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. 2005 Jan;79(2):927-33.
doi: 10.1128/JVI.79.2.927-933.2005.

Transformation of madin-darby canine kidney epithelial cells by sheep retrovirus envelope proteins

Shan-Lu Liu  1 , A Dusty Miller
Affiliations

Transformation of madin-darby canine kidney epithelial cells by sheep retrovirus envelope proteins

Shan-Lu Liu et al. J Virol. 2005 Jan.

Abstract

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) induce epithelial tumors in the airways of sheep and goats. In both of these simple retroviruses, the envelope (Env) protein is the active oncogene. Furthermore, JSRV Env can transform cultured cells by two distinct mechanisms. In rat and mouse fibroblasts, the cytoplasmic tail of JSRV Env is essential for transformation, which involves activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and the virus receptor hyaluronidase 2 (Hyal2) is not involved. In contrast, in the BEAS-2B human bronchial epithelial cell line, transformation is mediated by JSRV Env binding to Hyal2 followed by Hyal2 degradation and activation of the receptor tyrosine kinase RON, the activity of which is normally suppressed by Hyal2. Here we show that JSRV and ENTV Env proteins can also transform Madin-Darby canine kidney (MDCK) epithelial cells, but by a mechanism similar to that observed in fibroblast cell lines. In particular, the cytoplasmic tail of Env is required for transformation, the PI3K/Akt pathway is activated, expression of RON (which is not normally expressed in MDCK cells) does not affect transformation, and canine Hyal2 appears uninvolved. These results show that the JSRV and ENTV Env proteins can transform epithelial cells besides BEAS-2B cells and argue against a model for Env transformation involving different pathways that are uniquely active in fibroblasts or epithelial cells.

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Figures

FIG. 1.
FIG. 1.
Transformation of MDCK cells by JSRV and ENTV Env proteins. MDCK cells were transfected with plasmids encoding the Env proteins of JSRV (JSRV Env) or ENTV (ENTV Env) by using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) or were transduced by retroviral vectors encoding AP (LAPSN) or JSRV Env (LJeSN). The next day cells were trypsinized and seeded at a 1:2 dilution into 6-cm-diameter dishes in the presence (for JSRV Env and ENTV Env) or absence (for LJeSN and LAPSN) of G418 (600-μg/ml active concentration). The culture medium was replaced every 3 days, and transformed foci were photographed 14 days after DNA transfection or viral transduction (top four panels). The lower panels show the parental MDCK cells (left) and the JSRV Env-transformed MDCK cells (right) seeded at low density and photographed 4 days later.
FIG. 2.
FIG. 2.
Akt is activated in MDCK cells transformed by JSRV or ENTV Env proteins, and Akt activation is PI3K dependent. MDCK cells transformed by JSRV or ENTV Env protein were grown overnight in serum-free DMEM and were then treated with LY at the indicated concentrations for 1 h. Cells were lysed and were subjected to SDS-PAGE followed by IB with antibodies against phospho-Akt (upper panel). The PVDF membrane was stripped and reblotted with anti-Akt (lower panel). In both blots, the PVDF membrane was blocked with 1% nonfat milk at room temperature for 1 h, followed by incubation overnight with anti-phospho-Akt or anti-Akt antibodies diluted 1:1,000 in 5% bovine serum albumin. The ECL-Plus detection system (Amersham BioSciences) was used for antibody detection.
FIG. 3.
FIG. 3.
Akt is activated in MDCK cells transformed by YXXM mutants of JSRV/ENTV Env. Measurement of Akt activation (pAkt) and total Akt was performed as described in the legend to Fig. 2. The lower panel shows an immunoblot using anti-FLAG antibodies to detect FLAG-tagged Env proteins. The FLAG-tagged Env protein expressed in each cell line is listed above. Note that the full-length and SU portion of ENTV Env were barely detected by these methods.
FIG. 4.
FIG. 4.
JSRV Env protein is not tyrosine phosphorylated in the transformed MDCK cells. (A) MDCK cells transformed by the JSRV Env protein and the parental MDCK cells were grown in serum-free medium overnight. Cell lysates were prepared and portions were subjected to immunoprecipitation with anti-FLAG antibody (IP: anti-FLAG). Lysates and immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting, using anti-phosphotyrosine antibody 4G10. The lane labeled A431+EGF contained lysate from A431 cells that had been stimulated by epidermal growth factor (EGF; provided by Upstate Biotechnology and used as a positive control for 4G10 antibody binding). (B) The same PVDF membrane was stripped and reblotted with anti-FLAG antibody. The TM subunit and full-length JSRV Env proteins are indicated by arrows, while the SU and full-length ENTV Env proteins were not detected in this assay. IgG H indicates the IgG heavy chain.
FIG. 5.
FIG. 5.
JSRV Env protein does not coimmunoprecipitate with PI3K (p85) in the transformed MDCK cells. Preparation of samples was the same as described in the legend to Fig. 4. The resulting immunoprecipitates and cell lysates were subjected to SDS-PAGE, immunoblotted with anti-p85 (A), and then stripped and reblotted with anti-FLAG (B). IgG H and IgG L indicate the IgG heavy and light chains, respectively.
FIG. 6.
FIG. 6.
RON expression and phosphorylation in MDCK cells expressing RON and/or JSRV Env (Jenv). Cell lysates were subjected to IB using 4G10 antibody (anti pY) or RON antibody (anti-RON). The proteins expressed in the MDCK cells are indicated with the clone numbers for the cells expressing RON. The last two lanes show lysates from a previously isolated cell line that expresses a small amount of RON (RE7) (32) after incubation with or without 1.1 nM MSP for 20 min.
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