UMR1225 IHAP ENVT, 23 chemin des Capelles, BP 87614, 31076 Toulouse CEDEX, France
Correspondence
Frédérique L. Messud-Petit
f.messudpetit{at}envt.fr
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ABSTRACT |
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INTRODUCTION |
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Among the poxvirus family, only three viruses are known to be responsible for tumorigenesis: Shope fibroma virus (SFV), molluscum contagiosum virus (MCV) and Yaba monkey tumor virus (YMTV). Rabbits infected with SFV develop benign cutaneous tumours, MCV is associated with an increasing number of epidermal tumours in immunocompromised patients and YMTV is responsible for subcutaneous histiocytomas in monkeys (Shope, 1932a, b
; Bearcroft & Jamieson, 1958
; Schwartz & Myskowski, 1992
; Bugert & Darai, 1997
).
How poxviruses induce tumours remains unknown, as no similarity has been found between poxviruses genomes and oncogenes that exist in other viral families. However, many poxviruses encode a homologue of the epidermal growth factor (EGF) and transforming growth factor alpha (TGF-). This homologue is best characterized in vaccinia virus, myxoma virus (MV) and SFV, where it is refered to as VGF, MGF and SFGF, respectively (Brown et al., 1985
; Chang et al., 1987
; Upton et al., 1987
). Vaccinia virus is an orthopoxvirus and was used to eradicate smallpox. MV and SFV are two closely related leporipoxviruses. The former is the agent of myxomatosis, a non-proliferative, fatal disease of the European rabbit, whereas the latter induces cutaneous tumours in its host.
As EGF is responsible for cellular proliferation, its viral homologues are good candidates as tumour inducers. It was shown that synthetic peptides mimicking poxviral homologues bind to the same family of receptors as EGF (tyrosine kinase receptors of the ErbB family) and are capable of inducing cell proliferation (Tzahar et al., 1998). Indeed, it was proposed that EGF homologues are responsible for poxvirus tumorigenesis (Marechal & Piolot, 1999
), despite the fact that neither vaccinia virus nor MV is tumorigenic. Moreover, no such homologue has been found in YMTV or MCV (Senkevich et al., 1996
; Brunetti et al., 2003
).
A synthetic peptide derived from SFGF competes with EGF for its receptor (ErbB1) and shares EGF activities, such as stimulation of [3H]thymidine uptake in normal rat kidney fibroblasts and induction of colony formation in soft agar (Lin et al., 1988). Its biological activity was also demonstrated in vivo (incisor eruption and eyelid-opening acceleration in newborn mice) (Ye et al., 1988
). Although SFGF binds less efficiently to ErbB1 than EGF and is less potent for tyrosine phosphorylation of the receptor, it induces a longer activation of the mitogen-activated protein kinase (MAPK) pathway and is more potent mitogenically than EGF (Tzahar et al., 1998
).
These conflicting data led us to investigate the role of growth factors in the context of a tumorigenic poxvirus. We characterized the phenotype of SFV infection both in vivo and ex vivo and confirmed its specific, tumorigenic properties. We then engineered an SFV mutant deleted in the sfgf gene, replaced it with the mgf gene of MV and compared the phenotypes of the recombinant and wild-type (wt) viruses. Our results indicated that SFGF is critical in cell transformation/proliferation and lesion maintenance and that MGF, even when expressed by SFV, cannot substitute for SFGF.
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METHODS |
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Ex vivo culture of rabbit fibromatous cells (RFCs).
One 5-week-old rabbit was infected intradermally on the back with 100 µl SFV at a concentration of 107 p.f.u. ml1. Twelve days later, when the tumour was of maximum size, the rabbit was sacrificed, the tumour removed and RFCs were treated as described above. RFCs were passaged 20 times.
Cells, viruses and serum.
The Kasza (provided by G. McFadden) and Boerlage (Dermyxovax; Merial) strains of SFV and the SFV-sfgf mutant virus were grown on rabbit kidney cells (RK13) maintained in Dulbecco's minimum essential medium (DMEM) supplemented with 10 % FBS. The revertant SFV-sfgf rev and the chimeric SFV-mgf viruses were grown in HGPRT (hypoxanthineguanine phosphoribosyl transferase) RK13 cells in DMEM supplemented with 10 % FBS and 2-amino-6-mercaptopurine (20 µg ml1). Conventional BALB/c mice were inoculated intradermally three times at 1 month intervals with 2·5x106 p.f.u. SFV (Boerlage strain). Blood samples were collected 2 weeks after the last immunization.
Amplification and sequencing of sfgf DNA sequences.
The sfgf ORFs of the Kasza and the Boerlage strains of SFV were amplified by using primers upstream of the promoter and downstream of the stop codon. After purification, uncloned PCR products were sequenced by using synthetic oligonucleotide primers with an automated sequencer.
Construction of SFV-sfgf mutant, SFV-sfgf rev and SFV-mgf viruses.
To evaluate the involvement of SFGF in SFV pathogenicity, the sfgf gene of the Boerlage strain was inactivated by deletion and disruption with the ecogpt gene, encoding Escherichia coli guanine phosphoribosyl transferase. Two sets of primers containing 5'-terminal extensions with restriction sites (underlined) for subsequent subcloning were used to amplify fragments immediately upstream and downstream of the sfgf gene. The following primers were used: Sfgf5'SacI (5'-CAGCAGGAGCTCGTCTAAAGGAGGTTGTCTAC-3') paired with Sfgf5'EcoRI (5'-CAGCAGAATTCGGCCACTAGGTTCCGCGTCG-3'), and Sfgf3'PstI (5'-CAGCAGCTGCAGTTACTTATTAAGTGATAACCATTGC-3') paired with Sfgf3'XbaI (5'-CAGCAGTCTAGATGTATCTACTTTGGATATGATGC-3'). Amplified Sfgf-5' and Sfgf-3' DNA was digested with SacI/EcoRI and PstI/XbaI, respectively, and inserted into pRBgpt as described previously (Messud-Petit et al., 1998). The resulting plasmid, called psfgf : : gpt, thus consisted of an antisense gpt gene replacing nt 25231 of sfgf. The s009L gene, downstream of sfgf, and the s011L gene, upstream and partially overlapping sfgf, were kept intact. After psfgf : : gpt was sequenced, it was used for transfection of SFV-infected RK13 cells. To select the SFV-
sfgf mutant virus, MXHAT solution (25 µg mycophenolic acid ml1, 250 µg xanthine ml1, 15 µg hypoxanthine ml1, 0·176 µg aminopterine ml1, 4 µg thymidine ml1) was added to the medium and SFV-
sfgf mutant viruses were selected for their mycophenolic acid resistance. PCR analysis of recombinant virus DNA was performed to confirm the absence of wt viruses in preparations of SFV-
sfgf mutant viruses. A revertant virus, SFV-sfgf rev, containing a wt SFGF ORF, was obtained by transfecting plasmid DNA containing the complete sfgf gene into SFV-
sfgf-infected RK13 cells.
To engineer the SFV-mgf recombinant virus, the MV strain T1 mgf gene and its own promoter were amplified by using primers containing 5'-terminal extensions with restriction sites (underlined) for subsequent subcloning. These were: Mgf5'PstI 5'-CAGCAGCTGCAGCGGTATTGTCGCGGAAG-3' and Mgf3'EcoRI 5'-CAGCAGAATTCATACATGTAAAACGGGTTAC-3'. After digestion with PstI and EcoRI, the fragment was cloned into the psfgf : : gpt plasmid, thus replacing the gpt gene. The resulting plasmid, called psfgf : : mgf, was sequenced and transfected into SFV-sfgf-infected RK13 cells. SFV-sfgf rev and SFV-mgf viruses were selected for 2-amino-6-mercaptopurine resistance in RK13 HGPRT cells. PCR analysis of recombinant virus DNA was performed to confirm the absence of SFV-
sfgf mutant viruses in preparations of SFV-sfgf rev or SFV-mgf viruses. Expression of MGF and SFGF was assessed in chimeric and revertant viruses by RT-PCR analysis in cell culture, using specific primers.
Infection of rabbits with SFV Kasza, wt Boerlage, SFV-sfgf mutant, SFV-sfgf rev and SFV-mgf viruses.
Six-week-old male New Zealand White (NZW) rabbits were obtained from a local supplier and housed in biocontainment facilities according to the guidelines of the European Community Council on Animal Care (European Council directive 86/609/ECC, 24 November 1986). All procedures on animals were performed by workers accredited by the French Ministry of Agriculture and were aimed at limiting animal pain and distress. Infections were performed intradermally on the back (after removal of the hair on a round surface of approx. 3 cm diameter) with 2·5x106 p.f.u. of either Kasza, wt Boerlage, SFV-sfgf mutant, SFV-sfgf rev or SFV-mgf viruses. The inoculation sites were monitored daily and colour and temperature were noted. Tumours were considered as cylinders and the diameter and height were measured with a cutimeter, allowing the determination of its volume. For histological analysis, two rabbits from each group were sacrificed by using T61 (Distrivet) at 3, 13 and 20 days post-infection (p.i.) and three rabbits at critical times (7 and 10 days p.i.). Two mock-infected rabbits were sacrificed and used as controls.
Histological examination.
Tissue material from the injection site was taken and stored in 10 % neutral formalin for further analysis. After fixation, tissues were processed routinely in paraffin blocks, sectioned at 4 µm and stained with haematoxylin and eosin (H&E) for microscopic examination. Lesions were assessed histologically and graded from 0 to 2 or 3, according to their severity. For each animal, the histopathological analysis was repeated three times to ensure accuracy of results. Modifications were observed at the centre of the lesion and scored as follows. (i) Fibroblastic density. Normal (0), mucinosis occupies a much larger space than fibroblastic cells; moderately increased (1), mucinosis and fibroblastic cells occupy an equivalent space; markedly increased (2), fibroblastic cells occupy a much larger space than mucinosis. (ii) Mitosis. Rare (0), less than three mitoses in each high-power field (x400); moderate (1), three to six mitoses in each high-power field; numerous (2), more than six mitoses in each high-power field. (iii) Pleomorphism. None (0), nuclear isocaryosis; minimal (1), light anisocaryosis, with a maximal variation for the nuclear diameter of less than x1·5; moderate (2), anisocaryosis with a variation for the nuclear diameter of greater than x1·5; marked (3), anisocaryosis with a variation for the nuclear diameter of greater than x1·5 plus megalocytosis plus multinucleated cells.
Immunostaining of viral antigens.
Paraffin-embedded sections of lesions were treated with 0·1 % trypsin in PBS (pH 7·6) for 30 min at 37 °C and viral antigens were detected as described elsewhere (Guerin et al., 2001), using mouse hyperimmune anti-SFV serum as primary antibody and peroxidase goat anti-mouse immunoglobulin G (N-Histofine simple stain MAX PO; Nichirei). Bound antibody was revealed by DAB (3,3'-diaminobenzidine tetrahydrochloride), which formed a brown precipitate. The level of virus replication in lesions was quantified by counting the number of infected cells mm2, each lesion being cut medially. Three sections per lesion were observed.
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RESULTS |
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Twelve 6-week-old NZW rabbits were inoculated intradermally on the back with 2·5x106 p.f.u. SFV strain Boerlage and observed daily. At day 1 p.i., the inoculum was completely resorbed and no lesion was observed. As early as day 2 p.i., a cutaneous nodule was palpable at the site of inoculation. The following day, the nodule measured 1 cm3 and it grew until day 10 p.i., when it reached its maximum size (approx. 9 cm3) (Fig. 3). The lesion was limited to the skin and not adherent to subjacent tissues; it was firm, hot when handled and erythematous. A cutaneous ulceration, which appeared at day 7, had turned scabby by day 10 (Fig. 4
). On day 10, the lesion began regressing, becoming less firm. By day 13, pus was present under the scab. By day 20, the lesion had regressed almost completely, with only a 2 cm3 volume left (Fig. 3
). In comparison, 4 days after injection with MV, myxomas are raised, soft and red lesions, which have disseminated all over the body by day 8 and turned necrotic by day 12 (Joubert et al., 1972b
).
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Simultaneously, the lesion began regressing, due to severe necrosis. The scabby epithelium detached. In the deep part of the tumour, the oedema disappeared and was replaced by a fibroblastic and inflammatory cellular infiltrate. Healing by connective-tissue replacement took place, with the formation of granulation tissue that showed marked proliferation of fibroblasts and newly formed small blood vessels.
This complete description confirms that SFV induces a rapidly expanding, fibroblastic proliferation, associated with various and frequent cellular abnormal morphological features (cell size, nuclear and nucleolar modifications), which are all characteristic features of tumours. No metastasis was observed, suggesting that the lesions were benign and justifying their designation as fibromas. However, the fibromas, unlike most tumours, regressed and disappeared spontaneously in the rabbit. These lesions can be distinguished microscopically from myxomas, which are composed mainly of interstitial mucinosis due to activated fibroblasts and where cytological abnormalities are rare and less intense.
Involvement of SFGF in SFV tumorigenesis
Rabbits inoculated with the Kasza and Boerlage strains of SFV both developed cutaneous tumours that reached approximately 2·53 cm in diameter by day 10 p.i., but with the former, they were thinner (approx. 68 mm) and firmer than with the Boerlage strain (1215 mm; data not shown). In order to determine whether the phenotypic differences correlated with differences in viral growth factor expression or composition, we performed a PCR amplification of sfgf genes of both strains. After cloning, the PCR products were sequenced and revealed 100 % identity in their nucleic acid sequences, including the promoter region (data not shown). This indicated that SFGF is not the only determinant in the tumorigenic phenotype of SFV infection.
We then investigated the role of SFGF in the genesis and development of tumours induced by the Boerlage strain of SFV. We engineered a mutant SFV with a disruption in the sfgf gene. sfgf is present as a single copy in the SFV genome (Willer et al., 1999), with the s011L gene partially overlapping its ORF. After disruption of the sfgf gene with the ecogpt gene, we checked the integrity of adjacent genes (see Methods). A mycophenolic acid-resistant SFV-
sfgf mutant was isolated. As a control, we constructed a revertant virus derived from SFV-
sfgf, in which the sfgf gene was restored. A revertant SFV-sfgf rev virus was isolated by using reverse gpt selection. In vitro growth curves of the wt and mutant virus were similar in RK13 cells (data not shown). The in vivo phenotype of infections with the SFV-
sfgf and SFV-sfgf rev viruses was then assessed. The macroscopic evolution and microscopic analysis of lesions induced with the SFV-sfgf rev virus were similar to those observed with wt SFV. In contrast, lesions associated with the SFV-
sfgf virus were smaller, peaking at 1 cm3 on day 7 (Fig. 3
). Inflammation was moderate and the lesion was cold, soft and lightly erythematous. Ulcers were less developed than with wt SFV or the SFV-sfgf rev viruses (Fig. 4
).
Lesions induced by SFV-sfgf presented the same histological modifications as with wt SFV or the SFV-sfgf rev virus, but were extremely attenuated. Briefly, by day 7 p.i., the main differences consisted of less fibroblastic proliferation (Fig. 6
, top) and less severe morphological abnormalities: only a few large, hyperchromatic nuclei were noted, but these were neither very large nuclei nor multinucleated cells (Fig. 6
, bottom). Vascular modifications were rare and these were limited to the deep side of the dermis. The general lesional architecture was conserved, consisting of dermal oedema, mucinosis and activated fibroblasts surrounded by an inflammatory crown. Only at 10 days p.i. did the lesional intensity approach the level observed with wt SFV (Fig. 7
). Morphological abnormalities of fibroblasts were more frequent, but mitosis and monstrous cells could not match the rate observed in the presence of SFGF. Necrosis, thrombosis and haemorrhages were absent. From days 13 to 20, inflammation decreased and the tissue healed (fibroplasia). Hence, the absence of SFGF led to the same histological and cytological modifications as with wt SFV, but their onset was delayed, their expression less pronounced and their decrease earlier.
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Virus load in tissues
Immunostaining of viral antigens in histological sections, performed at days 3 and 7 p.i., revealed no difference among SFV, SFV-sfgf and SFV-mgf strains (data not shown). This semi-quantitative result suggested that these strains replicated at comparable levels in the skin. Hence, the phenotypic differences are not likely to be attributed to different viral behaviours in vivo, but rather to the effects of the viral growth factors.
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DISCUSSION |
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Rabbits infected with SFV (strain Boerlage) all developed a similar lesion, which was very inflamed, not adherent to subjacent tissues and grew fast, reaching a volume of 9 cm3 in only 10 days. The lesion then started regressing as necrosis and an ulcer developed; it had disappeared completely in approximately 1 month. Histological analysis revealed an oedematous dermal inflammation, with proliferating fibroblasts and abundant mucinosis. Mucinosis is commonly observed in myxomas; however, specific features of tumours that were absent in MV infections were a rapid and intense fibroblastic proliferation, cells presenting a strong pleomorphism and marked morphological alterations (cellular shape modifications, very large nuclei, multiple nuclei and plurinucleolation). Mitotic activity was intense, most of it being abnormal and leading to aberrant cells. Thus, the lesion showed many characteristics of a fibroblastic tumour. Despite cytological abnormalities, the term fibroma was justified because of its spontaneous involution. Our observations correlate with and give more details about previous descriptions of fibromas (Smith et al., 1973; Sell & Scott, 1981
; Strayer et al., 1984
). However, we could not reproduce results obtained by some authors, who claimed that SFV is tumorigenic in mice (Obom & Pogo, 1989
); in our hands, all attempts to induce visible tumours in conventional BALB/c mice and nude mice failed (data not shown). This is consistent with a host specificity of SFV for the rabbit as reported elsewhere (Joubert et al., 1972a
). Myxomas are quite different lesions: cell proliferation is far less intense and abundant mucinosis (secreted by activated fibroblasts) accounts for the volume of the lesions. Abnormal cellular features are exceptional (Joubert et al., 1972b
; Guerin et al., 2001
, 2002
). These observations justify the definition of SFV, but not of MV, as tumorigenic.
Poxviruses are large DNA viruses that replicate exclusively in the cell cytoplasm; the mechanisms that some of them use for the induction of tumours are still unknown. To date, no parallel has been made with strategies used by other tumorigenic viruses. However, poxviral tumorigenic properties are often attributed to the EGF homologues that they encode (Marechal & Piolot, 1999). SFGF has been the topic of a few papers, where a synthetic substrate mimicking SFGF displayed EGF-like activities (Lin et al., 1988
; Ye et al., 1988
; Tzahar et al., 1998
). Surprisingly, its impact in the context of a tumorigenic poxvirus has been neglected. To investigate the contribution of SFGF to the development of tumours by SFV, we engineered a mutant virus in which sfgf was disrupted (SFV-
sfgf) and observed its phenotype in the European rabbit. In the absence of SFGF, there was an obvious reduction in SFV pathogenesis: the lesions were smaller, reaching a maximal volume of 1 cm3 at day 7 p.i. Inflammation was markedly reduced and almost absent after 10 days p.i. and the lesions regressed spontaneously, without necrosis or ulceration, and were cleared sooner than with wt SFV. The same histological and cytological modifications and alterations were found in SFV-
sfgf lesions, but they were more transient and less marked than with the wt strain. Mitosis was less intense and more normal and, hence, fibroblast density was reduced. Activated fibroblasts secreting mucin had morphological alterations (size increases), but multinucleation and multinucleolation were absent. Thus, SFGF is a major factor in the pathogenesis of SFV; as SFV-
sfgf was not impaired significantly in its replicative capacity, our observations can be correlated directly with the absence of this viral growth factor.
Our observations demonstrated that SFGF is a major partner in the proliferative and atypical features of fibromas. This is in accordance with the known in vitro biological activities of poxviral growth factors, which serve as ligands for receptor tyrosine kinases of the ErbB family (ErbB1, 2, 3 and 4) and activate the MAPK pathways (Tzahar et al., 1998). A synthetic peptide derived from SFGF binds ErbB1, the same receptor as EGF, induces a longer activation of the MAPK pathway and is more potent mitogenically than EGF (Tzahar et al., 1998
). We can thus hypothesize that SFGF stimulates fibroblast proliferation by activating their ErbB receptors. However, the absence of reagents that cross-react with rabbit cellular enzymes prevented us from further exploring this hypothesis.
However, poxviral growth factors are found in non-tumorigenic poxviruses (vaccinia virus and MV), where they are virulence factors (Buller et al., 1988a, b
; Opgenorth et al., 1992a
, 1993
). A recombinant MV expressing VGF, SFGF or TGF-
produced infections that were clinically and histologically similar to wt myxomatosis (Opgenorth et al., 1993
), suggesting similar functions for these growth factors in MV. Here, we investigated whether MGF could replace SFGF in SFV. To do this, we created a chimeric virus in which mgf replaced the sfgf gene; the new virus was designated SFV-mgf. MGF enabled SFV lacking sfgf to recover part of its virulence: the lesional intensity of the SFV-mgf virus was intermediate between those obtained with wt SFV and SFV-
sfgf. Fibroblasts were less mitotic (and hence less numerous in the lesion) than with the original virus, but still were pleomorphic. Thus, MGF and SFGF are not entirely equivalent, a fact that might have been masked in a cytocidal virus such as MV. Our observation that SFGF is more efficient than MGF for the onset and maintenance of fibromas might be explained by the fact that both factors do not bind the same receptors of the ErbB family: SFGF binds every dimer of ErbB receptors, whereas MGF only binds ErbB2/ErbB3 heterodimers (Tzahar et al., 1998
).
In conclusion, we have demonstrated that SFGF is critical for the proliferative phenotype associated with SFV infections. MGF could not replace SFGF, indicating that the latter possesses specific biological activities that are necessary for the complete phenotypic expression of SFV. However, viral growth factors cannot explain all the morphological aberrations observed with tumorigenic poxviruses because: (i) when SFGF is not expressed by SFV, some cellular morphological modifications are still present; (ii) two strains of SFV (Kasza and Boerlage), although possessing identical growth factors, have different clinical outcomes in the European rabbit; and (iii), more importantly, no growth factor homologue was found in MCV or YMTV (Senkevich et al., 1996, 1997
; Brunetti et al., 2003
). For these viruses, it is possible that there are mechanisms in which ErbB receptors or homologues are activated. Tumours induced by poxviruses are probably dependent upon multiple mechanisms, which are presumably less trivial than is sometimes stated. It is time to carry out a thorough examination of this insufficiently investigated area.
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ACKNOWLEDGEMENTS |
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Received 21 May 2004;
accepted 9 August 2004.