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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.


Related in: MedlinePlus

Colony formation assay. (a) Primary WT mesothelial cells were able to form colonies in plates with soft agar, as well as the human MM-derived cell line MSTO-211H (b) used as a positive control (derived from a biphasic mesothelioma). Selected colonies typically representing the morphological appearance of immortalized cells iMeso-WT1 (c), and iMeso-NF3 (d) are shown. Scale bar: 250 μm.
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Fig4: Colony formation assay. (a) Primary WT mesothelial cells were able to form colonies in plates with soft agar, as well as the human MM-derived cell line MSTO-211H (b) used as a positive control (derived from a biphasic mesothelioma). Selected colonies typically representing the morphological appearance of immortalized cells iMeso-WT1 (c), and iMeso-NF3 (d) are shown. Scale bar: 250 μm.

Mentions: Since immortalized mesothelial cell clones of both genotypes (WT, Nf2+/−) also proliferated in low-serum (2%) conditions, a feature often observed in transformed cells, we investigated another aspect related to cell transformation, i.e., their ability to form anchorage-independent colonies in a semisolid medium. Cells from the various clones grew in soft agar, and images were taken after 17 d (Fig. 4). We then also tested primary mesothelial cells from WT mice; they were also able to form colonies of ≥250 μm diameter after 17 d of growth in anchorage-independent conditions (Fig. 4). These findings are in line with results on human primary mesothelial cells, where colony formation in semisolid medium has been reported before (La Rocca and Rheinwald 1985). Thus, anchorage-independent growth of primary mesothelial cells appears not to be restricted to cells of human origin; moreover, the soft agar assay does not allow to distinguish primary from immortalized or even transformed (RN5) cells. As a positive control, the human MM cell line MSTO-211H was used. The sole purpose of these experiments was to test whether or not murine mesothelial cells (primary, immortalized) have the capability of growing in soft agar in an anchorage-independent way. No attempt was made to correlate the mesothelial cell’s nature (primary vs. immortalized vs. transformed, from WT vs. transgenic mice) with the size of the colonies.Figure 4.


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)

Colony formation assay. (a) Primary WT mesothelial cells were able to form colonies in plates with soft agar, as well as the human MM-derived cell line MSTO-211H (b) used as a positive control (derived from a biphasic mesothelioma). Selected colonies typically representing the morphological appearance of immortalized cells iMeso-WT1 (c), and iMeso-NF3 (d) are shown. Scale bar: 250 μm.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig4: Colony formation assay. (a) Primary WT mesothelial cells were able to form colonies in plates with soft agar, as well as the human MM-derived cell line MSTO-211H (b) used as a positive control (derived from a biphasic mesothelioma). Selected colonies typically representing the morphological appearance of immortalized cells iMeso-WT1 (c), and iMeso-NF3 (d) are shown. Scale bar: 250 μm.
Mentions: Since immortalized mesothelial cell clones of both genotypes (WT, Nf2+/−) also proliferated in low-serum (2%) conditions, a feature often observed in transformed cells, we investigated another aspect related to cell transformation, i.e., their ability to form anchorage-independent colonies in a semisolid medium. Cells from the various clones grew in soft agar, and images were taken after 17 d (Fig. 4). We then also tested primary mesothelial cells from WT mice; they were also able to form colonies of ≥250 μm diameter after 17 d of growth in anchorage-independent conditions (Fig. 4). These findings are in line with results on human primary mesothelial cells, where colony formation in semisolid medium has been reported before (La Rocca and Rheinwald 1985). Thus, anchorage-independent growth of primary mesothelial cells appears not to be restricted to cells of human origin; moreover, the soft agar assay does not allow to distinguish primary from immortalized or even transformed (RN5) cells. As a positive control, the human MM cell line MSTO-211H was used. The sole purpose of these experiments was to test whether or not murine mesothelial cells (primary, immortalized) have the capability of growing in soft agar in an anchorage-independent way. No attempt was made to correlate the mesothelial cell’s nature (primary vs. immortalized vs. transformed, from WT vs. transgenic mice) with the size of the colonies.Figure 4.

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