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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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DNA double strand breaks (DSBs) measured by detection of γH2AX induction in late PD HMECs by mammogram X-rays last longer than in early PD HMECs.A. Time-dependence of γH2AX foci formation and disappearance measured by direct microscopic visualization. The foci were counted in 1000 cells per each time and cell subpopulation group. Error bars signify standard error. Simple asterisk (*) refers to statistically significant difference p<0.05 and double asterisk (**) refers to highly significant difference p<0.0001. Mann Whitney test was performed in all samples. B. Time-dependent flow cytometric bivariant detection of propidium iodide (PI)-and γH2AX. Histogram showing the kinetics of γH2AX appearance and disappearance measured by flow cytometry. For early PD HMECs (green), the maximum percentage of γH2AX positive G1-stage cells was reached at 30 minutes after irradiation, whereas in vitro aged HMECs (red) do not reach maximum until 90 min time-point. A minimum of 10,000 cells and two replicas were analyzed by bivariate flow cytometry for each time-point and cell subpopulation. C. Representative dot-plots illustrate that γH2AX labeling lasts longer in the in vitro aged epithelial cells (red dots indicate positive and blue negative γH2AX labeled cells). Cell cycle is displayed using contour plots and γH2AX labeling using pseudo-color plots. Gating set at 3% increment over unspecific background fluorescence for both control and irradiated HMECs. Percentages of positive γH2AX cells at the G1 cell cycle stage are indicated in each dot-plot. For cytometric analysis, early and late PD HMECs were irradiated with 1Gy γ-rays. D. Mean incidence of micronuclei per cell in early and late PD HMECs derived from three different donors and exposed to 0, 2 and 10 automatic X-ray shots under a mammogram device (24 h after irradiation). The micronuclei were counted in 2000 (donor 1) and 500 (donor 2 and 3) binucleates 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<0.05 and double asterisk (**) refers to highly significant difference p<0.0001.
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pone-0063052-g002: DNA double strand breaks (DSBs) measured by detection of γH2AX induction in late PD HMECs by mammogram X-rays last longer than in early PD HMECs.A. Time-dependence of γH2AX foci formation and disappearance measured by direct microscopic visualization. The foci were counted in 1000 cells per each time and cell subpopulation group. Error bars signify standard error. Simple asterisk (*) refers to statistically significant difference p<0.05 and double asterisk (**) refers to highly significant difference p<0.0001. Mann Whitney test was performed in all samples. B. Time-dependent flow cytometric bivariant detection of propidium iodide (PI)-and γH2AX. Histogram showing the kinetics of γH2AX appearance and disappearance measured by flow cytometry. For early PD HMECs (green), the maximum percentage of γH2AX positive G1-stage cells was reached at 30 minutes after irradiation, whereas in vitro aged HMECs (red) do not reach maximum until 90 min time-point. A minimum of 10,000 cells and two replicas were analyzed by bivariate flow cytometry for each time-point and cell subpopulation. C. Representative dot-plots illustrate that γH2AX labeling lasts longer in the in vitro aged epithelial cells (red dots indicate positive and blue negative γH2AX labeled cells). Cell cycle is displayed using contour plots and γH2AX labeling using pseudo-color plots. Gating set at 3% increment over unspecific background fluorescence for both control and irradiated HMECs. Percentages of positive γH2AX cells at the G1 cell cycle stage are indicated in each dot-plot. For cytometric analysis, early and late PD HMECs were irradiated with 1Gy γ-rays. D. Mean incidence of micronuclei per cell in early and late PD HMECs derived from three different donors and exposed to 0, 2 and 10 automatic X-ray shots under a mammogram device (24 h after irradiation). The micronuclei were counted in 2000 (donor 1) and 500 (donor 2 and 3) binucleates 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<0.05 and double asterisk (**) refers to highly significant difference p<0.0001.

Mentions: In order to broaden our understanding of the age cell-dependence of low-dose radiation sensitivity, we carried out a time-course experiment to score the number of γH2AX foci at 6 different times post-IR in early and late PD HMECs. It is accepted that rapid loss of γH2AX is contingent upon functional and efficient DSB repair [20]. The results of γH2AX foci dynamics after 10 automatic-shot X-rays under a mammogram device are shown in Figure 2A. It can be deduced from these experiments that the disappearance of γH2AX foci is strongly dependent on the cell’s PD. Early PD HMECs have a maximum peak of γH2AX foci 90 min post-irradiation, with the number of foci decreasing significantly thereafter. In contrast, the mean number of γH2AX foci in late PD increases throughout the whole experiment (4 hours), suggesting that DSBs remain unrepaired for a more prolonged time in the in vitro aged mammary epithelial cells than in their young counterparts.


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)

DNA double strand breaks (DSBs) measured by detection of γH2AX induction in late PD HMECs by mammogram X-rays last longer than in early PD HMECs.A. Time-dependence of γH2AX foci formation and disappearance measured by direct microscopic visualization. The foci were counted in 1000 cells per each time and cell subpopulation group. Error bars signify standard error. Simple asterisk (*) refers to statistically significant difference p<0.05 and double asterisk (**) refers to highly significant difference p<0.0001. Mann Whitney test was performed in all samples. B. Time-dependent flow cytometric bivariant detection of propidium iodide (PI)-and γH2AX. Histogram showing the kinetics of γH2AX appearance and disappearance measured by flow cytometry. For early PD HMECs (green), the maximum percentage of γH2AX positive G1-stage cells was reached at 30 minutes after irradiation, whereas in vitro aged HMECs (red) do not reach maximum until 90 min time-point. A minimum of 10,000 cells and two replicas were analyzed by bivariate flow cytometry for each time-point and cell subpopulation. C. Representative dot-plots illustrate that γH2AX labeling lasts longer in the in vitro aged epithelial cells (red dots indicate positive and blue negative γH2AX labeled cells). Cell cycle is displayed using contour plots and γH2AX labeling using pseudo-color plots. Gating set at 3% increment over unspecific background fluorescence for both control and irradiated HMECs. Percentages of positive γH2AX cells at the G1 cell cycle stage are indicated in each dot-plot. For cytometric analysis, early and late PD HMECs were irradiated with 1Gy γ-rays. D. Mean incidence of micronuclei per cell in early and late PD HMECs derived from three different donors and exposed to 0, 2 and 10 automatic X-ray shots under a mammogram device (24 h after irradiation). The micronuclei were counted in 2000 (donor 1) and 500 (donor 2 and 3) binucleates 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<0.05 and double asterisk (**) refers to highly significant difference p<0.0001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646886&req=5

pone-0063052-g002: DNA double strand breaks (DSBs) measured by detection of γH2AX induction in late PD HMECs by mammogram X-rays last longer than in early PD HMECs.A. Time-dependence of γH2AX foci formation and disappearance measured by direct microscopic visualization. The foci were counted in 1000 cells per each time and cell subpopulation group. Error bars signify standard error. Simple asterisk (*) refers to statistically significant difference p<0.05 and double asterisk (**) refers to highly significant difference p<0.0001. Mann Whitney test was performed in all samples. B. Time-dependent flow cytometric bivariant detection of propidium iodide (PI)-and γH2AX. Histogram showing the kinetics of γH2AX appearance and disappearance measured by flow cytometry. For early PD HMECs (green), the maximum percentage of γH2AX positive G1-stage cells was reached at 30 minutes after irradiation, whereas in vitro aged HMECs (red) do not reach maximum until 90 min time-point. A minimum of 10,000 cells and two replicas were analyzed by bivariate flow cytometry for each time-point and cell subpopulation. C. Representative dot-plots illustrate that γH2AX labeling lasts longer in the in vitro aged epithelial cells (red dots indicate positive and blue negative γH2AX labeled cells). Cell cycle is displayed using contour plots and γH2AX labeling using pseudo-color plots. Gating set at 3% increment over unspecific background fluorescence for both control and irradiated HMECs. Percentages of positive γH2AX cells at the G1 cell cycle stage are indicated in each dot-plot. For cytometric analysis, early and late PD HMECs were irradiated with 1Gy γ-rays. D. Mean incidence of micronuclei per cell in early and late PD HMECs derived from three different donors and exposed to 0, 2 and 10 automatic X-ray shots under a mammogram device (24 h after irradiation). The micronuclei were counted in 2000 (donor 1) and 500 (donor 2 and 3) binucleates 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<0.05 and double asterisk (**) refers to highly significant difference p<0.0001.
Mentions: In order to broaden our understanding of the age cell-dependence of low-dose radiation sensitivity, we carried out a time-course experiment to score the number of γH2AX foci at 6 different times post-IR in early and late PD HMECs. It is accepted that rapid loss of γH2AX is contingent upon functional and efficient DSB repair [20]. The results of γH2AX foci dynamics after 10 automatic-shot X-rays under a mammogram device are shown in Figure 2A. It can be deduced from these experiments that the disappearance of γH2AX foci is strongly dependent on the cell’s PD. Early PD HMECs have a maximum peak of γH2AX foci 90 min post-irradiation, with the number of foci decreasing significantly thereafter. In contrast, the mean number of γH2AX foci in late PD increases throughout the whole experiment (4 hours), suggesting that DSBs remain unrepaired for a more prolonged time in the in vitro aged mammary epithelial cells than in their young counterparts.

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