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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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Restoration of telomerase activity in the late PD HMEC samples does not rescue their delayed DSB disappearance phenotype.Results obtained from donor 1 samples. A. Western blot showing that transduction of late PD HMECs with hTERT gene restituted telomerase protein subunit expression. B. Metaphase plates of a late PD non-transduced (top) and hTERT-transduced (bottom) HMECs. Telomeric (red) and centromeric (green) sequences detected with fluorescent in situ hybridization procedures. Telomerase restoration resulted in a reduction of telomere-signal-free ends (white arrow heads) and end-to-end fusions (Dic) as a consequence of telomere elongation. C. Bar diagram showing average frequencies of telomere-signal-free ends in non-transduced and hTERT-transduced HMECs. D. Histogram showing the kinetics of γH2AX appearance and disappearance measured by flow cytometric analysis. hTERT-transduced late PD HMECs (brown) reach its maximum percentage of γH2AX positive cells 90 min post-irradiation, which is delayed as compared to the early PD HMECs (green), but is the same time point as non-transduced late PD HMECs (red). A minimum of 10,000 cells and two replicas were analyzed by bivariate flow cytometry for each time-point and cell subpopulation.
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pone-0063052-g003: Restoration of telomerase activity in the late PD HMEC samples does not rescue their delayed DSB disappearance phenotype.Results obtained from donor 1 samples. A. Western blot showing that transduction of late PD HMECs with hTERT gene restituted telomerase protein subunit expression. B. Metaphase plates of a late PD non-transduced (top) and hTERT-transduced (bottom) HMECs. Telomeric (red) and centromeric (green) sequences detected with fluorescent in situ hybridization procedures. Telomerase restoration resulted in a reduction of telomere-signal-free ends (white arrow heads) and end-to-end fusions (Dic) as a consequence of telomere elongation. C. Bar diagram showing average frequencies of telomere-signal-free ends in non-transduced and hTERT-transduced HMECs. D. Histogram showing the kinetics of γH2AX appearance and disappearance measured by flow cytometric analysis. hTERT-transduced late PD HMECs (brown) reach its maximum percentage of γH2AX positive cells 90 min post-irradiation, which is delayed as compared to the early PD HMECs (green), but is the same time point as non-transduced late PD HMECs (red). A minimum of 10,000 cells and two replicas were analyzed by bivariate flow cytometry for each time-point and cell subpopulation.

Mentions: The presence of dysfunctional telomeres in aged proliferative cells is associated with increased radiosensitivity as they interfere with the correct joining of DNA strand breaks [14]. We therefore speculated on whether the observed delay in DSB disappearance in the late PD cell samples was caused by the presence of dysfunctional telomeres or, instead, whether it was a direct consequence of cell aging. For this purpose, we investigated whether DSB repair kinetics–as measured by γH2AX and PI bivariant flow cytometry– was influenced by the restoration of telomerase through transduction of hTERT gene in late PD HMECs derived from donor 1 (Figure 3A). To ensure we had transduced the cells properly, instead of measuring the average telomere lengthening, we scored the frequency of chromosome ends without visible telomere signals (probably uncapped ends). We observed a significant reduction in the frequency of chromosome ends with no visible telomeric signal after transduction of cells with hTERT (Figure 3B and 3C). Regardless of having been transduced (or not) with hTERT, all late PD HMECs reached the maximum percentage of γH2AX positive G1-stage cells with delay, at 90 minutes after irradiation (Figure 3D). Taken as a whole, this would suggest that rather than a consequence of the presence of dysfunctional telomeres, the delayed disappearance of DSBs in the in vitro aged samples is directly associated with chronological cell aging.


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)

Restoration of telomerase activity in the late PD HMEC samples does not rescue their delayed DSB disappearance phenotype.Results obtained from donor 1 samples. A. Western blot showing that transduction of late PD HMECs with hTERT gene restituted telomerase protein subunit expression. B. Metaphase plates of a late PD non-transduced (top) and hTERT-transduced (bottom) HMECs. Telomeric (red) and centromeric (green) sequences detected with fluorescent in situ hybridization procedures. Telomerase restoration resulted in a reduction of telomere-signal-free ends (white arrow heads) and end-to-end fusions (Dic) as a consequence of telomere elongation. C. Bar diagram showing average frequencies of telomere-signal-free ends in non-transduced and hTERT-transduced HMECs. D. Histogram showing the kinetics of γH2AX appearance and disappearance measured by flow cytometric analysis. hTERT-transduced late PD HMECs (brown) reach its maximum percentage of γH2AX positive cells 90 min post-irradiation, which is delayed as compared to the early PD HMECs (green), but is the same time point as non-transduced late PD HMECs (red). A minimum of 10,000 cells and two replicas were analyzed by bivariate flow cytometry for each time-point and cell subpopulation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646886&req=5

pone-0063052-g003: Restoration of telomerase activity in the late PD HMEC samples does not rescue their delayed DSB disappearance phenotype.Results obtained from donor 1 samples. A. Western blot showing that transduction of late PD HMECs with hTERT gene restituted telomerase protein subunit expression. B. Metaphase plates of a late PD non-transduced (top) and hTERT-transduced (bottom) HMECs. Telomeric (red) and centromeric (green) sequences detected with fluorescent in situ hybridization procedures. Telomerase restoration resulted in a reduction of telomere-signal-free ends (white arrow heads) and end-to-end fusions (Dic) as a consequence of telomere elongation. C. Bar diagram showing average frequencies of telomere-signal-free ends in non-transduced and hTERT-transduced HMECs. D. Histogram showing the kinetics of γH2AX appearance and disappearance measured by flow cytometric analysis. hTERT-transduced late PD HMECs (brown) reach its maximum percentage of γH2AX positive cells 90 min post-irradiation, which is delayed as compared to the early PD HMECs (green), but is the same time point as non-transduced late PD HMECs (red). A minimum of 10,000 cells and two replicas were analyzed by bivariate flow cytometry for each time-point and cell subpopulation.
Mentions: The presence of dysfunctional telomeres in aged proliferative cells is associated with increased radiosensitivity as they interfere with the correct joining of DNA strand breaks [14]. We therefore speculated on whether the observed delay in DSB disappearance in the late PD cell samples was caused by the presence of dysfunctional telomeres or, instead, whether it was a direct consequence of cell aging. For this purpose, we investigated whether DSB repair kinetics–as measured by γH2AX and PI bivariant flow cytometry– was influenced by the restoration of telomerase through transduction of hTERT gene in late PD HMECs derived from donor 1 (Figure 3A). To ensure we had transduced the cells properly, instead of measuring the average telomere lengthening, we scored the frequency of chromosome ends without visible telomere signals (probably uncapped ends). We observed a significant reduction in the frequency of chromosome ends with no visible telomeric signal after transduction of cells with hTERT (Figure 3B and 3C). Regardless of having been transduced (or not) with hTERT, all late PD HMECs reached the maximum percentage of γH2AX positive G1-stage cells with delay, at 90 minutes after irradiation (Figure 3D). Taken as a whole, this would suggest that rather than a consequence of the presence of dysfunctional telomeres, the delayed disappearance of DSBs in the in vitro aged samples is directly associated with chronological cell aging.

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