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

<div><p>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.</p></div

Differential expression of cadherin 13 and cadherin 4 proteins in 46BR.1G1 and 7A3 cells.

<p>(A) Cell lysates from 46BR.1G1 and 7A3 cells were analyzed by Western blotting with anti-cadherin 13, anti-cadherin 4, and anti-α-tubulin antibodies. (B) Quantification of the assay was performed by densitometric analysis with NIH ImageJ 1.43 program. Bars show mean ± SEM of three independent experiments.</p

Analysis of vinculin, vimentin and cadherins gene expression by real time RT-PCR.

<p>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.</p

Differential expression of cadherin 13 and cadherin 4 proteins in 46BR.1G1 and 31W cells.

<p>Cell lysates from 46BR.1G1 and 31W cells were analyzed by Western blotting with antibodies against the indicated proteins.</p

Enrichment analysis of IPA molecular function categories.

<p>Enrichment analysis of IPA molecular function categories.</p

Correction of LigI defect affects cell morphology.

<p>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.</p

LigI-deficient 46BR.1G1 cells adhere more efficiently to the plate than complemented 7A3 cells.

<p>Cells were plated on 96-well plate and allowed to adhere for 30 minutes before fixing. Cells were stained with Crystal Violet, solubilized with acetic acid and quantified by measuring the OD at 620 nm. Data are shown as mean ± SEM of four independent experiments.</p

LigI-deficiency affects directional migration.

<p>A) Wound-healing assay. The same number of 46BR.1G1 and 7A3 cells were seeded in each side of an Ibidi culture insert and incubated for 24 h. Cells were photographed at the time of insert removal (0 h), 8 h and 16 h after. Magnification: 4x. B) The percentage of the scratched area at each time point was calculated with the WimScratch tool (Wimasis Image Analysis). Data are shown as mean ± SEM of three independent experiments. C) Representative images of 7A3 and 46BR.1G1 directional migration in the scratched area (magnification 10x).</p