[Changes in the Expression of AKT and ERK after the JSRV-Env-Induced Transfection of TC-1 and TC-1-Hyal2 Cells].

This study aims to explore the tumorigenic mechanism of the target cells following JSRV interaction with its receptor. We transfected mouse lung epithelial cells (TC-1) and mouse lung epithelial cells stably expressing sheep Hyal-2(TC-1-Hyal2)with JSRV-Env eukaryotic expression vector, measured the changes in the mRNA and protein expression of AKT(serine/threonine kinase)and ERK(extracellular signal-regulated kinase)in cellular signal transduction pathways, and analyzed the role of sheep Hyal-2in JSRV-Env-induced transformation of TC-1cells.First,TC-1and TC-1-Hyal2 cells were cultured in vitro and were each divided into pEGFP-C1-env transfection group,pEGFP-C1 transfection group, and untransfected group. The expression of key enzymes was determined by PCR and Western blotting. qPCR showed that, for both cell lines, compared with untransfected cells, the expression of AKT and ERK1/2mRNA was significantly increased in the pEGFP-C1-env transfected cells(P<0.05).Western blotting showed that, relative to untransfected cells, transfection with pEGFP-C1-env significantly increased p-Akt (S473)protein expression in both cell lines(P<0.05).Moreover, p-Akt (T308)and p-Erk1/2protein expression was increased significantly in the pEGFP-C1-env transfected TC-1cells(P<0.05),and very significantly in the pEGFP-C1-env transfected TC-1-Hyal2cells(P<0.01).Cells of each type transfected with the empty vector pEGFP-C1 and the untransfected cells did not show significant differences in their mRNA and protein levels of AKT and ERK(P >0.05).Thus, the expression of JSRV-Env in the cell lines TC-1and TC-1-Hyal2 activated the cellular signal transduction pathways Ras-Raf-MAPK and PI3K-Akt.The expression of AKT and ERK was significantly increased in pEGFP-C1-env transfected TC-1and TC-1-Hyal2 cells, but a greater increase was seen in the TC-1-Hyal2 cells.We speculate that Hyal2 plays a catalytic role in JSRV-Env-induced transformation of TC-1cells.

Expression of endogenous beta retroviruses and Hyal-2 mRNA in immune organs of fetuses and lambs.

Endogenous beta retroviruses (enJSRV) are highly homologous with Jaagsiekte sheep retrovirus (exJSRV), this exogenous retrovirus is the aetiological agent of ovine pulmonary adenocarcinoma (OPA). The aim of this study was to clarify the function of enJSRV and the immunological mechanisms of its corresponding antibody, that is undetectable in JSRV-infected ovine serum. The expression of enJSRV envelope protein and Hyal-2 mRNA in immune organs and lungs of ovine fetuses and lambs were analyzed by Real-Time reverse transcription PCR and In Situ Hybridization using specific probes. In Situ Hybridization results indicated that the enJSRV envelope protein and Hyal-2 mRNA were expressed in thymus, spleen, mesenteric lymph nodes and lungs at different times, while no positive signals were detected in the negative controls. On the other hand, results from Real-Time reverse transcription PCR analysis showed that in 130d fetuses and 3d newborn lambs the enJSRV mRNA levels were much higher in organs associated with the immune system than that in lungs, especially in the thymus and spleen, but levels of Hyal-2 mRNA expression was not significantly different in all collected tissue. These results provided evidence from an immunology point of view to understand why the circulating antibodies against exJSRV are undetectable in JSRV-infected ovine, and will help to unravel the pathogenesis of JSRV-infected ovine.

Overexpression of aldolase A and cytokeratin 19 in ovine pulmonary adenocarcinoma.

Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer of sheep caused by jaagsiekte sheep retrovirus (JSRV). In the present study the protein profiles of five neoplastic and three non-neoplastic sheep lung tissues were examined for the identification of proteins overexpressed in ovine pulmonary adenocarcinoma. Lung sections of the experimental group of sheep were collected during necropsies for proteomic and immunohistochemical examination. Two dimensional electrophoresis (2DE) was performed using gel strips with immobilized pH gradient 3-10. As a result of 2DE gel analysis 14 spots characterized by over 2-fold higher expression in tumour proteomes were selected for mass spectrometry. In eleven spots more than one polypeptide was identified indicating overlapping of proteins in gels. In two spots demonstrating over 3-fold higher expression in OPA proteomes, single proteins: cytokerarin 19 (CK19) and aldolase A were identified. Immunohistochemical studies revealed that CK19 and aldolase A were expressed in the cytoplasm of epithelial cells of bronchioles in non-neoplastic lung sections, as well as epithelial cells of bronchioles and neoplastic cells in lung sections of OPA affected sheep. The results indicate that the overexpression of the two proteins reflects the presence of neoplastic cells in the lungs of OPA affected sheep.

In-situ demonstration of mitogen-activated protein kinase Erk 1/2 signalling pathway in contagious respiratory tumours of sheep and goats.

Ovine pulmonary adenocarcinoma (OPA) and enzootic nasal adenocarcinoma (ENA) are two contagious neoplastic diseases of secretory epithelial cells in the respiratory system of sheep and goats. Jaagsiekte sheep retrovirus (JSRV) is the aetiological agent of OPA, and enzootic nasal tumour virus (ENTV) is associated with ENA. The genomes of these retroviruses do not contain known oncogenes but products of the env gene are important in the generation of transforming stimuli. However, the cell signalling pathways activated in vivo are not completely understood. This study was based on the use of activation stage antibodies specifically detecting proteins of the extracellular signal regulated kinase Erk 1/2 cell signalling pathway and transcription factors. Tissue sections were collected from four natural cases of OPA, four experimentally induced OPA tumours, four ENA tumours in sheep, four ENA tumours in goats, two normal sheep lungs and two lungs with chronic inflammation. Routine immunohistochemical procedures with phosphorylation stage-specific antibodies were carried out. Representative proteins of the Erk1/2 pathway (Raf-1, Mek1/2 and p44/42MAPK) were activated in natural cases of OPA and ENA in sheep and goats and also in experimentally induced OPA. Transcription factors 90Rsk and Elk-1 were activated in OPA and ENA tumours. However, c-Myc was activated only in OPA tumours. In contagious respiratory neoplasms of sheep and goats the Erk1/2 pathway appears to be important for the in-vivo generation of the transforming stimuli.

Further evidence for a retrovirus as the aetiological agent of sheep pulmonary adenomatosis (jaagsiekte).

The infective agent of jaagsiekte was shown to be present in the fluid which accumulates in the respiratory tract of sheep during the terminal stages of the disease. The fluid also contained reverse transcriptase (RT) activity which showed a clear preference for a ribonucleic acid synthetic template over the corresponding deoxyribonucleic acid template and which utilised the RT specific template/primer poly (2'-0-methylcytidylate) oligodeoxyguanylate. This enzyme activity was associated with a particle which had typical retroviral buoyant densities in a range of gradient media.

[Ovine pulmonary adenomatosis]. / Plicní adenomatóza ovcí

Lung adenomatosis was histologically demonstrated in seven sheep coming from one flock in western Bohemia. On the basis of morphological characteristics and biological properties the disease can be considered as a neoplasma. It is capable of forming metastases and calls forth a systemic reaction of the organism. Lund adenomatosis can be experimentally transferred and the present knowledge speaks in favour of virus etiology of the disease.