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Published ahead of print on December 7, 2006, doi:10.1165/rcmb.2006-0285OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 36, pp. 534-540, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0285OC

Alveolar Type II Cells Isolated from Pulmonary Adenocarcinoma

A Model for JSRV Expression In Vitro

Fabienne Archer, Emilie Jacquier, Monique Lyon, Joëlle Chastang, Vincent Cottin, Jean-Francçois Mornex and Caroline Leroux

Université de Lyon; INRA, UMR754, Rétrovirus et Pathologie Comparée; Ecole Pratique des Hautes Etudes; IFR 128; Department of Respiratory Diseases, Reference Center for Orphan Lung Diseases, Louis Pradel Hospital, Hospices Civils de Lyon, Lyon; and Ecole Nationale Vétérinaire de Lyon, Marcy L'Etoile, France

Correspondence and requests for reprints should be addressed to Caroline Leroux, UMR 754 INRA/ ENVL/UCBL, "Rétrovirus et Pathologie Comparée," IFR 128 BioSciences Lyon-Gerland, Université Claude Bernard, 50 avenue Tony Garnier, 69007 Lyon cedex 07, France. E-mail: caroline.leroux{at}univ-lyon1.fr


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring cancer in sheep, with clinical, radiologic, and histopathologic features similar to that of human pneumonic-type bronchioloalveolar carcinoma. JSRV (Jaagsiekte Sheep RetroVirus) is the etiologic agent of this contagious lung cancer in sheep. Cells involved in the tumor derive from alveolar type II cells and Clara cells, epithelial cells of the distal respiratory tract. These cells are the major site for viral expression in JSRV-infected animals. Recent studies clearly described the oncogenic properties of the JSRV envelope protein both in vitro and in vivo. Interestingly, the cellular pathways involved in the transformation process seem to be dependent of the origin and type of the cell used. In order to investigate the specific interactions between JSRV and alveolar type II cells, we developed an in vitro experimental model in which lung epithelial cells were isolated from OPA and control lungs. Cells in culture expressed alveolar type II cell specific markers such as surfactant protein (SP)-A, SP-C, and a high alkaline phosphatase activity. Alveolar Type II cells derived from tumoral lungs showed a proliferative advantage and expressed the JSRV virus. The reverse transcriptase activity decreased over passages in monolayer culture conditions, but was efficiently maintained in three-dimensional culture conditions. We thus report on the first in vitro system whereby alveolar type II cells from OPA were efficiently maintained in culture and stably expressed JSRV. This novel experimental model will set up the stage for elucidating lung epithelial transformation in the JSRV-induced tumor.

Key Words: lung • JSRV • alveolar type II cell • pneumocyte • bronchioloalveolar carcinoma



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We describe a new in vitro model to study molecular mechanisms of tumoral transformation of type II pneumocytes.

 
Lung cancer is the most common fatal malignancy worldwide. Approximately 80% of cases are non–small cell carcinoma; adenocarcinomas are the most frequent histopathologic type, accounting for 40% of all cases. Bronchioloalveolar carcinoma (BAC) is a rare tumor, representing less than 4% of all non–small cell lung carcinomas (1), that has always intrigued pathologists and chest physicians. The pneumonic presentation of BAC is less associated with cigarette smoking than other forms of lung cancer (2). Its similarities with the natural and worldwide occurring ovine pulmonary adenocarcinoma (OPA) were stressed as early as 1939 (3). Both the pneumonic form of human lung adenocarcinoma and OPA are described as mixed-type adenocarcinoma, with bronchioloalveolar predominance, and present multifocal nodules evolving toward pneumonia, associated with pulmonary shunting and bronchorrhea.

OPA was described in 1915, in South Africa. The causative agent is a retrovirus called JSRV (Jaagsiekte Sheep RetroVirus) (4). Whether JSRV or a related human retrovirus may be involved in some forms of human lung cancer such as BAC remains to be determined. Studies are arguing for or against potential presence of retroviral genomic sequences in human BAC (5–7). Nevertheless, OPA offers an invaluable model for studying the molecular mechanisms of lung epithelial transformation occurring in lung cancer.

It is generally admitted that epithelial tumoral cells in the lungs are the major site of JSRV replication (8). Immunohistochemical studies on natural and experimentally induced OPA have shown that JSRV-infected tumoral cells derived predominantly from alveolar type II cells and Clara cells (9). This observation supports the selective transformation of alveolar type II cells by JSRV in lung adenocarcinoma. Several groups have demonstrated that the envelope protein of JSRV can transform mouse (10), rat (11), and chicken fibroblasts (12, 13), as well as human bronchial (14) and canine (15) epithelial cells. Those studies give an interesting view of the transformation mechanisms, with different signaling cascades involved depending on the cell lines studied. They also stress the need for an in vitro cellular system closer to the cells at the origin of the tumor in vivo. Over time, several attempts were made to derive epithelial cell lines from OPA; unfortunately, they hardly maintained their alveolar type II phenotype in vitro and did not efficiently produce virus (16, 17). We therefore developed an in vitro system whereby alveolar type II cells from OPA were efficiently maintained in culture over several passages and stably expressed JSRV. This novel experimental model will allow further studies on transformation mechanisms of epithelial cells and set up the stage for elucidating lung cancer tumorigenesis in response to retrovirus infection.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animal Source, Cell Isolation, and Culture of Alveolar Epithelial Cells
Sixteen sheep within domestic sheep populations, from different areas in France, were clinically diagnosed for signs of respiratory distress after full examination by a veterinarian (Figure 1A). At necropsy, the collected lung showed typical pattern of diffuse tumor. The cut surface of the neoplasic masses had a wet appearance and abundant fluid filling the airways. Histopathologic examination confirmed the clinical evaluation and the OPA diagnosis. Eight lungs were collected from apparently healthy animals at a local slaughterhouse. Post-necropsy macroscopic exam did not evidence the presence of tumors; histopathologic examination confirmed the absence of tumoral lesions and these samples were considered as control lungs. Lung tissues were minced and transferred into Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO) containing type Ia collagenase (0.025%; Sigma), DNase I (10 µg/ml; Sigma), protease XIV (1 mg/ml; Sigma), and penicillin/streptomycin (100 U/100 µg; Sigma) for 24 h. After thorough shaking, free cells were separated from tissue by sequential filtration through sterile gauze and 40-µm nylon mesh. Cells were washed with PBS, plated on collagen I (40 µg/ml; Upstate-Chemicon, Hampshire, UK) and fibronectin (10 ng/ml; Sigma)-coated dishes, and maintained in selective epithelial medium Quantum 286 (PAA, Pasching, Austria) complemented with keratinocyte growth factor (KGF, 10 ng/ml; Abcys, Paris, France), hepatocyte growth factor (HGF 5 ng/ml; Abcys), and penicillin/streptomycin, and cultured in 5% CO2 at 37°C. Cells were passaged every 5 d and cultured directly on plastic dishes, on coated membrane inserts with fibronectin 10 µg/ml (Sigma) and collagen I (Upstate-Chemicon) or on Matrigel (BD-Biosciences, Bedford, MA)-coated dishes.


Figure 1
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  		Figure 1. Isolation of primary epithelial cells from tumoral or nontumoral lungs. (A) Ovine lung tissues were analyzed by PCR for the presence of JSRV proviral genome and by histopathologic examination for the presence of specific lesions of OPA. Primary cultures were derived from the same tissues and the numbers of passages they reached was reported as + : < 2 passages; ++ : 3–10 passages; +++ : > 10 passages. (B) Primary cultures were enriched in epithelial cells. Cells displayed a typical macroscopic epithelial phenotype with small cubical adjacent cells (left panel; magnification: x100) and expressed cytokeratin (right panel; magnification: x400). Nuclei were stained with DAPI.

 
Immunocytochemistry
Cells were fixed with methanol and incubated with primary antibodies overnight at 4°C in PBS/bovine serum albumin 1%. Anti-cytokeratin 8.13 at 1/100 (Sigma), anti–SP-A at 1/500 (Chemicon), anti-pro–SP-C at 1/300 (Chemicon), and anti–thyroid transcription factor (TTF)-1 at 1/20 (Dako, Glostrup, Denmark) were used. After washings with PBS, cells were incubated with the secondary antibodies coupled with fluorescein isothiocyanate (at 1/50; Sigma), Alexa-488 at 1/500, or Alexa-633 at 1/300 (Molecular Probes, Invitrogen, Cergy-Pontoise, France) for 1 h at room temperature, washed, and stained with DAPI when necessary. As negative controls, labeling was conducted without primary antibodies. The slides were observed on a confocal Axioplan2 LSM 510 microscope (Carl Zeiss, Le Pecq, France).

Endogenous Alkaline Phosphatase Activity
Fixed cells were incubated for 20 min at room temperature with 2 mg naphtol AS-MX phosphate (Sigma) in 0.2ml N,N-dimethylformamide, diluted in 0.1 M Tris buffer completed with 1 mg/ml Fast Blue BB. After washing with water, cells were stained with 0.1% Neutral red.

Transmission Electron Microscopy
After 4–7 d in culture, cells were fixed with 2% glutaraldehyde for 1 min, then in 4% glutaraldehyde/0.2 M cacodylate pH7.4 (vol/vol) for 30 min. Washings were done in 0.4 M saccharose/0.2 M cacodylate p H7.4 (vol/vol). Post-fixation was realized in 2% osmium/0.3 M cacodylate pH 7.4 (vol/vol) for 45 min. After a quick wash with water, cells were dehydrated by serial ethanol bath: 30, 50, 70, 95, and 100. All those steps were realized at 4. Ethanol 100 was then substituted by Epon/ethanol 100 (vol/vol) for 1 h at room temperature. Impregnation was realized in Epon for 1.5 d at room temperature. Cells were then soaked 1 h in Epon A+B plus DMP 30 at 1.7% and incubated for 72 h at 60°C. After being embedded in epoxy resin, ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined with a JEOL TEM. Cells from two nontumoral and six tumoral primary cultures were analyzed.

PCR Detection of Retroviral Sequences
Genomic DNAs were extracted from primary cells using the "QIAamp DNA mini kit" (Qiagen, Courtaboeuf, France) or from tissues using the Fast DNA kit and the Fastprep device (Qbiogene, Montreuil, France), following the recommendations of the supplier. A hemi-nested PCR protocol was used to detect JSRV proviral DNA. Briefly, reaction mixtures contained 500 ng total genomic DNA, 1x PCR buffer (Eurobio, Les Ulis, France) (67 mM Tris-HCl [pH 8.8], 16 mM (NH4)2SO4, 0.01% Tween 20), 0.2 mM each deoxynucleotide triphosphate, 0.2 mM each primer, 1.5 mM MgCl2, and 1.25 U EuroBlue Taq polymerase (Eurobio). To amplify the full-length env gene, the first round of PCR was performed with primers JSRV1 (5'-ATCACACTGCGGACGTTC-3') located in the pol gene and JSRV53 (5'-GGATTCTTACACAATCACC-3') located in the U3 LTR. The second round of PCR was performed with 1–5 µl PCR product using JSRV1 and JSRV52 (5'-CACCGGATTCTTATATAATC-3') located in the U3 region of the LTR. The 2,833-pb (JSRV1–JSRV53) and 2,818-pb (JSRV1–JSRV52) fragments were amplified as follows: 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 58°C, and 2 min at 72°C. Alternatively, JSRV provirus was detected by a hemi-nested PCR in the 3' part of the env gene. Briefly, primers JSRV98 (5'-GAGTTGAAATGCTGCATATG-3') located in the env gene and JSRV53 were used. Then the product of this first reaction was amplified with primers JSRV98 and JSRV52. The 290-pb (JSRV98–JSRV53) and 274-pb (JSRV98–JSRV52) fragments were amplified as follows: 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 55°C, and 1 min at 72°C. Specificity of the PCR products was controlled by hybridization with a 326-pb digoxygenin-labelled env probe. Detection of SRLV (Small Ruminant LentiViruses) proviral DNA was performed as previously described with primers located in the region of the region of the pol gene coding for the RT (reverse transcriptase); the specificity of the PCR products was controlled by hybridization with a digoxygenin-labeled pol probe (18).

Measure of RT Activity
The RT activity was measured using a chemiluminescent assay kit (Roche, Meyland, France) following the manufacturer instructions. Briefly, 2 ml of cell culture supernatant were concentrated 50 times by centrifugation at 14,000 x g for 90 min and the pellets were suspended in 40 µl lysis buffer. A mix of digoxigenin and biotin-labeled nucleotides was incorporated, and then the amount of DNA synthesized by the reverse transcriptase was quantified by ELISA as recommended. Purified CAEVCo (Caprine Arthritis Encephalitis Virus strain Cork) virus was used as positive control and lysis buffer as negative controls.

Detection of JSRV Capsid Expression
Cells were lysed in 250 µl lysis buffer (0.5 M Tris pH 8.0, 10% glycerol, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1 mM Na3VO4, 1 mM phenylmethylsulphonylfluoride, 10 mg/ml leupeptin, 10 mg/ml aprotinin), homogenized, and incubated for 30 min on ice. The lysates were centrifuged at 18,000 x g for 30 min at 4°C, and 50 µg protein were separated on a 12% SDS-Page and transferred onto a 0.2-µm nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The membranes were pre-incubated with TSBT (25 mM Tris pH 7.6, 0.15 M NaCl, 0.05% Tween 20) containing 5% nonfat dry milk for 1 h at room temperature. After three washes in TSBT, the membranes were incubated for 1 h at room temperature with 1:10,000 diluted rabbit polyclonal antibody directed against JSRV capsid protein (generously provided by J. C. De Martini, Dept. of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO). Membranes were washed three times for 5 min at room temperature with TBST and incubated with horseradish peroxidase–labeled anti-rabbit immunoglobulin G (Sigma) diluted in TBST containing 5% nonfat dry milk (1:10,000) for 1 h at room temperature. The immunoreactive bands were detected using an enhanced chemiluminescence detection kit (Perbio Science, Bezons, France).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Derivation of Primary Cultures of Ovine Airway Epithelial Cells
All tumoral lungs tested by PCR were positive for JSRV provirus DNA (Figure 1A). Diagnosis of OPA was confirmed by histopathologic examination. In control animals with no detectable clinical signs, the JSRV provirus was isolated in only 1/8 samples; histopathologic exam did not evidence any tumoral lesions in this sheep. Primary cultures were isolated from tumoral or nontumoral lung tissues. Isolated ovine alveolar type II cells were grown in a selective synthetic medium, complemented with KGF and HGF. Fifteen primary cultures were maintained for 7–10 passages when derived from tumoral lungs, including one cell line, 1014, reaching 20 passages. Cultures from control lungs (n = 8) could only be maintained for two or three passages. Clonal isolation was unsuccessful (data not shown). The proportion of epithelial cell observed in tumor-derived cell lines, once plated into dishes, was about 95% according to the Cytokeratin 8.13 staining (Figure 1B). Two types of cuboidal cells were observed: small rapidly dividing cells and large slowly dividing cells. These larger cells did not adhere to the plate after trypsination. The higher number of passage was observed for the culture presenting the higher proportion of small cuboidal cell. Fewer than 5% of spindle-shaped fibroblasts were present at the first passages. The selective culture medium (Q286 supplemented with KGF and HGF) efficiently depressed their growth while promoting the growth of the epithelial cells, thus increasing the proportion of alveolar type II cells over passages.

Characterization of the Alveolar Type II Cells
Ultrastructural study of epithelial monolayers showed polarized cells with numerous microvilli at the apical side covered by proteoglycans, basement membrane at the basal side, desmosomes, and a round-shaped nucleus (Figures 2A–2C). Cytoplasm was rich in rough endoplasmic reticulum. Cytoplasmic vacuoles were abundant in tumoral alveolar type II cells. In monolayer culture, typical lamellar bodies were observed in normal alveolar type II cells, while only rough-shaped bodies (nascent lamellar bodies) were observed in tumoral alveolar type II cells (Figure 2D). Cells maintained onto inserts were more cubical and more differentiated than the ones cultured onto plastic.


Figure 2
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  		Figure 2. Ultrastructure of ovine alveolar type II cells. Alveolar type II cells at the first passage (4–7 d of culture) maintained on coated inserts or plastic dishes were fixed and analyzed by transmission electron microscopy. (A) Primary cells derived from nontumoral lungs showed lamellar bodies (LB) with typical concentric lamellae (arrows). (B) Desmosomes. (C) Enriched rough endoplasmic reticulum (RER) cytoplasm. (D) Primary cells derived from tumoral lungs showed numerous rough shaped lamellar bodies with poorly organized lamellae (arrows).

 
Airway epithelial cultures derived from tumoral and nontumoral lung expressed alveolar type II cell specific surfactant proteins (SP)-A and proSP-C (Figures 3A–3B). Expression of those markers was maintained over passages, with proSP-C becoming more diffuse. A high level of alkaline phosphatase activity was evidenced at the membrane level of the cells (Figure 3C). Alveolar type II cell cultures expressed TTF-1, a tumoral alveolar type II cell marker (Figure 3D); CD44v6 protein (not shown); and ZO-1, a tight junction protein (Figures 3E–3G). ZO-1 expression demonstrates the capacity of ovine tumoral alveolar type II cells to polarize when cultured onto coated insert. Taken together, these results confirm the alveolar type II cell phenotype of cultures derived from ovine lungs. We previously described that tumoral alveolar type II primary cells had a proliferative advantage over those derived from normal lung (19).


Figure 3
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  		Figure 3. Expression of alveolar type II cell markers. Primary cells derived from tumoral lungs expressed surfactant proteins such as SP-A (A) and pro–SP-C (B) and displayed alkalin phosphatase activity (C). (D) Expression of TTF-1 was detected in the nucleus. Tight junctions were present in the cultures, as evidenced by the detection of ZO-1 (E–G). Magnification: x400.

 
Alveolar Type II Cells Are Productively Infected by JSRV
To investigate whether virus production occurred in in vitro derived alveolar type II cells, we measured the RT activity in the culture supernatants. The RT is a DNA-polymerase RNA dependent, involved in the first step of the retrovirus cycle. Its presence in cell surpernatants marks the production of virus by the cells. Enzymatic activity was detectable in 3 of the 11 tested tumoral type II cell cultures (deriving from 11 animals) and in none of the normal ones (Figure 4). This enzymatic activity evidenced the retrovirus production by the tumoral cells. To confirm the nature of the retrovirus, we looked for the presence of JSRV provirus by PCR and hybridization. All the tumoral alveolar type II cells harbored the JSRV provirus genome (Figure 5A), while the nontumoral cell lines were negative. These results are in accordance with the PCR detection from the tissues at the origin of the cultures (Figure 1A) and show the successful in vitro isolation of the JSRV-infected cells from the tumor. Ultrastructural studies revealed budding of retroviral particles at the apical side of tumoral alveolar type II cells (Figure 5B). Taken together, these results clearly showed that alveolar type II cells derived from lungs were infected by and expressed JSRV in vitro.


Figure 4
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  		Figure 4. Detection of reverse transcriptase (RT) activity in supernatants of tumoral alveolar type II cells. RT activity was measured in culture supernatants of tumoral (bold characters) and nontumoral alveolar type II cells at different passages (p0, p1 ...). NT, nontumoral; T, tumoral. C+: positive control made of purified CAEV, Cork strain.

 

Figure 5
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  		Figure 5. Detection of JSRV in tumoral alveolar type II cells. (A) After PCR amplification of the env gene, JSRV proviral genome was only detected in alveolar type II cells derived from tumoral lungs (bold characters). (B) Viral budding was observed at the apical side of alveolar type II cells in one of the five tumoral cultures examined by transmission electron microscopy. NT, nontumoral; T, tumoral.

 
SRLV, members of the retrovirus family, are common viral pathogens of sheep. We controlled their presence in the alveolar type II cell cultures by PCR and hybridization. Interestingly, SRLV provirus genome was detected in two tumoral cultures showing a clear type II phenotype (SP-C and SP-A expression). These preliminary results suggest that alveolar type II cells have a cellular SRLV receptor and may be a reservoir of SRLV infection in vivo.

JSRV-Infected Cells Have a Limited Lifespan In Vitro
We observed a decrease of the RT activity over passages (Figure 4). For example, RT was only detected in the very first passages of the 1014 alveolar type II cell line, while JSRV provirus DNA was present up to the seventh passage (Figure 6). These observations suggest a block in JSRV expression in alveolar type II cells. Interestingly, we observed the same disappearance of the infected-cell population over passages for two other cell lines (998 and 1013) (Figure 6). We confirmed by immunocytohemistry using anti–SP-C antibodies that these cells conserved a phenotype compatible with alveolar type II pneumocytes. These results suggest the in vitro coexistence of two subpopulations of tumoral alveolar type II pneumocytes: a proliferative JSRV-positive (+) cell subpopulation, not maintained over the seventh passage, and a highly proliferative JSRV-negative (–) cell subpopulation, characterized by a long-term maintenance in vitro.


Figure 6
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  		Figure 6. Decrease of viral expression over passages. (A) The subpopulation of alveolar type II cells infected by JSRV (as evidenced by PCR amplification of the env gene followed by Southern blot hybridization of the amplicons) disappeared over passages. JSRV proviral DNA was no longer detectable in late passages. (B) A longitudinal follow-up of the presence of JSRV proviral genome (detected by env PCR) over successive passages (0–17) for three tumoral alveolar type II cell lines (1013, 1014, 998) showed the loss of the infected subpopulation. +, presence of JSRV genome; –, absence of JSRV genome; C+, positive control.

 
We compared the number of JSRV(–)-infected cells in the native cell population (once the dissociated lung tissues were plated onto culture dishes, passage p0) and in the long-term cultures. The DNA concentration was measured and serial dilutions were used to detect JSRV provirus by PCR. At p0, the JSRV genome was found at a dilution of 10 cell equivalent (CE), suggesting that at least one copy of JSRV genome was present out of 10 cells. At a later passage (p5), JSRV provirus was detected at only one copy for 100,000 cells.

Three-Dimensional Culture Conditions Maintain a Better Virus Activity
As polarization of alveolar type II cells onto insert do not allow the maintenance of viral expression, we investigated the effect of three-dimensional culture conditions on duration of alveolar type II cell growth and viral production. Cells were cultured on a thick layer of Matrigel, a complex synthetic extracellular matrix. On Matrigel, cells formed numerous spheroids of variable size (Figure 7A). They were passaged every week and maintained on 3D Matrigel for five passages. The level of differentiation of alveolar type II cells was studied. Ultrastructural analysis revealed the presence of numerous lamellar bodies, showing the presence of well-differentiated alveolar type II cells (Figure 7A). Three-dimensionally organized cells expressed SP-C (not shown). JSRV genome was detected from passages 0–5, in monolayer as in spheroids (not shown). Interestingly, RT activity was better maintained in three-dimensional conditions (up to the fifth passage) than in monolayer (up to the second passage). JSRV capsid was detectable in cells maintained in three-dimensional culture (Figure 7C). Taken together, three-dimensional culture conditions seemed to greatly improve maintenance of virus expression in tumoral alveolar type II cell cultures.


Figure 7
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  		Figure 7. Persistence of viral expression in alveolar type II cells cultured in three-dimensional matrix. (A) Tumoral ovine alveolar type II cells grown in three-dimensional matrix formed typical spheroid (left panel; magnification: x100). Transmission electron microscopy revealed the presence of well-differentiated alveolar type II cells (center panel; bar: 10 µm) with numerous lamellar bodies (LB), shown in the enlarged view (right panel; bar: 1 µm). (B) RT activity, as an indicator of virus production in the supernatant, was maintained longer in three-dimensional culture (up to the fifth passage) in the 1045 alveolar type II cells (derived from the tumoral lung 1045) than in the same cells maintained in monolayer culture (only at the first passage). (C) JSRV capsid protein (27 kD) was expressed at high level in three-dimensionally cultured alveolar type II cells. NT, nontumoral; T, tumoral; 1012, 1013, 1014 ..., alveolar type II cells isolated from different sheep; C+, positive control.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In this study, we report the isolation and characterization of alveolar type II cells from ovine tumoral lungs diagnosed with OPA. Alveolar type II cells are well known for being difficult to maintain in vitro because of their low rate of division and their tendency to transdifferentiate into alveolar type I cells. Our results showed that we could readily and reproducibly isolate alveolar type II cells from tumoral OPA lungs (n = 15) and from normal lungs (n = 8). Their characterization evidenced the presence of lamellar bodies, the expression of the surfactant proteins SP-A and SP-C, TTF-1, and a high alkaline phosphatase activity. SP-A and SP-C expression was clearly maintained over passages, up to 20 passages for some of the cultures. When grown as monolayer, alveolar type II cell cultures were well differentiated and polarized, as evidenced by desmosomes assembly and ZO-1 expression. In three-dimensional conditions, alveolar type II cells were enriched in lamellar bodies. Interestingly, we observed the stabilization of viral expression in alveolar type II cells maintained in three-dimensional conditions, suggesting a role of cell polarization and differentiation for virus production. Recent works demonstrate the improvement of physiology, metabolism, interaction and processes of cells when maintained in a three-dimensional environment mimicking physiologic conditions. In our study, electron microscopy demonstrated the enrichment in well-differentiated lamellar bodies in three-dimensional cultures, while cells polarized on insert only shown few rough shaped lamellar bodies. We are now exploring a possible role of these vesicular trafficking components for efficient viral expression and/or budding.

We previously showed a proliferative advantage, measured by MTT assay, of OPA-derived alveolar type II cells over normal type II cells (19). Cell cycle analysis using flow cytometry indicated that the proportion of cells in the S phase was higher in tumoral cells than in control cells (19). We demonstrated in this study that JSRV-infected tumoral type II cells could be maintained for a large number of passages (5–20), while normal alveolar type II cells dedifferentiate and quickly lose replicative abilities. One out of eight alveolar type II cell cultures isolated from normal lungs was infected by the virus. This is in accordance with in vivo observations in flocks, relating the presence of the virus in animals free of clinical signs of OPA (20). Not all the infected animals will develop OPA during their commercial life.

Interestingly, we observed that cultures isolated from tumoral lungs were composed of two subpopulations of alveolar type II cells: JSRV-infected and noninfected cells. We observed that JSRV-infected alveolar type II cells have an increased lifespan compared to ovine normal type II cells, but a reduced lifespan compared to a subpopulation of tumoral JSRV-negative cells. The latter kept an alveolar type II cell phenotype (SP-C expression) and had a fast growing rate while not being infected by the virus. These results are in accordance with previous works describing the isolation of tumoral cells from OPA lesions 30 years ago (16, 17, 21). Those results taken together suggest that (1) the disappearance of viral production (RT activity) is mainly due to the loss of the alveolar type II cell subpopulation that has integrated the retrovirus more than to the loss of the differentiated phenotype in vitro, and (2) long-lasting JSRV-free alveolar type II cells are transformed while not carrying the JSRV genome. Going back to previous in vivo observations, immunohistochemical studies on natural and experimentally induced OPA have showed that JSRV-infected tumoral cells derived predominantly from alveolar type II cell and Clara cells and that not all of the tumoral cells were expressing the JSRV capsid protein (9). When looking at naturally occurring OPA, the tumor is characterized by large monocentric coalescing nodules (22), suggesting a slow transformation process such as insertional mutagenesis. When looking at experimentally induced OPA, tumors are constituted of small disseminated tumor nodules (23–25), suggesting an acutely transforming mechanism through a viral protein. Thus, JSRV may induce OPA by both acute and nonacute retroviral oncogenesis. The tumor development is probably the result of a multistep process involving viral protein–induced cell proliferation and provirus-induced insertional mutagenesis (26). This could explain our finding of two subpopulations of tumoral alveolar type II cells. We described a subpopulation of cells replicating JSRV in the first passages and characterized by a limited lifespan. This population carries the integrated JSRV genome and may have been transformed by a slow mechanism such as insertional mutagenesis. We showed a second population that did not carry the integrated JSRV genome and had a longer lifespan; these cells may have been acutely transformed in vivo or right in vitro, by an acute mechanism involving a viral protein–induced cell proliferation; such as the envelope expressed by the JSRV-replicating population. More studies are necessary to investigate these possible different steps leading to the alveolar type II cell transformation. We are currently exploring the cell death process of the early JSRV-infected and the late JSRV-free cells. Tumorigenic properties of those cells will be evaluated by in vivo transfer into nude mice. Whether JSRV is deleterious for the infected cell is not known and should be determined.

Mechanisms of transformation by JSRV have being actively studied over the last years. Recent studies evidenced the in vivo oncogenic property of JSRV envelope protein in mice (27) and sheep (28). Before that, several groups had demonstrated that the envelope can efficiently transform fibroblast cell lines derived from mice (10), rats (11), and chicken (12, 13), and epithelial cell lines such as immortalized human bronchial epithelial cell lines (14) and canine kidney epithelial cells (15). Cell types and their origins seem to be an important determinant for the signaling pathways stimulated by the JSRV envelope, and those interesting results stressed the need for an in vitro cellular model, closer to the cells involved in vivo. The ovine alveolar type II cells we developed in this study provide an invaluable tool, and signaling pathways involved in infected versus noninfected alveolar type II cells, and in transformed versus normal alveolar type II cells, are currently being explored. We have already showed dysregulation of the replicative senescence and Akt pathway (19). We described telomerase activation in alveolar type II cells isolated from OPA lungs (19), suggesting that inhibition of cell senescence may be involved in OPA tumorigenesis. We have also showed high levels of phosphorylated Akt in OPA tumors, with abolition of Akt activation in response to epidermal growth factor stimulation in alveolar type II cells in vitro (19), suggesting dysregulation of the Akt pathway.

For one of the alveolar type II cell culture cultured in a three-dimensional environment, we were able to simultaneously detect expression of both SP-C and CC-10, specific markers of alveolar type II cells and of Clara cells, respectively, in the same cell. This preliminary result raises the question of the possible stem-cell origin of OPA. Several lung progenitor cells have been identified; however, there is limited data regarding the lineage relationships of these cells. A recent study reports on isolation of a bronchioloalveolar stem cell population called BASCs (29). BASCs express alveolar type II cell marker (SP-C) as well as Clara cell marker (CC-10), and the authors propose that they may be at the origin of lung adenocarcinoma. A putative progenitor subpopulation of alveolar type II cells has also been identified (30). Those cells are E-cadherin–negative, proliferate, and express high levels of telomerase activity. Alveolar type II cell lines isolated from OPA versus normal lung give us the opportunity to better elucidate the origin of tumoral cell in lung adenocarcinoma, OPA, and BAC, and to look for the role of stem cells.


   Acknowledgments
 
The authors thank the "Centre de microscopie - IFR128 Biosciences Lyon Gerland" and the "Centre d'imagerie de Laennec"- IFR 62 Lyon (Simone Peyrol) for their technical support, and Prof. Francçoise Thivolet-Bejui for her anatomopathologic expertise on lung tissues.


   Footnotes
 
This work was supported by the Rhône-Alpes Region, departmental committees of Ligue Nationale contre le cancer (Loire, Ardèche and Drôme) and the INRA (Institut National de la Recherche Agronomique).

Originally Published in Press as DOI: 10.1165/rcmb.2006-0285OC on December 7, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 7, 2006

Accepted in final form October 23, 2006


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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