of PyLMP1B95-8 line 53 compared with transgene-positive siblings of the line (1+, 2+, and tg+). A, 15% gel, epidermal and dermal dorsal skin samples). B, 7.5% gel (epidermal samples), and following electrophoretic transfer, blots were probed with antisera directed against TGF-{alpha} (A) and total EGFR (B). The latter blot was stripped and reprobed with an antiserum directed against phospho-(Tyr845)-EGFR (C). D and E, extracts from ears of transgene-positive animals of line 117 of phenotypic stages I to III (st1-st3) as indicated, compared with a transgene-negative control (c1) matched to the stage I phenotypic animal (7.5% gels). Blots were probed with antisera directed against total EGFR (D) or phospho-(Tyr845)-EGFR (E). Both blots were stripped and reprobed with an antiserum directed against ß-tubulin as a loading indicator (below each). F and G, extracts from murine carcinoma cell lines from transgene-positive (tg+) or negative (tg–) mice of lines 53, 105B, and 117 and a control murine carcinoma cell line CarB. The blot was first probed with antiserum directed against total EGFR (F), then against phospho-(Tyr845)-EGFR (G) and then against ß-tubulin as a loading indicator (bottom). Specific bands are indicated (arrows). Protein marker sizes are indicated in kDa.

 
Expression of EGFR and its phosphorylated forms were also examined. The level of total EGFR was seen to be diminished in the PyLMP1B95-8.53 transgenic epidermis compared with controls, whereas reprobing of the same blot revealed a higher level of smaller products reacting with the anti-phospho-EGFR antibodies (both specific for TyrP845 [data shown] and TyrP1068 [data not shown]), which may reflect turnover of the activated receptor (ref. 33; Fig. 5B and C). Examination of EGFR expression and activation in the phenotypic ears of mice of line L2LMP1CAO.117 revealed that whereas levels of total EGFR may be slightly elevated at stage I (1.36-fold compared with controls, normalized against ß-tubulin), this decreases with advancing phenotypic stage (stage II, 1.1-fold; stage III, 0.85-fold) concomitant with the appearance of the related smaller products (Fig. 5D) and phospho-EGFR (Fig. 5E). In contrast to these data, expression of LMP1 in cultured carcinoma cells has been found to increase EGFR expression levels (15, 34), correlating with the observation that EGFR is overexpressed in nasopharyngeal carcinoma (35). We therefore examined the levels of EGFR in cell lines derived from carcinomas that developed on these mice (both transgene positive and negative) following chemical carcinogen treatment (23). In these cells lines, levels of total EGFR were found to be equivalent or higher in the LMP1-positive cell lines compared with negative, as was detection of phospho-EGFR and the related smaller products (Fig. 5F and G).

Levels of transformation-related proteins in the skin of LMP1CAO mice. Western blot analysis of proteins extracted from the ear tissue of progressive phenotypic stage from mice of lines 105B and 117 was employed to assess the levels of proteins encoded by a number of genes that have been shown to be influenced by LMP1 in other systems. The panel of proteins studied included MMP9 (found to be up-regulated by LMP1 in C33A cells; ref. 36), VEGF (shown to be up-regulated by LMP1 via cyclooxygenase-2; ref. 37), and the tumor suppressor p16INK4a, which has been reported to be down-regulated by LMP1 (38, 39).

Of the several forms of VEGF, a 15-kDa product (possibly VEGF121) was clearly detectable in all transgene-positive samples but not in the transgene-negative samples (Fig. 4), and other VEGF variants may also be differentially regulated. Neither MMP9 nor p16INK4a were detected in transgene-negative extracts, but both were detected in four of six of the transgene-positive samples (Fig. 4). These four samples were derived from older animals with a more advanced ear phenotype (but not carcinoma) from both lines 105B and 117. The timing of these changes suggest that, like TGF-{alpha}, induction of the 15-kDa VEGF form results as an immediate consequence of LMP1 expression, whereas expression of MMP9 and p16INK4a are secondary changes induced in response to the developing phenotype.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report the generation of several lines of transgenic mice expressing LMP1 under the control of the EBV ED-L2 promoter. The ED-L2 promoter has been shown previously to direct expression to stratified squamous epithelia in transgenic mice (21, 24, 40, 41) and here, whereas expression was detected in several tissues, the skin showed the strongest phenotypic consequence of this expression. All founders and lines generated displayed a phenotype to some degree, showing the unusual efficacy of this promoter in the transgenic context.

Both LMP1CAO and LMP1B95-8 variants were used in the generation of mice; however, only two founders were produced following L2LMP1B95-8 microinjection both dying soon after birth. Three of the eight founders/lines produced with L2LMP1CAO also showed early lethality (Table 1). It may be coincidence that the L2LMP1B95-8 founders showed the most severe phenotype; alternatively, it is possible that the B95-8 form of LMP1 is more "potent" than the CAO variant and in the transgenic context this leads to lethality (the numbers generated are too few to be conclusive). This may reflect the greater capacity of LMP1B95-8 to induce cell growth inhibition and changes in cellular gene expression compared with LMP1CAO (13, 16). Indeed, whereas it has been reported that there is a selection against immunogenic forms of LMP1 (9, 20), this raises the possibility that there might also be a selection against highly active forms in carcinogenesis. It has therefore not been possible in this study to directly compare the carcinogenic potential between these two variants in transgenic mice. Nevertheless, expression of LMP1CAO does lead to oncogenic changes in the transgenic epithelium, first hyperplasia and vascularization, then keratoacanthotic or papillomatous lesions that can progress to carcinoma.

Expression of LMP1 protein has not been detected in all nasopharyngeal carcinoma samples tested, although RT-PCR has shown expression in the majority of cases (42, 43). In the transgenic system described, LMP1 is clearly responsible for the ensuing phenotype, but it could not be readily detected without a vigorous extraction protocol. The same could be true for nasopharyngeal carcinoma tissues; thus, the proportion of nasopharyngeal carcinomas determined to be LMP1 positive may be an underestimate in terms of LMP1 functional contribution to the pathology. It is also possible that LMP1 is required early in tumor development and is expendable at later stages.

The phenotypes of mice of the L2LMP1CAO lines and the PyLMP1B95-8 transgenic mice previously generated in this laboratory (21) resemble those described for HK14.TGF-{alpha} and HK1.TGF-{alpha} mice (31, 32). In this regard, it is also noteworthy that the hyperplastic phenotype of the LMP1 mice resembles psoriasis, a human dermatologic condition strongly associated with increased levels of TGF-{alpha} (44). Accordingly, we found increased TGF-{alpha} levels in the preneoplastic phenotypic tissues of both PyLMP1B95-8 mice and L2LMP1CAO mice. The mature form of TGF-{alpha} can show an apparent size range from 5 to 20 kDa due to differential glycosylation and the membrane-bound precursor has been shown to be active (45). A 10-kDa product was detected in LMP1 tissues by Western blotting, a form which was also detected in transgenic TGF-{alpha} mice (33).

TGF-{alpha} is a ligand for EGFR and expression of the latter has been shown to be up-regulated by LMP1 in carcinoma cell lines (15, 34). Whereas levels of total, full-length EGFR diminished with increasing phenotypic stage in the preneoplastic LMP1 transgenic tissues, there was evidence of increased phospho-EGFR products. Overexpression of TGF-{alpha} in TGF-{alpha} transgenic animals is reported to decrease levels of EGFR protein (32, 33). This would suggest that in the nonneoplastic tissues of the LMP1 mice, the negative feedback loop on EGFR expression that operates when TGF-{alpha} binds to EGFR is intact, or that EGFR turnover is high. Furthermore, in the carcinoma cell lines derived from these mice, total, full-length EGFR expression was high, suggesting that this negative feedback loop has been disrupted in the progression to carcinoma.

LMP1 has been shown to activate signaling through the NF-{kappa}B and AP-1 pathways (4, 5). The 5' regulatory regions of the human (46) and mouse (47) TGF-{alpha} genes both have NF-{kappa}B and AP-1 sites in the upstream region (47). This would suggest a mechanism whereby LMP1 could lead to direct induction of TGF-{alpha} expression. It therefore seems likely that LMP1 induces TGF-{alpha} expression thus leading to tyrosine-phosphorylation and activation of EGFR and subsequent signaling in the epidermis of LMP1 animals. One predicted effect of this is an increase in proliferation, and this is indeed evident upon PCNA-staining of the mouse ears.

Increased VEGF levels and vascularization in the ears of transgenic animals is further indication that signaling via EGFR might be important in this system. EGFR has been shown to be required for up-regulation of VEGF and for angiogenesis in mouse skin carcinogenesis (48). In addition, like the TGF-{alpha} promoter, the VEGF promoter has NF-{kappa}B and AP-1 sites. It is possible therefore that the VEGF promoter could be responsive to direct signaling from LMP1.

It has been shown that ras signaling can induce antiproliferative defense mechanisms by up-regulating the tumor suppressor p16INK4a (49). A role for p16INK4a in blocking aberrant proliferation is suggested by our findings as levels of p16INK4a protein are clearly increased in severely phenotypic, preneoplastic tissues. However, because p16INK4a is not induced in the early stages of the mildly phenotypic tissues (when TGF-{alpha} expression is induced), it would suggest the response is not a direct consequence of LMP1 expression. In relation to this, chemical carcinogenesis experiments conducted on PyLMP1 mice revealed a block in the growth of lesions (compared with nontransgenic mice) which was lifted in an INK4a knockout strain background (22, 23). The study showed that whereas LMP1 could promote lesion formation, the block in the growth of these lesions was mediated by products of the INK4a locus and is therefore consistent with the increased levels of p16INK4a reported here. This may occur as a protective measure in response to aberrant mitogenic signaling (reviewed in ref. 50). This interpretation of events would explain the selection for frequent loss of p16INK4a expression in nasopharyngeal carcinoma (51, 52). The loss of the chromosomal region encoding p16INK4a in a significant proportion of asymptomatic individuals in southern China suggested that loss of the INK4a locus may be an early event in the development of nasopharyngeal carcinoma and precede EBV infection (53). Consistent with our murine model, loss of p16INK4a would make epithelial cells in the nasopharynx susceptible to growth transformation upon EBV infection and concomitant latent expression of LMP1. There is compelling evidence that LMP1 down-regulates the expression of p16INK4a in fibroblasts and blocks downstream mediators of p16INK4a action (38, 39). However, the induction of p16INK4a observed in the tissues of the LMP1CAO mice shows that the ability of LMP1 to down-regulate the expression of p16INK4a either does not occur in this tissue, or does not occur in the same cells of the tissue, or cannot override the induction in response to aberrant proliferation. p16INK4a expression in the presence of LMP1 has also been noted in LCLs (54).

As with p16INK4a, induction of MMP9 expression was observed only in the more severe stages of the phenotype. This therefore reflects the progressive nature of the phenotype in vivo.

In conclusion, we have generated transgenic mice expressing LMP1CAO that exhibit severe hyperplasia, spontaneous papilloma, and keratoacanthoma development, which can progress to carcinoma. The striking similarity of phenotype to TGF-{alpha} transgenic lines allied with the observed increase in TGF-{alpha} levels and apparent EGFR activation implies that a major feature of LMP1 action, initiated early in carcinogenesis, is increased signaling through EGFR. This could occur by virtue of up-regulation of TGF-{alpha} expression via AP-1 and NF-{kappa}B elements upstream of the TGF-{alpha} gene. Similarly, VEGF may be up-regulated and could provide one route to increased vascularization observed in these animals and in nasopharyngeal carcinoma. The increase in proliferation could act as a stimulus for the induction of p16INK4a as a counterbalance during tissue homeostasis. Activating ras mutations are found in many human cancers (55) and have been sought in nasopharyngeal carcinoma but not detected (56). A large number of human cancers that do not exhibit activating ras mutations have instead bypassed or usurped ras signaling pathways by other means, and LMP1 may perform this role in nasopharyngeal carcinoma. Figure 6 outlines a proposed model to explain the phenotype and detected levels of various proteins using our data and published information on LMP1 signaling and genomic sequence information. Such events may well play an important role in the initiation and progression of nasopharyngeal carcinoma.



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  		Figure 6. A model for the action of LMP1 in the first stages of carcinogenesis. Up-regulation of TGF-{alpha} by LMP1 could be achieved through the activation of NF-{kappa}B and AP-1 as could VEGF. TGF-{alpha} activates EGFR, which leads to increased proliferation and its own down-regulation. Induction of p16INK4a and MMP9 are subsequent events. During carcinogenic progression, the negative feedback loop upon EGFR fails and loss of expression of p16INK4a (which could happen before EBV infection) removes the block to lesion expansion.

 

   Acknowledgments
 
Grant support: AICR grant 97-44.

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.

We thank Willie Thomson for assistance with tissue sectioning, Adrian Philbey for expert histopathologic analyses, George Klein (Microbiology and Tumor Biology Centre, Karolinska Institute, Stockholm, Sweden) for the kind gift of the LMP1CAO containing plasmid, and Friedrich Grässer (Department of Virology, Institute for Medical Microbiology and Hygiene, University Hospital, Homburg/Saar, Germany) for an unfailing supply of anti-LMP1 antiserum.

Received 2/23/05. Revised 5/18/05. Accepted 7/22/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. IARC. Epstein-Barr virus and Kaposi's sarcoma herpesvirus/human herpesvirus 8. Monographs on the evaluation of carcinogenic risks to humans 70. Lyon: WHO; 1997.
  2. Wang D, Liebowitz D, Kieff E. An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell 1985;43:831–40.[CrossRef][Medline]
  3. Kaye KM, Izumi KM, Kieff E. Epstein-Barr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation. Proc Natl Acad Sci U S A 1993;90:9150–4.[Abstract/Free Full Text]
  4. Hammarskjold ML, Simurda MC. Epstein-Barr virus latent membrane protein transactivates the human immunodeficiency virus type 1 long terminal repeat through induction of NF-{kappa} B activity. J Virol 1992;66:6496–501.[Abstract/Free Full Text]
  5. 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 1997;16:6478–85.[CrossRef][Medline]
  6. Eliopoulos AG, Gallagher NJ, Blake SM, Dawson CW, Young LS. Activation of the p38 mitogen-activated protein kinase pathway by Epstein-Barr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production. J Biol Chem 1999;274:16085–96.[Abstract/Free Full Text]
  7. Gires O, Kohlhuber F, Kilger E, et al. Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins. EMBO J 1999;18:3064–73.[CrossRef][Medline]
  8. Knecht H, Bachmann E, Brousset P, et al. Deletions within the LMP1 oncogene of Epstein-Barr virus are clustered in Hodgkin's disease and identical to those observed in nasopharyngeal carcinoma. Blood 1993;82:2937–42.[Abstract/Free Full Text]
  9. Edwards RH, Sitki-Green D, Moore DT, Raab-Traub N. Potential selection of LMP1 variants in nasopharyngeal carcinoma. J Virol 2004;78:868–81.[Abstract/Free Full Text]
  10. Hu LF, Zabarovsky ER, Chen F, et al. Isolation and sequencing of the Epstein-Barr virus BNLF-1 gene (LMP1) from a Chinese nasopharyngeal carcinoma. J Gen Virol 1991;72:2399–409.[Abstract/Free Full Text]
  11. Huen DS, Henderson SA, 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 1995;10:549–60.[Medline]
  12. Li SN, Chang YS, Liu ST. Effect of a 10-amino acid deletion on the oncogenic activity of latent membrane protein 1 of Epstein-Barr virus. Oncogene 1996;12:2129–35.[Medline]
  13. Dawson CW, Eliopoulos AG, Blake SM, Barker R, Young LS. Identification of functional differences between prototype Epstein-Barr virus-encoded LMP1 and a nasopharyngeal carcinoma-derived LMP1 in human epithelial cells. Virology 2000;272:204–17.[CrossRef][Medline]
  14. Blake SM, Eliopoulos AG, Dawson CW, Young LS. The transmembrane domains of the EBV-encoded latent membrane protein 1 (LMP1) variant CAO regulate enhanced signalling activity. Virology 2001;282:278–87.[CrossRef][Medline]
  15. Miller WE, Cheshire JL, Baldwin AS, Jr., Raab-Traub N. The NPC derived C15 LMP1 protein confers enhanced activation of NF-{kappa}B and induction of the EGFR in epithelial cells. Oncogene 1998;16:1869–77.[CrossRef][Medline]
  16. Johnson RJ, Stack M, Hazlewood SA, et al. The 30-base-pair deletion in Chinese variants of the Epstein-Barr virus LMP1 gene is not the major effector of functional differences between variant LMP1 genes in human lymphocytes. J Virol 1998;72:4038–48.[Abstract/Free Full Text]
  17. Khanim F, Yao QY, Niedobitek G, Sihota S, Rickinson AB, Young LS. Analysis of Epstein-Barr virus gene polymorphisms in normal donors and in virus-associated tumors from different geographic locations. Blood 1996;88:3491–501.[Abstract/Free Full Text]
  18. Hahn P, Novikova E, Scherback L, et al. The LMP1 gene isolated from Russian nasopharyngeal carcinoma has no 30-bp deletion. Int J Cancer 2001;91:815–21.[CrossRef][Medline]
  19. Trivedi P, Hu LF, Chen F, et al. Epstein-Barr virus (EBV)-encoded membrane protein LMP1 from a nasopharyngeal carcinoma is non-immunogenic in a murine model system, in contrast to a B cell-derived homologue. Eur J Cancer 1994;30A:84–8.[CrossRef]
  20. Hu L, Troyanovsky B, Zhang X, Trivedi P, Ernberg I, Klein G. Differences in the immunogenicity of latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus genomes derived from LMP1-positive and -negative nasopharyngeal carcinoma. Cancer Res 2000;60:5589–93.[Abstract/Free Full Text]
  21. Wilson JB, Weinberg W, Johnson R, Yuspa S, Levine AJ. 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 1990;61:1315–27.[CrossRef][Medline]
  22. Curran JA, Laverty FS, Campbell D, Macdiarmid J, Wilson JB. Epstein-Barr virus encoded latent membrane protein-1 induces epithelial cell proliferation and sensitizes transgenic mice to chemical carcinogenesis. Cancer Res 2001;61:6730–8.[Abstract/Free Full Text]
  23. Macdiarmid J, Stevenson D, Campbell DH, Wilson JB. The latent membrane protein 1 of Epstein-Barr virus and loss of the INK4a locus: paradoxes resolve to cooperation in carcinogenesis in vivo. Carcinogenesis 2003;24:1209–18.[Abstract/Free Full Text]
  24. Nakagawa H, Wang TC, Zukerberg L, et al. The targeting of the cyclin D1 oncogene by an Epstein-Barr virus promoter in transgenic mice causes dysplasia in the tongue, esophagus and forestomach. Oncogene 1997;14:1185–90.[CrossRef][Medline]
  25. Wilson JB, Drotar ME. Considerations in generating transgenic mice: DNA, RNA and protein extractions from tissues: rapid and effective blotting. Epstein-Barr virus protocols. In: Wilson JB, May GHW, editors. Methods in molecular biology, 174. Totowa (NJ): The Humana Press Inc.; 2001. p. 361–77.
  26. Macdiarmid J, Wilson JB. Separation of epidermal tissue from underlying dermis and primary keratinocyte culture. Epstein-Barr virus protocols. In: Wilson JB, May GHW, editors. Methods in molecular biology, 174. Totowa (NJ): The Humana Press Inc.; 2001. p. 401–10.
  27. Mann KP, Staunton D, Thorley-Lawson DA. Epstein-Barr virus-encoded protein found in plasma membranes of transformed cells. J Virol 1985;55:710–20.[Abstract/Free Full Text]
  28. Nicholls J, Hahn P, Kremmer E, et al. Detection of wild type and deleted latent membrane protein 1 (LMP1) of Epstein-Barr virus in clinical biopsy material. J Virol Methods 2004;116:79–88.[CrossRef][Medline]
  29. Winter H, Schweizer J, Goerttler K. Keratins as markers of malignancy in mouse epidermal tumors. Carcinogenesis 1980;1:391–8.[Abstract/Free Full Text]
  30. Moorthy R, Thorley-Lawson DA. Processing of the Epstein-Barr virus-encoded latent membrane protein p63/LMP. J Virol 1990;64:829–37.[Abstract/Free Full Text]
  31. Vassar R, Fuchs E. Transgenic mice provide new insights into the role of TGF-{alpha} during epidermal development and differentiation. Genes Dev 1991;5:714–27.[Abstract/Free Full Text]
  32. Dominey AM, Wang XJ, King LE, Jr., 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 1993;4:1071–82.[Abstract]
  33. Shibata MA, Ward JM, Green JE, Merlino G. Enhanced sensitivity to tumor growth and development in multistage skin carcinogenesis by transforming growth factor-{alpha}-induced epidermal growth factor receptor activation but not p53 inactivation. Mol Carcinog 1997;18:160–70.[CrossRef][Medline]
  34. Miller WE, Earp HS, Raab-Traub N. The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor. J Virol 1995;69:4390–8.[Abstract]
  35. 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 1994;30B:290–5.[CrossRef]
  36. Yoshizaki T, Sato H, Furukawa M, Pagano JS. The expression of matrix metalloproteinase 9 is enhanced by Epstein-Barr virus latent membrane protein 1. Proc Natl Acad Sci U S A 1998;95:3621–6.[Abstract/Free Full Text]
  37. Murono S, Inoue H, Tanabe T, et al. Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells. Proc Natl Acad Sci U S A 2001;98:6905–10.[Abstract/Free Full Text]
  38. Yang X, He Z, Xin B, Cao L. LMP1 of Epstein-Barr virus suppresses cellular senescence associated with the inhibition of p16INK4a expression. Oncogene 2000;19:2002–13.[CrossRef][Medline]
  39. Ohtani N, Brennan P, Gaubatz S, et al. Epstein-Barr virus LMP1 blocks p16INK4a-RB pathway by promoting nuclear export of E2F4/5. J Cell Biol 2003;162:173–83.[Abstract/Free Full Text]
  40. Nakagawa H, Inomoto T, Rustgi AK. A CACCC box-like cis-regulatory element of the Epstein-Barr virus ED-L2 promoter interacts with a novel transcriptional factor in tissue-specific squamous epithelia. J Biol Chem 1997;272:16688–99.[Abstract/Free Full Text]
  41. Jenkins TD, Opitz OG, Okano J, Rustgi AK. Transactivation of the human keratin 4 and Epstein-Barr virus ED-L2 promoters by gut-enriched Kruppel-like factor. J Biol Chem 1998;273:10747–54.[Abstract/Free Full Text]
  42. Brooks L, Yao QY, Rickinson AB, Young LS. Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP1, and LMP2 transcripts. J Virol 1992;66:2689–97.[Abstract/Free Full Text]
  43. Sheen TS, Huang YT, Chang YL, et al. Epstein-Barr virus-encoded latent membrane protein 1 co-expresses with epidermal growth factor receptor in nasopharyngeal carcinoma. Jpn J Cancer Res 1999;90:1285–92.[Medline]
  44. Sergi C, Kahl P, Otto HF. Immunohistochemical localization of transforming growth factor-{alpha} and epithelial growth factor receptor in human fetal developing skin, psoriasis and restrictive dermopathy. Pathol Oncol Res 2000;6:250–5.[Medline]
  45. Imamoto A, Beltran LM, DiGiovanni J. Evidence for autocrine/paracrine growth stimulation by transforming growth factor-{alpha} during the process of skin tumor promotion. Mol Carcinog 1991;4:52–60.[Medline]
  46. Jakobovits EB, Schlokat U, Vannice JL, Derynck R, Levinson AD. The human transforming growth factor {alpha} promoter directs transcription initiation from a single site in the absence of a TATA sequence. Mol Cell Biol 1988;8:5549–54.[Abstract/Free Full Text]
  47. Berkowitz EA, Seroogy KB, Schroeder JA, et al. Characterization of the mouse transforming growth factor {alpha} gene: its expression during eyelid development and in waved 1 tissues. Cell Growth Differ 1996;7:1271–82.[Abstract]
  48. Casanova ML, Larcher F, Casanova B, et al. A critical role for ras-mediated, epidermal growth factor receptor-dependent angiogenesis in mouse skin carcinogenesis. Cancer Res 2002;62:3402–7.[Abstract/Free Full Text]
  49. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 1997;88:593–602.[CrossRef][Medline]
  50. Prenzel N, Fischer OM, Streit S, Hart S, Ullrich A. The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification. Endocr Relat Cancer 2001;8:11–31.[Abstract]
  51. Lo KW, Huang DP, Lau KM. p16 gene alterations in nasopharyngeal carcinoma. Cancer Res 1995;55:2039–43.[Abstract/Free Full Text]
  52. Lo KW, Cheung ST, Leung SF, et al. Hypermethylation of the p16 gene in nasopharyngeal carcinoma. Cancer Res 1996;56:2721–5.[Abstract/Free Full Text]
  53. Chan AS, To KF, Lo KW, et al. Frequent chromosome 9p losses in histologically normal nasopharyngeal epithelia from southern Chinese. Int J Cancer 2002;102:300–3.[CrossRef][Medline]
  54. Hayes MJ, Koundouris A, Gruis N, Bergman W, Peters GG, Sinclair AJ. p16(INK4A)-independence of Epstein-Barr virus-induced cell proliferation and virus latency. J Gen Virol 2004;85:1381–6.[Abstract/Free Full Text]
  55. Bos JL. ras oncogenes in human cancer: a review. Cancer Res 1989;49:4682–9.[Abstract/Free Full Text]
  56. Yung WC, Sham JS, Choy DT. Ng MH, ras mutations are uncommon in nasopharyngeal carcinoma. Eur J Cancer B Oral Oncol 1995;31B:399–400.[CrossRef]



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