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. Author manuscript; available in PMC: 2015 Jul 2.
Published in final edited form as: Virology. 2011 Feb 12;412(2):349–356. doi: 10.1016/j.virol.2011.01.022

Enhanced proliferation of primary rat type II pneumocytes by Jaagsiekte sheep retrovirus envelope protein

Chassidy Johnson a, Sohail Jahid a, Dennis R Voelker b, Hung Fan a,*
PMCID: PMC4489409  NIHMSID: NIHMS695000  PMID: 21316726

Abstract

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep. The envelope protein (Env) is the oncogene, as it can transform cell lines in culture and induce tumors in animals, although the mechanisms for transformation are not yet clear because a system to perform transformation assays in differentiated type II pneumocytes does not exist. In this study we report culture of primary rat type II pneumocytes in conditions that favor prolonged expression of markers for type II pneumocytes. Env-expressing cultures formed more colonies that were larger in size and were viable for longer periods of time compared to vector control samples. The cells that remained in culture longer were confirmed to be derived from type II pneumocytes because they expressed surfactant protein C, cytokeratin, displayed alkaline phosphatase activity and were positive for Nile red. This system will be useful to study JSRV Env in the targets of transformation.

Keywords: Jaagsiekte sheep retrovirus, Env, Transformation, Type II pneumocyte

Introduction

Sheep infected with Jaagsiekte sheep retrovirus (JSRV) can development ovine pulmonary adenocarcinoma (OPA) (Palmarini and Fan, 2001). OPA shares histological similarities with some forms of human lung cancers and is defined as adenocarcinoma of mixed type with a bronchioloalveolar carcinoma (BAC) component. Similarities between OPA and human pulmonary adenocarcinoma makes studying JSRV and OPA a useful model system to further understand lung cancers in humans.

JSRV and the closely related retrovirus, enzootic nasal tumor virus (ENTV), are unique retroviruses because one of the structural proteins, envelope (Env, encoded by the env gene), also functions as an oncogene. We and others have shown that expression of Env alone can transform murine, rat, canine and human cells in culture (Allen et al., 2002; Danilkovitch-Miagkova et al., 2003; Liu et al., 2003b; Liu and Miller, 2005; Maeda et al., 2001; Palmarini and Fan, 2001; Rai et al., 2001) as well as induce tumors in animals (Caporale et al., 2006; Wootton et al., 2005). The env gene is expressed as a polyprotein that is post-translationally cleaved into surface (SU) and transmembrane (TM) proteins. We and others have demonstrated that the C-terminal cytoplasmic tail of TM is crucial for transformation (Chow et al., 2003; Hofacre and Fan, 2004; Hull and Fan, 2006; Palmarini et al., 2001). However, other domains of Env, including SU are also important for transformation (Hofacre and Fan, 2004). JSRV Env transformation has been shown to activate the PI3K–Akt–mTOR and Ras–Raf–MEK–MAPK pathways (Liu et al., 2003b; Liu and Miller, 2005; Maeda et al., 2005; Palmarini et al., 2001; Zavala et al., 2003). However, these pathways are not required in all cell lines and culture conditions (Johnson et al., 2010; Maeda et al., 2003). The differences in pathways used in Env transformation may be attributed to species, cell type and/or the differentiation state.

Human pulmonary adenocarcinoma and OPA arise from secretory epithelial cells of the distal airway, type II pneumocytes and less frequently Clara cells (Sharp and DeMartini, 2003). Ideally it would be desirable to study JSRV transformation in the target cells for tumorigenesis. Cell lines have been derived from tumors of type II pneumocytes and Clara cells but they are already transformed and furthermore do not maintain the complete differentiated state (Dobbs, 1990; Dobbs et al., 1985; Manzer et al., 2006; Mason et al., 1977; Wang et al., 2006). Studying JSRV Env transformation in the targets of transformation would help clarify the requirements for transformation. To date, JSRV Env transformation has not been studied in normal lung epithelial cells because a system has not been available.

When cultured on plastic substrata, primary type II pneumocytes no longer express surfactant proteins and can transdifferentiate into type I pneumocytes (Dobbs, 1990; Dobbs et al., 1985; Manzer et al., 2006; Mason et al., 1977; Wang et al., 2006). Several strategies have been described to maintain the differentiation state of type II pneumocytes for limited periods of time. Culture of primary rat type II pneumocytes in rat serum provides exogenous fatty acids and increases lipid synthesis required for surfactant production (Borok et al., 1995; Cott et al., 1987; Leslie et al., 1985), while culture in fetal bovine serum increases cellular spreading, contributing to transdifferentiation. Addition of keratinocyte growth factor (KGF) stimulates DNA synthesis and helps to maintain the differentiation state (Wang et al., 2006). Providing an air–liquid interface to the apical side of primary type II pneumocytes also helps to maintain the differentiation state (Dobbs et al., 1997; Gruenert et al., 1995; Whitcutt et al., 1988; Yamaya et al., 1992). Primary rat type II pneumocytes cultured on Engelbreth–Holm–Swarm (EHS; Matrigel) tumor matrix or contracted collagen gels maintain the phenotype for longer periods of time and in some studies this treatment reverts the transdifferentiated phenotype (Danto et al., 1995; Olsen et al., 2005; Shannon et al., 1990, 1987). Growth on EHS alone results in spherical aggregates of differentiated type II pneumocytes while culture on adherent collagen gels results in growth of transdifferentiated cells (Shannon et al., 1990, 1987; Wang et al., 2006).

In these studies JSRV Env transformation was studied in purified primary rat type II pneumocytes. The cells were cultured on a mixture of Matrigel and rat tail collagen at an air–liquid interface to favor growth of differentiated cells. In addition the cells were cultured in medium that favored growth of airway epithelial cells and disfavors growth of contaminating fibroblasts. We performed transformation assays by DNA transfection or transduction-mediated delivery of JSRV Env. Results indicated that JSRV Env provided a growth advantage to type II pneumocytes and the differentiation state of the cells was maintained for the duration of the assay.

Results

A culture system to grow purified primary rat type II pneumocytes (RTII) under conditions that favor prolonged expression of differentiation markers of type II pneumocytes was developed. It employed several previously established approaches. The cells were cultured in transwells (6 well plates) on a mixture of polymerized rat tail collagen and Matrigel (80:20) matrix in airway epithelial culture media (AEC). AEC is optimized for growth of airway epithelial cells and the lack of serum disfavors growth of contaminating fibroblasts. A small volume of culture medium was added to the inside of the transwell inserts so that with continuous rocking an air–liquid interface at the apical region of the cells was maintained. The cultures were initially seeded at 5 × 105 cells/well for freshly isolated cells and 1 × 106/well for frozen cells. Seeding at these densities favored growth of well-isolated colonies that allowed quantification of transformation assays.

The RTII cultures were transfected with a JSRV Env expression plasmid, pCMVJS21ΔGP (ΔGP) (Maeda et al., 2001) or a Flag epitope-tagged version (ΔGPflag). For comparison, cells were also transfected with the pcDNA3.1 backbone plasmid as a negative control. The transfected RTII cells grew as well-isolated colonies that could be counted. The number of colonies that formed at 7, 14 or 21 days following transfection is shown in Table 1. For all cultures, the number of colonies decreased over time, which reflected cell death and deterioration of the colonies. However at all times the ΔGP and ΔGPflag-transfected cultures showed more colonies than pcDNA3.1-transfected cultures.

Table 1.

Number of colonies in transfected primary rat type II pneumocytes cultures.

Transfected
DNA		 Number of colonies
Day 7 Day 14 Day 21
Experiment 1 pcDNA3.1 116 54 8
ΔGP 207 157 60
Experiment 2 pcDNA3.1 49 21
ΔGPflag 117 47
Experiment 3 pcDNA3.1 45 13
ΔGP 72 22
Experiment 4 pcDNA3.1 11
ΔGP 34
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RTII cultures were transfected with the indicated DNA as described in Materials and methods. The total number of colonies was counted at days 7, 14 and 21 after transfection. The numbers reflect the total of all colonies in the 6 well plate.

The sizes of colonies in the transformation assays were scored at 7 and 28 days following transfection, and the distributions of colony sizes are shown in Fig. 1. At day 7, the majority of the colonies were small and no difference was observed between pcDNA3.1 and ΔGP-transfected cultures (Fig. 1A). At 28 days, the remaining colonies had increased in size for both pcDNA3.1- and ΔGP-transfected cultures, indicating that cell division was occurring. Moreover at later times the colonies in the ΔGP-transfected cultures were larger than those in the pcDNA3.1-transfected cultures. At 28 days, 46.7% ± 4.9% of colonies in ΔGPflag-transfected samples were >660 μM in diameter (large) compared to 12.5% ± 4.3% for pcDNA3.1-transfected samples (Fig. 1B). The fact that ΔGP-transfected cultures formed more and larger colonies than pcDNA3.1-transfected cells suggested that Env provided increased growth potential of primary rat type II pneumocytes. Representative colonies from pcDNA3.1- and ΔGPflag-transfected samples are shown in Fig. 2. ΔGP-transfected cultures showed larger colonies than pcDNA3.1-transfected samples (Fig. 2A vs. B). Cells in the colonies were cuboidal-shaped, which was consistent with the morphology of type II pneumocytes (Fig. 2C and D).

Fig. 1.

Fig. 1

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Effect of JSRV Env on colony size. RTII cell cultures were transfected with pcDNA3.1 or ΔGP as described in Materials and methods. At day 7 (A) and day 28 (B) following transfection, all of the colonies were scored by size and presented as the percent of the total (means ± standard errors) colonies that fell into the indicated size categories (small, medium or large). The results are representative of at least four independent experiments. The size categories were defined as small (10–330 μM), medium (331–660 μM) and large (≥661 μM).

Fig. 2.

Fig. 2

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Colonies in transfected primary rat type II pneumocyte cultures. Light microscopy photographs of pcDNA3.1- (A and C) and ΔGPflag (B and D)-transfected cultures at 21 days following transfection. Magification, ×40 (A and B) and ×100 (C and D); bar, 200 μm.

While Env expression apparently increased the growth potential of the RTIIs, it did not result in immortalization and all of the cells in the cultures eventually died. However, expression of Env extended the length of time that the RTIIs could be cultured as shown in Table 2. The transfected cultures were incubated until the colonies grew and reached confluency (if they could). At that point the cultures were transferred as described in Materials and methods, and this was continued until the cultures died out. The number of transfers attainable with each culture is shown in Table 2, along with the total number of days the culture remained viable. pcDNA3.1-transfected (and untreated) cultures generally could not be passaged because they never reached confluency at the density seeded, the colonies deteriorated or the cells did not survive the passage. In contrast, ΔGP and pCMVJS21-transfected cells could be passaged multiple times (Table 2). In experiment 2, ΔGP-transfected cells termed 12AF could be passaged at least 6 times and were viable in culture for >134 days. At that point they were frozen; they could be thawed and the cultures remained viable for a limited period of time. However, the thawed 12AF cells could not be further passaged after freezing. We attempted to establish an immortalized cell line by co-transfecting rat type II pneumocytes with ΔGP or ΔGPflag along with a second gene known to be important in immortalization. Co-transfection of ΔGPflag with expression plasmids for activated H-Ras, mutant p53 (R175H), mTert, hTert or SV40LT did not yield an immortal cell line.

Table 2.

Enhanced replication in transfected primary rat type II pneumocyte cultures.

Transfected DNA Transfer numbera Days in culture
Experiment 1 pcDNA3.1 1 32
ΔGP 4 50
Experiment 2 pcDNA3.1 0 10
ΔGPb 6 >134
Experiment 3 pcDNA3.1 0 27
ΔGP 2 59
pCMVJS21 2 61
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RTII cultures were transfected with the indicated DNA as described in Materials and methods. When cells reached confluency the cultures were transferred as described, and this was repeated until the cultures showed no additional growth potential. The number of transfers is indicated, as well as the total days in culture (of all transfers). The results are representative of 8 independent experiments.

a

The initial transfected cultures were transfer 0.

b

Cells from this experiment (12AF) were passaged 6 times and then frozen at 134 days for further experiments.

The results shown in Table 2 likely under-represented the enhanced growth potential of the Env-transfected RTII cultures. The recovery of cells from the collagen-Matrigel transwells was in fact inefficient, and the seeding at 1:2 actually resulted in substantially fewer cells seeded than would have been expected. The number of cells recovered by dispase treatment from any individual transwell was so low that that they could not be accurately counted, but we estimate that the transferred cells were seeded at less than 20% the density compared to the seeding concentrations of the original cultures. Additionally, the viability of seeded cells was low. Thus multiple rounds of division were required between transfers.

We next tested if JSRV Env could be introduced into the RTIIs by retroviral vector transduction. RTII cultures were infected with a murine leukemia virus-based vector expressing JSRV Env with or without a C-terminal HA tag (LXSNEnvHA and LXSNEnv; respectively). After 14 days in culture, the number of colonies that formed in retroviral vector transduced samples was counted. JSRV Env- and EnvHA-transduced cells formed many more colonies than cells transduced by the empty LXSN vector (Table 3). The morphology of the LXSN- and LXSNEnvHA-transduced samples after 21 days of culture are depicted (Fig. 3). LXSN-transduced cells maintained a low density and distinct colonies were no longer observed as the cells deteriorated (Fig. 3A). In contrast, LXSNEnvHA-transduced samples formed a monolayer and in some areas the cells grew on top of each other forming foci (Fig. 3B). In general, cells in the LXSNEnvHA-transduced cultures were cuboidal in shape, characteristic of epithelial cells (Fig. 3B). The results suggested that an M-MuLV-based vector expressing JSRV Env can infect primary rat type II pneumocytes and that effects on their growth properties were more efficient than DNA transfection-mediated delivery of Env. The greatly increased number of colonies after LXSNEnvHA transduction might reflect higher efficiency of Env gene delivery for the retroviral vector. Nevertheless cultures transduced with LXSNEnv or LXSNEnvHA did not yield an immortalized cell line.

Table 3.

Number of colonies in retroviral vector transduced primary rat type II pneumocytes.

DNA Number of colonies
Experiment 1 LXSN 45.5±8.8
LXSNEnvHA ≥325*
Experiment 2 LXSN 4.5±3.5
LXSNEnv 70±15.4
LXSNEnvHA TMTC
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RTII were infected with the indicated retroviral vectors as described in Materials and methods. The number of colonies that formed at 14 days following transduction is indicated. The results are the average number of colonies±standard deviation. Experiments were performed in triplicate and the data are representative of 3 independent experiments. TMTC, too many to count.

*

Asterisk indicates the average number of colonies counted but may represent an undercount of the total number of colonies because of the density of the colonies.

Fig. 3.

Fig. 3

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Morphology of transduced primary rat type II pneumocytes. RTII cultures were infected with (A) LXSN or (B) LXSNEnvHA vectors and light microscopy of cells at 21 days of culture are depicted. Magnification, ×100; scale bar, 200 μm.

While these experiments indicated that introduction of JSRV Env into primary rat type II pneumocytes extended their proliferative capacity in the culture conditions employed, it was important to verify that the replicating cells were indeed derived from type II pneumocytes, and that they were expressing Env protein. A particular concern was more rapidly growing fibroblasts that can contaminate type II pneumocyte preparations, even though their growth would not be favored by the medium used (no serum). Transformation of contaminating fibroblasts could lead to a reduced growth factor requirement and consequent replication in the serum-free conditions. After cells in control RTII cultures (pcDNA-transfected or LXSN-transduced) were no longer viable, JSRV Env-transfected or -transduced cultures were analyzed for markers used to distinguish type II pneumocytes: pro-surfactant protein C (SP-C), alkaline phosphatase activity (AP) and Nile red staining (Fig. 4). Cells in the LXSNEnvHA-transduced cultures were positive for pro-SP-C (Fig. 4C), alkaline phosphatase activity (Fig. 4E) and Nile red (Fig. 4F) by immunofluorescence. Two cells in the field of view (indicated with asterisks) were positive for Nile red (Fig. 4F). Additionally, the cells were positive for an epithelial cell marker, cytokeratin (Fig. 4G). ΔGP-transfected samples were also positive for pro-SP-C, alkaline phosphatase activity, Nile red and cytokeratin (data not shown). Together, the results indicate that the cells with enhanced growth potential were derived from type II pneumocytes and not contaminating cells. Furthermore, several properties of type II pneumocytes were maintained for the duration of the assay. To test for Env expression, cells were immunostained with an antibody to the epitope tags HA (Fig. 4H) and flag (not shown); they were positive for JSRV Env. Finally, we examined the differentiation status of the transfected 12AF cells that had been passaged 6 times and frozen after 134 days in culture. Frozen 12AF cells were thawed, initiated on matrix and analyzed for expression of pro-SP-C (Fig. 4D); the thawed cells were uniformly positive for pro-SP-C. Taken together, these results indicate that expression of JSRV Env in primary rat type II pneumocytes extends the replicative capacity of these cells, under the employed culture conditions.

Fig. 5.

Fig. 5

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Properties of Env-expressing cultures. Immunofluorescence for pro-SP-C (A–D) was performed on (A) normal mouse lung, (B) MDCK cells, (C) LXSNEnvHA-transduced RTII cells at passage 35 (transfer 1) when LXSN-transduced cultures were no longer viable and (D) frozen 12AF cells that were thawed and initiated on collagen–Matrigel matrices. LXSNEnvHA-transduced cultures (passage 35; transfer 1) were also immunostained for (E) alkaline phosphatase activity (AP), (F) Nile red and (G) cytokeratin and (H) HA. Samples were counter stained with DAPI (blue) except for (F) where DAPI and Nile red staining were incompatible, and analyzed by confocal microscopy. In panel F, there were two cells (*), and they were both positive for Nile red staining. Magnification, ×630 (A–D, F and G) and ×400 (E).

Discussion

To date, we and others have studied JSRV Env transformation in epithelial and fibroblastic cell lines derived from species other than sheep due to their availability. Although these studies have proven informative, they have limitations. The ideal in vitro system would be JSRV Env transformation of normal type II pneumocytes that have maintained the differentiation state. In this report, we describe a culture system to study JSRV Env in purified primary rat type II pneumocytes that maintain properties of differentiated type II pneumocytes for the duration of the assay. Although JSRV Env did not morphologically transform RTIIs, it increased the replicative capacity because more and larger colonies were observed in the cultures compared to control cultures. The cells were viable longer in culture and they could be passaged more times compared to vector controls. The replicating cells were confirmed to be of type II pneumocyte origin that maintained properties of differentiated cells and expressed Env. This mirrors the status of OPA tumor cells in JSRV-infected sheep. Thus the system described here can be used to study JSRV Env in vitro in the in vivo target of JSRV oncogenesis, the type II pneumocyte.

Much is known about JSRV Env transformation from previous in vitro studies. However, the domains of Env and pathways required for transformation may differ among cell lines. For example, the YXXM motif in the TM protein is absolutely required for transformation in some cell lines (NIH-3T3) but not others (DF-1), suggesting that the role of YXXM may differ among different cell types (Allen et al., 2002; Hofacre and Fan, 2004; Liu et al., 2003b; Palmarini et al., 2001; Zavala et al., 2003). In Env-transformed NIH-3T3 and RK3E cells, the Ras–Raf–MEK–MAPK pathway is activated and inhibition of MEK-1 (PD98059) and H/N-Ras (FTI-277) inhibits transformation (Maeda et al., 2005). In contrast, the Ras–Raf–MEK–MAPK pathway does not play a role in MDCK transformation in 3-D or monolayer culture (Johnson et al., 2010). Indeed, cell-type specific signaling has been reported for other viral oncogenes (Aftab et al., 1997). Therefore, clarifying the importance of cell-type and species differences will be critical to further understand JSRV Env transformation.

Type II pneumocytes derived from rats were chosen for JSRV Env transformation because methods for purification and culture of primary rat type II pneumocytes have been well-described, and culture conditions that favor maintenance of the differentiation state have been established (Dobbs and Mason, 1979; Dobbs et al., 1997; Gruenert et al., 1995; Leslie et al., 1993; Mason et al., 2002; Shannon et al., 1987; Sugahara et al., 1995; Xu et al., 1998). Also much is known about JSRV Env transformation in rat and mouse cell lines. Studying JSRV Env transformation in differentiated rat type II pneumoyctes vs. other epithelial and fibroblast cell lines will help to distinguish between species- and cell-type-specific differences in JSRV transformation. Ideally, we would like to study JSRV Env transformation in primary sheep type II pneumocytes. This is important because there are species-specific requirements for transformation by JSRV Env so there might be differences in the mechanism for JSRV Env transformation between rat and sheep cells. Archer et al. have reported that culture of primary cells from sheep OPA tumors on Matrigel maintained the differentiation state for limited periods of time and had enhanced growth compared to normal cells (Archer et al., 2007). Thus the culture model described here may be applicable to sheep type II pnuemocytes as well. However subtle differences in culture conditions may be required because species-specific requirements for culturing primary type II pneumocytes in the differentiated state are reported (Wang et al., 2007). In contrast to Archer et al., this study shows JSRV Env enhanced replicative capacity of normal primary type II pneumocytes in vitro (Archer et al., 2007). This will allow study of early events in oncogenic transformation that might be difficult to detect by study of end-stage tumor cells. Such early events might also be important for development of human adenocarcinoma.

These experiments employed culture conditions that favored maintenance of the differentiated state of primary rat type II pneumocytes. The conditions recapitulated an air–liquid interface, and extracellular matrix and/or basement membrane components provided by addition of Matrigel (EHS extract) to the collagen matrix. Thus the cell–matrix interactions that occur in vivo were recapitulated. These results contribute to a growing body of evidence that interactions taking place in the microenvironment are important for maintenance of the differentiation state and function of epithelial cells (Dobbs et al., 1997; Gruenert et al., 1995; Shannon et al., 1990, 1987; Whitcutt et al., 1988).

The finding that JSRV Env increased the growth potential of primary rat type II pneumocytes is consistent with previous reports that OPA-derived tumor cells and Env-transformed MDCK cells have a growth advantage compared to control Env-negative cells (Johnson et al., 2010; Suau et al., 2006). While JSRV Env morphologically transforms immortalized cell lines (which have already undergone several genetic changes) in vitro, it is possible that the enhanced proliferative potential in primary RTII cells described here may be more reflective of the effects of Env on type II pneumocytes in vivo. Enhanced target cell proliferation could facilitate viral replication and spread in the lung; oncogenic transformation may be a by-product.

OPA tumors have activated Tert and increased telomerase activity (Suau et al., 2006) making it likely that activation of Tert provides an immortalization event required for oncogenesis in vivo. However co-transfection of RTIIs with human or murine Tert expression plasmids along with ΔGP did not further increase the growth properties or result in an immortal cell line. We have not tested if expression of mTERT or rTERT by transfection or transduction in RTIIs transduced with Env-expressing retroviral vectors (which show more profound enhancement of growth potential) can result in immortalized cells. Alternatively Tert or the other oncogenes used in the co-transfection experiments cannot cooperate with plasmid borne JSRV Env to transform and immortalize primary rat type II pneumocytes.

MuLV-based retroviral vectors are relatively inefficient at infecting lung epithelial cells in vivo because the viral envelope protein is inactivated by the detergent-like properties of lung surfactant (Duncan et al., 1997; Zsengeller et al., 1999). However the studies presented here indicate that LXSNEnv and LXSNEnvHA efficiently infected RTII cultures in vitro. One possibility is that although the cells continue to express SP-C, the amount of surfactant released into the culture medium may be lower than the levels in the lungs in vivo. Secondly, since the vector producing cells would also express JSRV Env protein, there were likely JSRV pseudotypes in the vector stock. Coil et al. have shown that MuLV vectors pseudotyped with JSRV Env are relatively resistant to lung surfactant (Coil et al., 2001). However, the JSRV receptor, Hyal2, appears to be masked on rat cell lines because endogenous levels do not support entry of JSRV vectors (Liu et al., 2003a, 2003b).

In summary this report describes the establishment of in vitro conditions whereby JSRV Env can be shown to enhance the replicative potential of primary rat type II pneumocytes—the cell type that is the target for JSRV oncogenesis in sheep. These conditions allowed long-term culture of the Env-expressing cells, where properties of differentiated type II pneumocytes were maintained. On the other hand, immortalized type II pneumocyte cell lines were not obtained by expression of Env. Nevertheless, the enhanced replicative potential was extensive, which will allow for future biochemical and molecular studies to identify processes involved in JSRV oncogenesis in this in vitro system.

Materials and Methods

Isolation and culture of primary rat type II pneumocytes

Primary rat type II pneumocytes (RTII) were isolated from specific pathogen-free male Sprague–Dawley rats using techniques described previously (Dobbs and Mason, 1979). Briefly, lungs were perfused, removed, instilled with 4.3 U/ml elastase (Roche) and single cell suspensions were prepared by mincing and filtration through nylon cloth. The cell suspension was incubated with chlorofluorocarbon and macrophages were separated from the RTII cells by centrifugation through a metrizamide density gradient (OptiPrep; Accurate Chemical and Scientific Corp.). The purified RTII cells were used immediately or frozen for future experiments as described below. The culture system used in this study was modified from previously developed procedures (Xu et al., 1998). RTII cells were seeded on 30 mm, 0.4 μm transwell (Falcon) filter inserts coated with a collagen type I–Matrigel mixture (80:20) in rat seeding medium (RSM). RSM is low glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% rat serum (Pel-Freeze), penicillin (100 U/ml) and 2.5 μg/ml amphotericin B and 10 ng/ml keratinocyte growth factor (KGF; Research Diagnostic). Collagen type I was prepared from rat tails as previously described (Shannon et al., 1987), and Matrigel (EHS extract) was purchased from BD Biosciences. Cells were allowed to attach for 24–48 h after plating; following attachment, they were cultured in Airway Epithelial Cell Basal Medium (AEC, Promocell) supplemented with 0.4% Bovine Pituitary Extract (BPE), 0.5 ng/ml Epidermal Growth Factor, 5 μg/ml human recombinant Insulin, 0.5 μg/ml Hydrocortisone, 0.5 μg/ml Epinephrine, 6.5 ng/ml Triiodo-L-thyronine, 10 μg/ml human Transferrin, 0.1 ng/ml retinoic acid, penicillin (100 U/ml) and amphotericin B (2.5 μg/ml). 1 ml of AEC was placed in the upper transwell containing the cells, and 2 ml was placed in the lower well. Cultures were maintained on a rocking platform which provides an air–liquid interface. Freshly prepared RTII cells were frozen in 90% fetal bovine serum (FBS) supplemented with 10% DMSO and stored at −140 °C for later use. When the RTII cultures reached confluency they were passaged by incubating the collagen–Matrigel matrices in 2 ml of low glucose DMEM containing 75 U of dispase (BD Biosciences) and 2.5 mg/ml collagenase A (Roche) for 2 h at 37 °C with gentle shaking every 15 min. Released cells were harvested by overlaying on 1 ml of FBS followed by low speed centrifugation, resuspended in RSM and seeded at 1:2 into fresh transwells containing collagen–Matrigel and culture in AEC medium was continued.

Transfections and transductions

Freshly isolated and frozen cells were seeded at 5 × 105 or 1×106 cells per transwell insert as above. Cells were transfected with 1 μg of plasmid DNA per well using Fugene 6 (Roche) reagent following the manufacturer's protocol. pCMVJS21, pCMVJS21ΔGP (ΔGP) and pCMVJS21ΔGPflag (ΔGPflag) have been previously described (Maeda et al., 2005, 2001; Palmarini et al., 1999). Expression plasmids encoding activated H-Ras and human and murine telomerase reverse transcriptase (hTERT and mTERT) were kindly provided by Robert Weinberg. An SV40 large T-antigen (SV40LT) expression plasmid was provided by William Hahn. The mutant p53 construct, pCMVneoR175H, was provided by Rainner Brachmann. The JSRV envelope expressing vector (pLXSNenvHA (EnvHA)) and production of retroviral vectors has been previously described (Johnson et al., 2010). Infection with the vectors (1 × 104 CFU/ml) was performed in the presence of 4 μg/ml polybrene.

Immunofluoresence

Cultures were fixed in 10% neutral buffered formalin (Sigma-Aldrich) for 15 min and embedded in paraffin. 6 μm sections were cut on a microtome, mounted on glass slides and processed for immunofluoresence. Antigen retrieval was performed by heating slides in a microwave for 20 min in 100 mM sodium citrate (pH6). Samples were treated with 100 mM phosphate buffered saline (PBS)/glycine two times for 15 min followed by 0.5% triton x-100/PBS for 15 min. Samples were blocked for 1 h at room temperature in 3% bovine serum albumin (BSA)/PBS and probed overnight at 4 °C in 3% BSA/PBS. Antibodies used in analysis included rabbit anti-pro-SPC (1:200; Chemicon), rabbit anti-HA (1:200; Cell signaling) and goat anti-cytokeratin 8.13 (1:200; Sigma). Secondary antibodies were anti-rabbit Alexafluor-488 and -546 and anti-goat Alexa fluor-488 (Invitrogen). Alkaline phosphatase activity was determined using Vector Red Alkaline Phosphatase Substrate Kit I (Vector Laboratories). Nile red (Molecular Probes) was used at 5 μg/ml to detect lamellar bodies. Normal lung and MDCK cells were used as positive and negative controls, respectively. Samples were counterstained with Vectorsheild DAPI (Vector Laboratories; 4′, 6′-diamidino-2-phenylindole). Samples were analyzed by fluorescent and confocal microscopy.

Acknowledgments

This work was supported by NIH grants R01 CA94188 to HF and RO1CA59116 to DV. CJ was supported by a postdoctoral fellowship from the George E. Hewitt Foundation, and training grant T32 CA009054-32. The Optical Biology Shared Resource of the UCI Chao Family Comprehensive Cancer Center is acknowledged. This paper is dedicated to the memory of Amanda Evans.

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