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Increased mammogram-induced DNA damage in mammary epithelial cells aged in vitro.

Hernández L, Terradas M, Martín M, Feijoo P, Soler D, Tusell L, Genescà A - PLoS ONE (2013)

Bottom Line: We provide evidence that aged epithelial breast cells are more radiosensitive than younger ones.The result of this is a significant increase in micronuclei frequency in the in vitro aged mammary epithelial cells exposed to doses equivalent to a single mammogram X-ray exploration.Therefore, retarded DNA break repair is a direct consequence of cellular aging itself, rather than a consequence of the presence of dysfunctional telomeres.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Physiology, and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Spain.

ABSTRACT
Concerned about the risks of mammography screening in the adult population, we analyzed the ability of human mammary epithelial cells to cope with mammogram-induced DNA damage. Our study shows that an X-ray dose of 20 mGy, which is the standard dose received by the breast surface per two-view mammogram X-ray exploration, induces increased frequencies of DNA double-strand breaks to in vitro aged-but not to young-human mammary epithelial cells. We provide evidence that aged epithelial breast cells are more radiosensitive than younger ones. Our studies point to an inefficient damage response of aged cells to low-dose radiation, this being due to both delayed and incomplete mobilization of repair proteins to DNA strand breaks. This inefficient damage response is translated into an important delay in double-strand break disappearance and consequent accumulation of unrepaired DNA breaks. The result of this is a significant increase in micronuclei frequency in the in vitro aged mammary epithelial cells exposed to doses equivalent to a single mammogram X-ray exploration. Since our experiments were carried out in primary epithelial cell cultures in which cells age at the same time as they undergo replication-dependent telomere shortening, we needed to determine the contribution of these two factors to their phenotype. In this paper, we report that the exogenous expression of human telomerase retrotranscriptase in late population doubling epithelial cells does not rescue its delayed repair phenotype. Therefore, retarded DNA break repair is a direct consequence of cellular aging itself, rather than a consequence of the presence of dysfunctional telomeres. Our findings of long-lasting double strand breaks and incomplete DNA break repair in the in vitro aged epithelial cells are in line with the increased carcinogenic risks of radiation exposures at older ages revealed by epidemiologic studies.

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Detection of phosphorylated histone (γH2AX) foci in human mammary epithelial cells (HMECs) to estimate DNA damage.A. Endogenous γH2AX foci: the proportion of cells containing ≥1 foci in late PD cells is significantly higher than in early PD cells (p<0.0001, Chi-squared test). The foci were counted in 1000 cells per sample. B. Mean incidence of γH2AX foci per cell 2 h after mammogram X-ray exposures is greater in the late PD HMECs in each of the three donors analyzed. The foci were counted in 2000 cells (donor 1) and 1000 cells (donor 2 and 3) per group. Error bars signify standard error. Asterisk denotes statistically significant difference in a group of irradiated HMECs compared to the shamirradiated controls of each cell subpopulation (Mann Whitney test). Simple asterisk (*) refers to statistically significant difference p<0.01 and double asterisk (**) refers to highly significant difference p<0.0001. C. Representative images of γH2AX foci in HMECs from early and late PD exposed to 0, 2 and 10 automatic X-ray shots under a mammogram device (2 hours post-irradiation). Arrowheads point to the γH2AX foci stained in red. In vitro aged populations exhibited a higher number of γH2AX foci compared to the young counterparts.
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pone-0063052-g001: Detection of phosphorylated histone (γH2AX) foci in human mammary epithelial cells (HMECs) to estimate DNA damage.A. Endogenous γH2AX foci: the proportion of cells containing ≥1 foci in late PD cells is significantly higher than in early PD cells (p<0.0001, Chi-squared test). The foci were counted in 1000 cells per sample. B. Mean incidence of γH2AX foci per cell 2 h after mammogram X-ray exposures is greater in the late PD HMECs in each of the three donors analyzed. The foci were counted in 2000 cells (donor 1) and 1000 cells (donor 2 and 3) per group. Error bars signify standard error. Asterisk denotes statistically significant difference in a group of irradiated HMECs compared to the shamirradiated controls of each cell subpopulation (Mann Whitney test). Simple asterisk (*) refers to statistically significant difference p<0.01 and double asterisk (**) refers to highly significant difference p<0.0001. C. Representative images of γH2AX foci in HMECs from early and late PD exposed to 0, 2 and 10 automatic X-ray shots under a mammogram device (2 hours post-irradiation). Arrowheads point to the γH2AX foci stained in red. In vitro aged populations exhibited a higher number of γH2AX foci compared to the young counterparts.

Mentions: First, we used γH2AX (protein immunofluorescence) detection to identify and quantify basal levels of DSBs in epithelial mammary cells derived from three different healthy donors. The nucleosomal histone H2AX is phosphorylated on its Ser139 in large segments of DSB-flanking chromatin, which are visible by epifluorescent microscopy as nuclear foci [19], [20]. We observed that the proportion of cells containing endogenous γH2AX foci was higher in the in vitro aged cell subpopulations (Figure 1A shows results obtained in HMECs derived from donor 1; chi-squared test, p<0.0001). The higher levels of endogenous damage in late PD cells in comparison to their early counterparts may be revealing age-associated accumulation of irreparable DSBs [17], [21] and/or critical telomere erosion [22].


Increased mammogram-induced DNA damage in mammary epithelial cells aged in vitro.

Hernández L, Terradas M, Martín M, Feijoo P, Soler D, Tusell L, Genescà A - PLoS ONE (2013)

Detection of phosphorylated histone (γH2AX) foci in human mammary epithelial cells (HMECs) to estimate DNA damage.A. Endogenous γH2AX foci: the proportion of cells containing ≥1 foci in late PD cells is significantly higher than in early PD cells (p<0.0001, Chi-squared test). The foci were counted in 1000 cells per sample. B. Mean incidence of γH2AX foci per cell 2 h after mammogram X-ray exposures is greater in the late PD HMECs in each of the three donors analyzed. The foci were counted in 2000 cells (donor 1) and 1000 cells (donor 2 and 3) per group. Error bars signify standard error. Asterisk denotes statistically significant difference in a group of irradiated HMECs compared to the shamirradiated controls of each cell subpopulation (Mann Whitney test). Simple asterisk (*) refers to statistically significant difference p<0.01 and double asterisk (**) refers to highly significant difference p<0.0001. C. Representative images of γH2AX foci in HMECs from early and late PD exposed to 0, 2 and 10 automatic X-ray shots under a mammogram device (2 hours post-irradiation). Arrowheads point to the γH2AX foci stained in red. In vitro aged populations exhibited a higher number of γH2AX foci compared to the young counterparts.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646886&req=5

pone-0063052-g001: Detection of phosphorylated histone (γH2AX) foci in human mammary epithelial cells (HMECs) to estimate DNA damage.A. Endogenous γH2AX foci: the proportion of cells containing ≥1 foci in late PD cells is significantly higher than in early PD cells (p<0.0001, Chi-squared test). The foci were counted in 1000 cells per sample. B. Mean incidence of γH2AX foci per cell 2 h after mammogram X-ray exposures is greater in the late PD HMECs in each of the three donors analyzed. The foci were counted in 2000 cells (donor 1) and 1000 cells (donor 2 and 3) per group. Error bars signify standard error. Asterisk denotes statistically significant difference in a group of irradiated HMECs compared to the shamirradiated controls of each cell subpopulation (Mann Whitney test). Simple asterisk (*) refers to statistically significant difference p<0.01 and double asterisk (**) refers to highly significant difference p<0.0001. C. Representative images of γH2AX foci in HMECs from early and late PD exposed to 0, 2 and 10 automatic X-ray shots under a mammogram device (2 hours post-irradiation). Arrowheads point to the γH2AX foci stained in red. In vitro aged populations exhibited a higher number of γH2AX foci compared to the young counterparts.
Mentions: First, we used γH2AX (protein immunofluorescence) detection to identify and quantify basal levels of DSBs in epithelial mammary cells derived from three different healthy donors. The nucleosomal histone H2AX is phosphorylated on its Ser139 in large segments of DSB-flanking chromatin, which are visible by epifluorescent microscopy as nuclear foci [19], [20]. We observed that the proportion of cells containing endogenous γH2AX foci was higher in the in vitro aged cell subpopulations (Figure 1A shows results obtained in HMECs derived from donor 1; chi-squared test, p<0.0001). The higher levels of endogenous damage in late PD cells in comparison to their early counterparts may be revealing age-associated accumulation of irreparable DSBs [17], [21] and/or critical telomere erosion [22].

Bottom Line: We provide evidence that aged epithelial breast cells are more radiosensitive than younger ones.The result of this is a significant increase in micronuclei frequency in the in vitro aged mammary epithelial cells exposed to doses equivalent to a single mammogram X-ray exploration.Therefore, retarded DNA break repair is a direct consequence of cellular aging itself, rather than a consequence of the presence of dysfunctional telomeres.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Physiology, and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Spain.

ABSTRACT
Concerned about the risks of mammography screening in the adult population, we analyzed the ability of human mammary epithelial cells to cope with mammogram-induced DNA damage. Our study shows that an X-ray dose of 20 mGy, which is the standard dose received by the breast surface per two-view mammogram X-ray exploration, induces increased frequencies of DNA double-strand breaks to in vitro aged-but not to young-human mammary epithelial cells. We provide evidence that aged epithelial breast cells are more radiosensitive than younger ones. Our studies point to an inefficient damage response of aged cells to low-dose radiation, this being due to both delayed and incomplete mobilization of repair proteins to DNA strand breaks. This inefficient damage response is translated into an important delay in double-strand break disappearance and consequent accumulation of unrepaired DNA breaks. The result of this is a significant increase in micronuclei frequency in the in vitro aged mammary epithelial cells exposed to doses equivalent to a single mammogram X-ray exploration. Since our experiments were carried out in primary epithelial cell cultures in which cells age at the same time as they undergo replication-dependent telomere shortening, we needed to determine the contribution of these two factors to their phenotype. In this paper, we report that the exogenous expression of human telomerase retrotranscriptase in late population doubling epithelial cells does not rescue its delayed repair phenotype. Therefore, retarded DNA break repair is a direct consequence of cellular aging itself, rather than a consequence of the presence of dysfunctional telomeres. Our findings of long-lasting double strand breaks and incomplete DNA break repair in the in vitro aged epithelial cells are in line with the increased carcinogenic risks of radiation exposures at older ages revealed by epidemiologic studies.

Show MeSH
Related in: MedlinePlus