Limits...
Establishment of immortalized murine mesothelial cells and a novel mesothelioma cell line.

Blum W, Pecze L, Felley-Bosco E, Worthmüller-Rodriguez J, Wu L, Vrugt B, de Perrot M, Schwaller B - In Vitro Cell. Dev. Biol. Anim. (2015)

Bottom Line: All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum.Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate.RN5 cells are highly tumorigenic.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Fribourg, Route Albert-Gockel 1, 1700, Fribourg, Switzerland.

ABSTRACT
Mesothelial cells are susceptible to asbestos fiber-induced cytotoxicity and on longer time scales to transformation; the resulting mesothelioma is a highly aggressive neoplasm that is considered as incurable at the present time Zucali et al. (Cancer Treatment Reviews 37:543-558, 2011). Only few murine cell culture models of immortalized mesothelial cells and mesothelioma cell lines exist to date. We generated SV40-immortalized cell lines derived from wild-type (WT) and neurofibromatosis 2 (merlin) heterozygote (Nf2+/-) mice, both on a commonly used genetic background, C57Bl/6J. All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum. Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate. The tumor suppressor gene NF2 is one of the most frequently mutated genes in human mesothelioma, but its detailed function is still unknown. Thus, these genotypically distinct cell lines likely relevant for malignant mesothelioma formation are expected to serve as useful in vitro models, in particular to compare with in vivo studies in mice of the same genotype. Furthermore, we generated a novel murine mesothelioma cell line RN5 originating from an Nf2+/- mouse subjected to repeated crocidolite exposure. RN5 cells are highly tumorigenic.

No MeSH data available.


Related in: MedlinePlus

Western blot for SV40 large T antigen and mesothelin in WT and Nf2+/− cells in comparison of the primary immortalized clones from WT and Nf2+/− mesothelial cells. (a) Western blot signals from a representative experiment. The Western blot signals for β-actin were used for the normalization of the mesothelin expression levels. (b) Quantitative analysis of a representative Western blot (n = 3 samples per genotype, p < 0.05 grouped primary cells vs. grouped immortalized cell lines).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4539351&req=5

Fig3: Western blot for SV40 large T antigen and mesothelin in WT and Nf2+/− cells in comparison of the primary immortalized clones from WT and Nf2+/− mesothelial cells. (a) Western blot signals from a representative experiment. The Western blot signals for β-actin were used for the normalization of the mesothelin expression levels. (b) Quantitative analysis of a representative Western blot (n = 3 samples per genotype, p < 0.05 grouped primary cells vs. grouped immortalized cell lines).

Mentions: As expected, primary mesothelial cells of either genotype (WT, Nf2+/−), as well as the murine macrophage cell line RAW264.7 (Raschke et al.1978), did not express TAg evidenced by Western blotting (Fig. 3a). Strong TAg expression was observed in all primary mesothelial cell clones transfected with the lentivirus coding for TAg/tag (Fig. 3). All cells (primary, immortalized) of mesothelial origin were clearly positive for mesothelin, a mesothelial cell-specific marker. The single positive band on the Western blots had a relative molecular mass (Mr) of 69 kDa corresponding to the mesothelin precursor protein. Western blot quantification revealed decreased mesothelin levels in immortalized cell clones from both genotypes compared to primary cells WT mice (Fig. 3b). Immunohistochemical analysis showed homogeneous positive staining for desmin and mosaic-like expression for Pan-cytokeratin in all examined cell lines including the primary mesothelial cultures, the immortalized cells, and the tumor-derived RN5 cells. A typical example for RN5 cells is shown in Fig. 5c.Figure 3.


Establishment of immortalized murine mesothelial cells and a novel mesothelioma cell line.

Blum W, Pecze L, Felley-Bosco E, Worthmüller-Rodriguez J, Wu L, Vrugt B, de Perrot M, Schwaller B - In Vitro Cell. Dev. Biol. Anim. (2015)

Western blot for SV40 large T antigen and mesothelin in WT and Nf2+/− cells in comparison of the primary immortalized clones from WT and Nf2+/− mesothelial cells. (a) Western blot signals from a representative experiment. The Western blot signals for β-actin were used for the normalization of the mesothelin expression levels. (b) Quantitative analysis of a representative Western blot (n = 3 samples per genotype, p < 0.05 grouped primary cells vs. grouped immortalized cell lines).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4539351&req=5

Fig3: Western blot for SV40 large T antigen and mesothelin in WT and Nf2+/− cells in comparison of the primary immortalized clones from WT and Nf2+/− mesothelial cells. (a) Western blot signals from a representative experiment. The Western blot signals for β-actin were used for the normalization of the mesothelin expression levels. (b) Quantitative analysis of a representative Western blot (n = 3 samples per genotype, p < 0.05 grouped primary cells vs. grouped immortalized cell lines).
Mentions: As expected, primary mesothelial cells of either genotype (WT, Nf2+/−), as well as the murine macrophage cell line RAW264.7 (Raschke et al.1978), did not express TAg evidenced by Western blotting (Fig. 3a). Strong TAg expression was observed in all primary mesothelial cell clones transfected with the lentivirus coding for TAg/tag (Fig. 3). All cells (primary, immortalized) of mesothelial origin were clearly positive for mesothelin, a mesothelial cell-specific marker. The single positive band on the Western blots had a relative molecular mass (Mr) of 69 kDa corresponding to the mesothelin precursor protein. Western blot quantification revealed decreased mesothelin levels in immortalized cell clones from both genotypes compared to primary cells WT mice (Fig. 3b). Immunohistochemical analysis showed homogeneous positive staining for desmin and mosaic-like expression for Pan-cytokeratin in all examined cell lines including the primary mesothelial cultures, the immortalized cells, and the tumor-derived RN5 cells. A typical example for RN5 cells is shown in Fig. 5c.Figure 3.

Bottom Line: All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum.Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate.RN5 cells are highly tumorigenic.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Fribourg, Route Albert-Gockel 1, 1700, Fribourg, Switzerland.

ABSTRACT
Mesothelial cells are susceptible to asbestos fiber-induced cytotoxicity and on longer time scales to transformation; the resulting mesothelioma is a highly aggressive neoplasm that is considered as incurable at the present time Zucali et al. (Cancer Treatment Reviews 37:543-558, 2011). Only few murine cell culture models of immortalized mesothelial cells and mesothelioma cell lines exist to date. We generated SV40-immortalized cell lines derived from wild-type (WT) and neurofibromatosis 2 (merlin) heterozygote (Nf2+/-) mice, both on a commonly used genetic background, C57Bl/6J. All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum. Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate. The tumor suppressor gene NF2 is one of the most frequently mutated genes in human mesothelioma, but its detailed function is still unknown. Thus, these genotypically distinct cell lines likely relevant for malignant mesothelioma formation are expected to serve as useful in vitro models, in particular to compare with in vivo studies in mice of the same genotype. Furthermore, we generated a novel murine mesothelioma cell line RN5 originating from an Nf2+/- mouse subjected to repeated crocidolite exposure. RN5 cells are highly tumorigenic.

No MeSH data available.


Related in: MedlinePlus