Jaagsiekte sheep retrovirus transformation in Madin-Darby canine kidney epithelial cell three-dimensional culture

Abstract

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep that shares similarities with human bronchioloalveolar carcinoma (BAC). JSRV is unique because the envelope gene (env) is the oncogene, as it can transform cells in culture and induce tumors in animals. The phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR and H/N-Ras-MEK-mitogen-activated protein kinase (MAPK) pathways have been shown to be critical for Env transformation. However, the question still remains of how disruption of these pathways relates to tumor formation. To address this, JSRV Env transformation was studied in the context of epithelial structure, using the polarized Madin-Darby canine kidney (MDCK) epithelial cell three-dimensional (3-D) culture system. The results indicated that JSRV Env-transformed MDCK cells were larger and had full or multiple lumens, in contrast to the single lumens observed in controls. The altered phenotype was largely mediated by an increase in proliferation, in addition to overcoming the proliferative suppression signal. JSRV Env was not found to disrupt polarity or tight junctions or to inhibit lumen apoptosis. The PI3K-Akt-mTOR pathway was important for Env transformation in MDCK cells, although the mechanisms of action differed in 3-D and monolayer cultures. PI3K-dependent signaling to mTOR occurred in monolayers, while PI3K-independent signaling to mTOR occurred in 3-D culture. In contrast, the H/N-Ras-MEK-MAPK pathway was found to be inhibitory to transformation in both normal and transformed MDCK cells in 3-D culture. However, in monolayer culture, inhibition of MEK reverted the transformed phenotype, suggesting a different mechanism(s) of action in monolayer versus 3-D culture.

FIG. 1.

Stably transduced MDCK cells. MDCK cells were stably transduced with retroviral vector LXSN or LXSNenvHA and selected with G418 to create stably transduced lines. (A) SDS-PAGE and immunoblot analysis of cell lysates from MDCK cells stably transduced with LXSN or LXSNenvHA was performed using an antibody to the HA tag. The mobility of the posttranslationally cleaved Env protein (32 kDa) containing the HA tag is indicated. (B) The morphologies of stably transduced MDCK cell lines at low and high densities were examined by light microscopy. Photographs were taken 48 h (low density) and 20 days (high density) following plating. Magnification, ×100; bar, 200 μm.

FIG. 2.

Effect of JSRV Env on acinus size. LXSN- and LXSNenvHA-transduced MDCK cells were grown in 3-D culture. (A) Light microscopy photographs of acini at day 20 of culture. Bar, 200 μM. (B) Acini were stained with DAPI (left) and anti-HA (right) at day 20 of culture and then examined by confocal microscopy. Magnification, ×630. (C) The diameters through the centers of acini were measured at 20 days, grouped into the indicated size categories, and represented as percentages of the total (means ± standard errors). The results are representative of three independent experiments performed in triplicate, and more than 100 acini were counted.

FIG. 3.

Effect of JSRV Env on acinus structure and polarization. (A) LXSN- and LXSNenvHA-transduced MDCK cells were grown in 3-D culture and counterstained with DAPI (nuclei; blue) and phalloidin (actin; red) at the indicated time points. Shown are confocal cross sections through the center of the structures. (B) Acini were analyzed for hollowness at days 5 and 20, and the results are represented as percentages of total acini analyzed (≥100) that were hollow. The experiment was performed in duplicate, and the means (± standard errors) from 3 independent experiments are shown. Statistical significance was determined by Student's t test. *, P = 0.001; **, P = 0.00007. (C) Acini were immunostained with anti-GM130 (green) and DAPI (blue) at days 10 and 20 of culture and then analyzed by confocal microscopy. Higher-magnification images of the indicated areas (boxes) are shown in the insets. (D) Transduced MDCK cells were seeded in collagen (bottom) or Matrigel (top), immunostained with anti-GM130 (green), phalloidin (red), and DAPI (blue) at days 1, 2, and 5, and examined by confocal microscopy. Magnification, ×400; bars, 50 μm.

FIG. 4.

EnvHA structures are defective in responses to growth regulatory signals. (A) Confocal cross sections through the center of acini stained with phalloidin (red) and immunostained with anti-Ki-67 (green) at 10 and 20 days in culture. Magnification, ×400; bar, 50 μm. (B) Confocal cross sections through the center of acini stained with DAPI (blue) and phalloidin (red) and immunostained with anti-c-caspase-3 (green) at 5 and 20 days in culture. Magnification, ×400; bar, 50 μm. *, c-caspase-3-positive cells. (C) Cell death in acini was monitored by ethidium bromide (EtBr) staining (1 μM) at 15 days of culture. Acini were analyzed by fluorescence microscopy; representative images are shown. Magnification, ×200; bar, 100 μm. (D) Confocal cross sections of acini that were immunostained with an antibody to ZO-1 (green) and counterstained with DAPI (blue) are depicted. Magnification, ×400; bars, 50 μm.

FIG. 5.

EnvHA structures are hyperapoptotic and resistant to proliferative suppression signals. (A) Stably transduced MDCK cells were grown in 3-D culture and analyzed for Ki-67 expression at days 5 and 20, and the results are represented as percentages of the total acini analyzed (≥100) that were Ki-67 positive. The experiment was performed in duplicate, and data represent the means (± standard errors) for 3 independent experiments. Statistical significance was determined by Student's t test. *, P = 0.006. (B) Cell growth curve for transduced MDCK cells. A total of 1 × 10⁵ cells were seeded in a 6-well plate in duplicate, and the number of viable cells was determined by trypan blue exclusion over a period of 4 days. The results are representative of three independent experiments. *, P < 0.0001. (C) Transduced MDCK cells were grown in 3-D culture, and acini were analyzed for c-caspase-3 expression at days 5 and 20 of culture. The results are represented as percentages of the total acini analyzed (≥100) that were positive. The experiment was performed in duplicate, and data represent the means (± standard errors) for 3 independent experiments. *, P < 0.05.

FIG. 6.

Pathways of JSRV Env transformation. Stably transduced MDCK cells were serum starved for 24 h and then treated with DMSO, rapamycin (10 ng/ml), LY294002 (10 μM), PD98059 (20 μM), or FTI-277 (5 μg/ml) for 48 h, and light microscopy photographs were taken. Magnification, ×100; bar, 200 μm.

FIG. 7.

Pathways of Env transformation in 3-D culture. LXSN- and LXSNenvHA-transduced MDCK cells were grown in 3-D culture in the presence of the indicated inhibitors. Day 10 acini were counterstained with phalloidin (actin; red) and DAPI (nuclei; blue). Shown are confocal cross sections through the center of the structures.

FIG. 8.

Pathways of JSRV Env proliferation. Cell growth curves are shown for transduced MDCK cells cultured in the presence of rapamycin (A), LY294002 (B), or PD98059 (C). A total of 1 × 10⁵ cells were seeded in a 6-well plate in duplicate, and the number of viable cells was determined by trypan blue exclusion over a period of 4 days. The results are representative of three independent experiments. *, P < 0.05.