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Chronic Replication Problems Impact Cell Morphology and Adhesion of DNA Ligase I Defective Cells.

Cremaschi P, Oliverio M, Leva V, Bione S, Carriero R, Mazzucco G, Palamidessi A, Scita G, Biamonti G, Montecucco A - PLoS ONE (2015)

Bottom Line: This replication stress elicits a constitutive phosphorylation of the ataxia telangiectasia mutated (ATM) checkpoint kinase that fails to arrest cell cycle progression or to activate apoptosis or cell senescence.Here we show that parental 46BR.1G1 and 7A3 cells differ in important features such as cell morphology, adhesion and migration.Interestingly, ATM inhibition makes 46BR.1G1 more similar to 7A3 cells for what concerns morphology, adhesion and expression of cell-cell adhesion receptors.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (CNR), Pavia, Italy; Dipartimento di Biologia e Biotecnologie "L. Spallanzani", Università degli Studi di Pavia, Pavia, Italy.

ABSTRACT
Moderate DNA damage resulting from metabolic activities or sub-lethal doses of exogenous insults may eventually lead to cancer onset. Human 46BR.1G1 cells bear a mutation in replicative DNA ligase I (LigI) which results in low levels of replication-dependent DNA damage. This replication stress elicits a constitutive phosphorylation of the ataxia telangiectasia mutated (ATM) checkpoint kinase that fails to arrest cell cycle progression or to activate apoptosis or cell senescence. Stable transfection of wild type LigI, as in 7A3 cells, prevents DNA damage and ATM activation. Here we show that parental 46BR.1G1 and 7A3 cells differ in important features such as cell morphology, adhesion and migration. Comparison of gene expression profiles in the two cell lines detects Bio-Functional categories consistent with the morphological and migration properties of LigI deficient cells. Interestingly, ATM inhibition makes 46BR.1G1 more similar to 7A3 cells for what concerns morphology, adhesion and expression of cell-cell adhesion receptors. These observations extend the influence of the DNA damage response checkpoint pathways and unveil a role for ATM kinase activity in modulating cell biology parameters relevant to cancer progression.

No MeSH data available.


Related in: MedlinePlus

Correction of LigI defect affects cell morphology.A) Time-lapse imaging of cell migration. Cells were seeded at low density and monitored by time-lapse microscopy as described in Materials and Methods. Representative still images of control fibroblasts (GM847), complemented 7A3 expressing wild type LigI and LigI-deficient 46BR.1G1 cells are shown. B) Distribution of actin cytoskeleton. Cells were grown on coverslips and decorated with TRITC-conjugated phalloidin. Nuclei were counterstained with DAPI. C) Quantification of morphological differences between 46BR.1G1 and 7A3 cells was determined by measuring the average ratio between the short and long axes of the cell (circularity). Circularity was also measured in the presence (+) of caffeine and KU-55933 as described in Materials and Methods. At least 100 cells/conditions for each cell line were analysed. Bars show mean ± SEM. *** P < 0.001.
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pone.0130561.g001: Correction of LigI defect affects cell morphology.A) Time-lapse imaging of cell migration. Cells were seeded at low density and monitored by time-lapse microscopy as described in Materials and Methods. Representative still images of control fibroblasts (GM847), complemented 7A3 expressing wild type LigI and LigI-deficient 46BR.1G1 cells are shown. B) Distribution of actin cytoskeleton. Cells were grown on coverslips and decorated with TRITC-conjugated phalloidin. Nuclei were counterstained with DAPI. C) Quantification of morphological differences between 46BR.1G1 and 7A3 cells was determined by measuring the average ratio between the short and long axes of the cell (circularity). Circularity was also measured in the presence (+) of caffeine and KU-55933 as described in Materials and Methods. At least 100 cells/conditions for each cell line were analysed. Bars show mean ± SEM. *** P < 0.001.

Mentions: To more precisely characterize this aspect and to understand whether the effect on cell morphology involved the DDR, we monitored by time-lapse microscopy 46BR.1G1 and 7A3 in the presence or not of checkpoint inhibitors. We compared four different parameters: morphology, directionality, accumulated distance, and velocity. As shown in Fig 1A and S1, S2 and S3 Videos, 46BR.1G1 cells are significantly more rounded compared to 7A3 cells that express ectopic wild type (wt) LigI and show a fibroblast-like morphology. A similar difference was observed when 46BR.1G1 were compared to another independent clone (31W) expressing wt LigI (S1 Fig) confirming that the effect on cell morphology is not cell clone specific. This shape difference is accompanied by an altered distribution of the actin cytoskeleton. As expected for normal fibroblasts, 7A3 cells display long stress fibers, running along the entire length of the elongated cells. Conversely, in 46BR.1G1 actin stress fibers are mainly confined to a cortical rim, while only short actin filaments are detectable in the cytoplasm (Fig 1B).


Chronic Replication Problems Impact Cell Morphology and Adhesion of DNA Ligase I Defective Cells.

Cremaschi P, Oliverio M, Leva V, Bione S, Carriero R, Mazzucco G, Palamidessi A, Scita G, Biamonti G, Montecucco A - PLoS ONE (2015)

Correction of LigI defect affects cell morphology.A) Time-lapse imaging of cell migration. Cells were seeded at low density and monitored by time-lapse microscopy as described in Materials and Methods. Representative still images of control fibroblasts (GM847), complemented 7A3 expressing wild type LigI and LigI-deficient 46BR.1G1 cells are shown. B) Distribution of actin cytoskeleton. Cells were grown on coverslips and decorated with TRITC-conjugated phalloidin. Nuclei were counterstained with DAPI. C) Quantification of morphological differences between 46BR.1G1 and 7A3 cells was determined by measuring the average ratio between the short and long axes of the cell (circularity). Circularity was also measured in the presence (+) of caffeine and KU-55933 as described in Materials and Methods. At least 100 cells/conditions for each cell line were analysed. Bars show mean ± SEM. *** P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495043&req=5

pone.0130561.g001: Correction of LigI defect affects cell morphology.A) Time-lapse imaging of cell migration. Cells were seeded at low density and monitored by time-lapse microscopy as described in Materials and Methods. Representative still images of control fibroblasts (GM847), complemented 7A3 expressing wild type LigI and LigI-deficient 46BR.1G1 cells are shown. B) Distribution of actin cytoskeleton. Cells were grown on coverslips and decorated with TRITC-conjugated phalloidin. Nuclei were counterstained with DAPI. C) Quantification of morphological differences between 46BR.1G1 and 7A3 cells was determined by measuring the average ratio between the short and long axes of the cell (circularity). Circularity was also measured in the presence (+) of caffeine and KU-55933 as described in Materials and Methods. At least 100 cells/conditions for each cell line were analysed. Bars show mean ± SEM. *** P < 0.001.
Mentions: To more precisely characterize this aspect and to understand whether the effect on cell morphology involved the DDR, we monitored by time-lapse microscopy 46BR.1G1 and 7A3 in the presence or not of checkpoint inhibitors. We compared four different parameters: morphology, directionality, accumulated distance, and velocity. As shown in Fig 1A and S1, S2 and S3 Videos, 46BR.1G1 cells are significantly more rounded compared to 7A3 cells that express ectopic wild type (wt) LigI and show a fibroblast-like morphology. A similar difference was observed when 46BR.1G1 were compared to another independent clone (31W) expressing wt LigI (S1 Fig) confirming that the effect on cell morphology is not cell clone specific. This shape difference is accompanied by an altered distribution of the actin cytoskeleton. As expected for normal fibroblasts, 7A3 cells display long stress fibers, running along the entire length of the elongated cells. Conversely, in 46BR.1G1 actin stress fibers are mainly confined to a cortical rim, while only short actin filaments are detectable in the cytoplasm (Fig 1B).

Bottom Line: This replication stress elicits a constitutive phosphorylation of the ataxia telangiectasia mutated (ATM) checkpoint kinase that fails to arrest cell cycle progression or to activate apoptosis or cell senescence.Here we show that parental 46BR.1G1 and 7A3 cells differ in important features such as cell morphology, adhesion and migration.Interestingly, ATM inhibition makes 46BR.1G1 more similar to 7A3 cells for what concerns morphology, adhesion and expression of cell-cell adhesion receptors.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (CNR), Pavia, Italy; Dipartimento di Biologia e Biotecnologie "L. Spallanzani", Università degli Studi di Pavia, Pavia, Italy.

ABSTRACT
Moderate DNA damage resulting from metabolic activities or sub-lethal doses of exogenous insults may eventually lead to cancer onset. Human 46BR.1G1 cells bear a mutation in replicative DNA ligase I (LigI) which results in low levels of replication-dependent DNA damage. This replication stress elicits a constitutive phosphorylation of the ataxia telangiectasia mutated (ATM) checkpoint kinase that fails to arrest cell cycle progression or to activate apoptosis or cell senescence. Stable transfection of wild type LigI, as in 7A3 cells, prevents DNA damage and ATM activation. Here we show that parental 46BR.1G1 and 7A3 cells differ in important features such as cell morphology, adhesion and migration. Comparison of gene expression profiles in the two cell lines detects Bio-Functional categories consistent with the morphological and migration properties of LigI deficient cells. Interestingly, ATM inhibition makes 46BR.1G1 more similar to 7A3 cells for what concerns morphology, adhesion and expression of cell-cell adhesion receptors. These observations extend the influence of the DNA damage response checkpoint pathways and unveil a role for ATM kinase activity in modulating cell biology parameters relevant to cancer progression.

No MeSH data available.


Related in: MedlinePlus