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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.


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Morphology and growth of the murine mesothelioma cell lines RN5, AK7, and AB12 in vitro. (a) Epithelioid morphology of RN5 and AK7 compared to biphasic or mixed type morphology of AB12 cells. Cell size, morphology, and growth characteristics (b) are strikingly similar for RN5 and AK7 cells. (c) Immunofluorescene images of RN5 cells show a rather homogeneous staining for desmin (red). Pan-cytokeratin staining (green) shows a typical mosaic pattern; cell nuclei were labeled with DAPI (blue). (d) Western blot analysis (30 μg total protein/lane) showing the expression of mesothelin in the three murine mesothelioma cell lines RN5, AK7, and AB12.
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Fig5: Morphology and growth of the murine mesothelioma cell lines RN5, AK7, and AB12 in vitro. (a) Epithelioid morphology of RN5 and AK7 compared to biphasic or mixed type morphology of AB12 cells. Cell size, morphology, and growth characteristics (b) are strikingly similar for RN5 and AK7 cells. (c) Immunofluorescene images of RN5 cells show a rather homogeneous staining for desmin (red). Pan-cytokeratin staining (green) shows a typical mosaic pattern; cell nuclei were labeled with DAPI (blue). (d) Western blot analysis (30 μg total protein/lane) showing the expression of mesothelin in the three murine mesothelioma cell lines RN5, AK7, and AB12.

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)

Morphology and growth of the murine mesothelioma cell lines RN5, AK7, and AB12 in vitro. (a) Epithelioid morphology of RN5 and AK7 compared to biphasic or mixed type morphology of AB12 cells. Cell size, morphology, and growth characteristics (b) are strikingly similar for RN5 and AK7 cells. (c) Immunofluorescene images of RN5 cells show a rather homogeneous staining for desmin (red). Pan-cytokeratin staining (green) shows a typical mosaic pattern; cell nuclei were labeled with DAPI (blue). (d) Western blot analysis (30 μg total protein/lane) showing the expression of mesothelin in the three murine mesothelioma cell lines RN5, AK7, and AB12.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig5: Morphology and growth of the murine mesothelioma cell lines RN5, AK7, and AB12 in vitro. (a) Epithelioid morphology of RN5 and AK7 compared to biphasic or mixed type morphology of AB12 cells. Cell size, morphology, and growth characteristics (b) are strikingly similar for RN5 and AK7 cells. (c) Immunofluorescene images of RN5 cells show a rather homogeneous staining for desmin (red). Pan-cytokeratin staining (green) shows a typical mosaic pattern; cell nuclei were labeled with DAPI (blue). (d) Western blot analysis (30 μg total protein/lane) showing the expression of mesothelin in the three murine mesothelioma cell lines RN5, AK7, and AB12.
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