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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

Analysis of vinculin, vimentin and cadherins gene expression by real time RT-PCR.The panels show the relative expression levels of the indicated transcripts in 46BR.lG1 (gray bars) and 7A3 cells (black bars) before (-) and after (+) incubation with 10 μM KU-55933. Gene transcripts have been internally normalized versus RPLP0 expression levels. Data are shown as mean ± SEM of four independent experiments. CDH: cadherin, VCL: vinculin, VIM: vimentin. * P < 0 .05, ** P < 0.01, *** P < 0.001.
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pone.0130561.g004: Analysis of vinculin, vimentin and cadherins gene expression by real time RT-PCR.The panels show the relative expression levels of the indicated transcripts in 46BR.lG1 (gray bars) and 7A3 cells (black bars) before (-) and after (+) incubation with 10 μM KU-55933. Gene transcripts have been internally normalized versus RPLP0 expression levels. Data are shown as mean ± SEM of four independent experiments. CDH: cadherin, VCL: vinculin, VIM: vimentin. * P < 0 .05, ** P < 0.01, *** P < 0.001.

Mentions: As a further validation of the high-throughput analyses we decided to measure by qRT-PCR the expression of a few selected genes. IPA categories describing the process of cell migration include vinculin and some members of the cadherin superfamily involved in cell adhesion and migration [34]. We focused on genes of the cadherin family, some of which were detected as differentially expressed in 46BR.1G1 vs 7A3 cells by both microarray and RNA-Seq analyses. As shown in Fig 4, in agreement with the genome wide analyses, qRT-PCR measured statistically significant differences in the expression of cadherin 4 (CDH4 also called R-cadherin), cadherin 13 (CDH13, H-cadherin), cadherin 9 (CDH9, T1-cadherin) and cadherin 12 (CDH12, N-Cadherin 2). Notably CDH4 is a critical regulator of epithelial phenotype [35] and CDH13 levels are frequently down regulated in invasive carcinoma cells [36]. In order to verify the effect of this regulation at the protein level, cell extracts from 46BR.1G1 and 7A3 cells were immunoblotted with antibodies against CDH13 and CDH4, whose transcripts are overexpressed in LigI-deficient cells. Fig 5 shows that, in agreement with the qPCR analysis, both proteins are overexpressed in 46BR.1G1 cells. The down-regulation of CDH13 and CDH4 in LigI-proficient cells was also confirmed in 31W cells (Fig 6) ruling out the possibility that the observed change in gene expression was cell clone specific. Notably, the differential expression of these cadherins is consistent with the idea that LigI-deficiency may induce a shift toward an epithelial-like shape. In line with this hypothesis CDH9, which is up-regulated during EMT (epithelial to mesenchymal cell transition) of renal tubular epithelial cells [37], and CDH12, whose overexpression increases the invasive properties of salivary adenoid cystic carcinoma cells [38], are down-regulated in 46BR.1G1 cells. We also analyzed two members of the cadherin family whose expression is commonly used as a diagnostic marker of EMT events: CDH1 and CDH2 genes, which are respectively down and up regulated during EMT. The RNA-Seq, but not the microarray analysis, evidenced a moderate but statistically significant reduction of CDH2 mRNA in 46BR.1G1 cells (LFC = -0.66 p-value = 4x10-4) while both methods were unable to predict the behavior of CDH1 because its expression was too low to be analyzed under the experimental conditions used in this study. In agreement with RNA-Seq data, qRT-PCR analysis evidenced statistically significant down-regulation of CDH2 in LigI-deficient cells accompanied by a slight increase of CDH1 mRNA (Fig 4, panel B). In particular, CDH2 expression was reduced to about 50% in 46BR.1G1 cells, consistent with the difference estimated by RNA-Seq analysis. The differential expression between 7A3 and 46BR.1G1 of different cadherins is notable. It has been shown that the expression of several cadherin genes is differentially affected by epithelial as opposed to the mesenchymal phenotype. Within this framework, for example CDH9 and CDH12 are up regulated as expected in the more mesenchymal-like line 7A3. CDH1, the prototypical epithelial junctional protein, is elevated in LigI-deficient cells while CDH2 (the mesenchymal N-cadherin) is down regulated. The functional phenotypic consequences of other cadherins is less understood and would be interesting in future to explore their impact on the nature of epithelial vs mesenchymal phenotype. Altogether this analysis is consistent with the idea, suggested by the morphological data, that LigI deficiency induces a shift toward an epithelial-like morphology. Moreover, in agreement with the increase in adhesion properties (Fig 2), the vinculin (VCL) gene, which encodes a focal adhesion protein [39], is up-regulated in 46BR.1G1 cells (Fig 4 panel C). Up-regulation of vinculin was detected only by the micro-array and confirmed by qRT-PCR but not by the RNA-Seq analysis, once more pointing to the cautions that must be put in the interpretation of genome wide data, particularly when low number of reads are considered in RNA-Seq experiments. We also evaluated the expression of vimentin (VIM) a member of the intermediate filaments family of proteins responsible for maintaining cell shape, and whose expression is typically up regulated during EMT. In accord with microarray and RNA-Seq data, qPCR analysis detected a comparable expression of vimentin in 46BR.1G1 and 7A3 cells (Fig 4 panel C).


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)

Analysis of vinculin, vimentin and cadherins gene expression by real time RT-PCR.The panels show the relative expression levels of the indicated transcripts in 46BR.lG1 (gray bars) and 7A3 cells (black bars) before (-) and after (+) incubation with 10 μM KU-55933. Gene transcripts have been internally normalized versus RPLP0 expression levels. Data are shown as mean ± SEM of four independent experiments. CDH: cadherin, VCL: vinculin, VIM: vimentin. * P < 0 .05, ** P < 0.01, *** P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4495043&req=5

pone.0130561.g004: Analysis of vinculin, vimentin and cadherins gene expression by real time RT-PCR.The panels show the relative expression levels of the indicated transcripts in 46BR.lG1 (gray bars) and 7A3 cells (black bars) before (-) and after (+) incubation with 10 μM KU-55933. Gene transcripts have been internally normalized versus RPLP0 expression levels. Data are shown as mean ± SEM of four independent experiments. CDH: cadherin, VCL: vinculin, VIM: vimentin. * P < 0 .05, ** P < 0.01, *** P < 0.001.
Mentions: As a further validation of the high-throughput analyses we decided to measure by qRT-PCR the expression of a few selected genes. IPA categories describing the process of cell migration include vinculin and some members of the cadherin superfamily involved in cell adhesion and migration [34]. We focused on genes of the cadherin family, some of which were detected as differentially expressed in 46BR.1G1 vs 7A3 cells by both microarray and RNA-Seq analyses. As shown in Fig 4, in agreement with the genome wide analyses, qRT-PCR measured statistically significant differences in the expression of cadherin 4 (CDH4 also called R-cadherin), cadherin 13 (CDH13, H-cadherin), cadherin 9 (CDH9, T1-cadherin) and cadherin 12 (CDH12, N-Cadherin 2). Notably CDH4 is a critical regulator of epithelial phenotype [35] and CDH13 levels are frequently down regulated in invasive carcinoma cells [36]. In order to verify the effect of this regulation at the protein level, cell extracts from 46BR.1G1 and 7A3 cells were immunoblotted with antibodies against CDH13 and CDH4, whose transcripts are overexpressed in LigI-deficient cells. Fig 5 shows that, in agreement with the qPCR analysis, both proteins are overexpressed in 46BR.1G1 cells. The down-regulation of CDH13 and CDH4 in LigI-proficient cells was also confirmed in 31W cells (Fig 6) ruling out the possibility that the observed change in gene expression was cell clone specific. Notably, the differential expression of these cadherins is consistent with the idea that LigI-deficiency may induce a shift toward an epithelial-like shape. In line with this hypothesis CDH9, which is up-regulated during EMT (epithelial to mesenchymal cell transition) of renal tubular epithelial cells [37], and CDH12, whose overexpression increases the invasive properties of salivary adenoid cystic carcinoma cells [38], are down-regulated in 46BR.1G1 cells. We also analyzed two members of the cadherin family whose expression is commonly used as a diagnostic marker of EMT events: CDH1 and CDH2 genes, which are respectively down and up regulated during EMT. The RNA-Seq, but not the microarray analysis, evidenced a moderate but statistically significant reduction of CDH2 mRNA in 46BR.1G1 cells (LFC = -0.66 p-value = 4x10-4) while both methods were unable to predict the behavior of CDH1 because its expression was too low to be analyzed under the experimental conditions used in this study. In agreement with RNA-Seq data, qRT-PCR analysis evidenced statistically significant down-regulation of CDH2 in LigI-deficient cells accompanied by a slight increase of CDH1 mRNA (Fig 4, panel B). In particular, CDH2 expression was reduced to about 50% in 46BR.1G1 cells, consistent with the difference estimated by RNA-Seq analysis. The differential expression between 7A3 and 46BR.1G1 of different cadherins is notable. It has been shown that the expression of several cadherin genes is differentially affected by epithelial as opposed to the mesenchymal phenotype. Within this framework, for example CDH9 and CDH12 are up regulated as expected in the more mesenchymal-like line 7A3. CDH1, the prototypical epithelial junctional protein, is elevated in LigI-deficient cells while CDH2 (the mesenchymal N-cadherin) is down regulated. The functional phenotypic consequences of other cadherins is less understood and would be interesting in future to explore their impact on the nature of epithelial vs mesenchymal phenotype. Altogether this analysis is consistent with the idea, suggested by the morphological data, that LigI deficiency induces a shift toward an epithelial-like morphology. Moreover, in agreement with the increase in adhesion properties (Fig 2), the vinculin (VCL) gene, which encodes a focal adhesion protein [39], is up-regulated in 46BR.1G1 cells (Fig 4 panel C). Up-regulation of vinculin was detected only by the micro-array and confirmed by qRT-PCR but not by the RNA-Seq analysis, once more pointing to the cautions that must be put in the interpretation of genome wide data, particularly when low number of reads are considered in RNA-Seq experiments. We also evaluated the expression of vimentin (VIM) a member of the intermediate filaments family of proteins responsible for maintaining cell shape, and whose expression is typically up regulated during EMT. In accord with microarray and RNA-Seq data, qPCR analysis detected a comparable expression of vimentin in 46BR.1G1 and 7A3 cells (Fig 4 panel C).

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