Jaagsiekte Sheep Retrovirus Transformation in Madin-Darby Canine Kidney Epithelial Cell Three-Dimensional Culture

Abstract

Lung cancer is the leading cause of cancer deaths worldwide. While most cases are associated with cigarette smoking, 15% of cases in men and 53% in women occur in nonsmokers (39). Lung cancer in nonsmokers is most commonly diagnosed as adenocarcinoma (21). Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of a contagious neoplasm in sheep termed ovine pulmonary adenocarcinoma (OPA) (35). OPA is derived from secretory epithelial cells, type II pneumocytes, and Clara cells and closely resembles bronchioloalveolar carcinoma (BAC) in humans (36,41). The similarities between OPA and BAC make JSRV and OPA a useful animal model for studying lung carcinogenesis.JSRV is unique because the envelope protein (Env, encoded by the env gene) is the oncogene. Expression of Env can transform cells in culture (2, 30, 43) as well as induce tumors in animals (38, 51). Initial studies illustrated the importance of the phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR pathway in JSRV Env transformation. It is now clear that there are PI3K-dependent and -independent mechanisms of Akt-mTOR signaling. PI3K-dependent signaling was evident because JSRV Env-transformed cell lines have constitutively active Akt and inhibiting PI3K prevented transformation and caused reversion of the transformed phenotype (24,25,37,52). On the other hand, PI3K-independent Akt activation was later demonstrated, since cells in which PI3K was disabled could still be transformed by JSRV and, under these conditions, there was still activation of Akt (29).More recently, the H/N-Ras-MEK-mitogen-activated protein kinase (MAPK) pathway was found to play a more important role in JSRV Env transformation, since inhibiting MEK1 and H/N-Ras prevented transformation in NIH 3T3 and RK3E cell monolayers (28). Note that p38 MAPK has a negative role in JSRV Env transformation, as inhibiting p38 MAPK increases transformation. The finding that JSRV Env does not induce transformation solely through the PI3K-Akt-mTOR pathway is supported by the fact that not all OPA tissues demonstrate activated Akt (46). Together, these results underscore the complexity of JSRV Env transformation and the need to further understand the signaling pathways involved in transformation.Madin-Darby canine kidney (MDCK) epithelial cells grown in three-dimensional (3-D) culture by being embedded in Engelbreth-Holm-Swarm tumor extract (Matrigel) or collagen readily form spherical structures termed "acini." Acini grow as a single layer of polarized cells that are attached to the underlying basement membrane and enclose a central hollow lumen (7,26,33,34). Acinar structure and cell-cell and cell-environment interactions mimic what occurs in vivo. Maintenance of this highly ordered structure is critical for proper differentiated cell function, and when it is disrupted, malignancy can ensue. The events of MDCK acinus formation have been well defined and include cell polarization, secretion of basement membrane, formation of tight junctions, and responsiveness to apoptotic and proliferative suppressive si...

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

“…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.…”

“…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).…”

“…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 × 10 4 CFU/ml) was performed in the presence of 4 μg/ml polybrene.…”

“…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.…”

Three-dimensional culture of an ovine pulmonary adenocarcinoma-derived cell line results in re-expression of surfactant proteins and Jaagsiekte sheep retrovirus

“…Studying JSRV Env transformation in JS7 3-D cultures will allow us to study mechanisms of transformation in the context of epithelial structure and signaling of sheep type II pneumocytes. This is important because we previously reported different signaling pathways for transformation of MDCK (kidney epithelial) cells cultured in 3-D compared to 2-D (Johnson, Sanders, and Fan, 2010). …”