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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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Mammogram-induced DNA damage response in early and late PD HMECs.A. Mean incidence of 53BP1 foci per cell 120 min after mammogram X-ray exposures: weakened low-dose radiation response of late PD HMECs. The foci were counted in 2000 cells per group (donor 1). Error bars signify standard error. Asterisk denotes statistically significant difference in a group of irradiated HMECs compared to the shamirradiated controls of each subpopulation. B. Diagram showing the kinetics of 53BP1 foci formation. Late PD HMECs show a 100-minute delay in their peak of 53BP1 foci per cell as compared to early PD HMECs. The 53BP1 foci for kinetics analyses were counted in 1000 cells per time-point and cell subpopulation (donor 1). Cells were exposed to 10 automatic X-ray shots under a mammogram device. A & B. 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. C. Histogram showing the fraction of cells with full colocalitzation of γH2AX and 53BP1 for both early and late PD in time (donor 1). Late PD samples do not reach full colocalitzation even after 2 h post-irradiation, revealing a slower mobilization of repair proteins to the damaged site than early PD HMECs. D. Representative images of early and late PD HMECs immunostained for γH2AX and 53BP1 at various times post-IR. Cells were irradiated with 1Gy of γ-rays. Red (γH2AX) and green (53BP1) fluorochromes appear yellow where they coincide in the merged images. Post-IR mobilization of 53BP1 to the γH2AX nuclear foci follows different kinetics in early and late PD HMECs.
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pone-0063052-g004: Mammogram-induced DNA damage response in early and late PD HMECs.A. Mean incidence of 53BP1 foci per cell 120 min after mammogram X-ray exposures: weakened low-dose radiation response of late PD HMECs. The foci were counted in 2000 cells per group (donor 1). Error bars signify standard error. Asterisk denotes statistically significant difference in a group of irradiated HMECs compared to the shamirradiated controls of each subpopulation. B. Diagram showing the kinetics of 53BP1 foci formation. Late PD HMECs show a 100-minute delay in their peak of 53BP1 foci per cell as compared to early PD HMECs. The 53BP1 foci for kinetics analyses were counted in 1000 cells per time-point and cell subpopulation (donor 1). Cells were exposed to 10 automatic X-ray shots under a mammogram device. A & B. 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. C. Histogram showing the fraction of cells with full colocalitzation of γH2AX and 53BP1 for both early and late PD in time (donor 1). Late PD samples do not reach full colocalitzation even after 2 h post-irradiation, revealing a slower mobilization of repair proteins to the damaged site than early PD HMECs. D. Representative images of early and late PD HMECs immunostained for γH2AX and 53BP1 at various times post-IR. Cells were irradiated with 1Gy of γ-rays. Red (γH2AX) and green (53BP1) fluorochromes appear yellow where they coincide in the merged images. Post-IR mobilization of 53BP1 to the γH2AX nuclear foci follows different kinetics in early and late PD HMECs.

Mentions: To approach mechanistic clues underlying the observed radiation sensitivity of late PD epithelial mammary cells, we investigated their capacity to trigger an effective response when exposed to low doses of X-ray. The reduced amount of DSBs induced by mammograms may pose a serious threat to genome integrity if they are not sufficient to trigger DDR. We were therefore led to enquire whether mammary epithelial cells do indeed respond to the low number of DSBs induced by mammogram X-ray exposures, and whether early and late PD cells do this with the same efficacy. To answer these questions, we blindly analyzed the formation of 53BP1 foci, a protein that participates in the activation of factors involved in cell-cycle control and DNA repair if recruited at break sites [25]. In the early PD HMECs irradiated under the mammogram device (0, 1 and 10 shots), enumeration of 53BP1 discrete foci revealed a significantly increase with respect to the basal levels after 10 automatic shots (Mann Whitney<0.001; Figure 4A). This increase is in line with the observed increase of γH2AX foci (Figure 1B) thus providing evidence of an effective response despite the small number of DSBs induced. In contrast to the early passage cell samples, the previously observed increase in the number of γH2AX foci after two X-ray shots (Figure 1B) did not entail a significant increase of 53BP1 foci in late passage HMECs (Mann Whitney p>0.05; Figure 4A).


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)

Mammogram-induced DNA damage response in early and late PD HMECs.A. Mean incidence of 53BP1 foci per cell 120 min after mammogram X-ray exposures: weakened low-dose radiation response of late PD HMECs. The foci were counted in 2000 cells per group (donor 1). Error bars signify standard error. Asterisk denotes statistically significant difference in a group of irradiated HMECs compared to the shamirradiated controls of each subpopulation. B. Diagram showing the kinetics of 53BP1 foci formation. Late PD HMECs show a 100-minute delay in their peak of 53BP1 foci per cell as compared to early PD HMECs. The 53BP1 foci for kinetics analyses were counted in 1000 cells per time-point and cell subpopulation (donor 1). Cells were exposed to 10 automatic X-ray shots under a mammogram device. A & B. 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. C. Histogram showing the fraction of cells with full colocalitzation of γH2AX and 53BP1 for both early and late PD in time (donor 1). Late PD samples do not reach full colocalitzation even after 2 h post-irradiation, revealing a slower mobilization of repair proteins to the damaged site than early PD HMECs. D. Representative images of early and late PD HMECs immunostained for γH2AX and 53BP1 at various times post-IR. Cells were irradiated with 1Gy of γ-rays. Red (γH2AX) and green (53BP1) fluorochromes appear yellow where they coincide in the merged images. Post-IR mobilization of 53BP1 to the γH2AX nuclear foci follows different kinetics in early and late PD HMECs.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646886&req=5

pone-0063052-g004: Mammogram-induced DNA damage response in early and late PD HMECs.A. Mean incidence of 53BP1 foci per cell 120 min after mammogram X-ray exposures: weakened low-dose radiation response of late PD HMECs. The foci were counted in 2000 cells per group (donor 1). Error bars signify standard error. Asterisk denotes statistically significant difference in a group of irradiated HMECs compared to the shamirradiated controls of each subpopulation. B. Diagram showing the kinetics of 53BP1 foci formation. Late PD HMECs show a 100-minute delay in their peak of 53BP1 foci per cell as compared to early PD HMECs. The 53BP1 foci for kinetics analyses were counted in 1000 cells per time-point and cell subpopulation (donor 1). Cells were exposed to 10 automatic X-ray shots under a mammogram device. A & B. 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. C. Histogram showing the fraction of cells with full colocalitzation of γH2AX and 53BP1 for both early and late PD in time (donor 1). Late PD samples do not reach full colocalitzation even after 2 h post-irradiation, revealing a slower mobilization of repair proteins to the damaged site than early PD HMECs. D. Representative images of early and late PD HMECs immunostained for γH2AX and 53BP1 at various times post-IR. Cells were irradiated with 1Gy of γ-rays. Red (γH2AX) and green (53BP1) fluorochromes appear yellow where they coincide in the merged images. Post-IR mobilization of 53BP1 to the γH2AX nuclear foci follows different kinetics in early and late PD HMECs.
Mentions: To approach mechanistic clues underlying the observed radiation sensitivity of late PD epithelial mammary cells, we investigated their capacity to trigger an effective response when exposed to low doses of X-ray. The reduced amount of DSBs induced by mammograms may pose a serious threat to genome integrity if they are not sufficient to trigger DDR. We were therefore led to enquire whether mammary epithelial cells do indeed respond to the low number of DSBs induced by mammogram X-ray exposures, and whether early and late PD cells do this with the same efficacy. To answer these questions, we blindly analyzed the formation of 53BP1 foci, a protein that participates in the activation of factors involved in cell-cycle control and DNA repair if recruited at break sites [25]. In the early PD HMECs irradiated under the mammogram device (0, 1 and 10 shots), enumeration of 53BP1 discrete foci revealed a significantly increase with respect to the basal levels after 10 automatic shots (Mann Whitney<0.001; Figure 4A). This increase is in line with the observed increase of γH2AX foci (Figure 1B) thus providing evidence of an effective response despite the small number of DSBs induced. In contrast to the early passage cell samples, the previously observed increase in the number of γH2AX foci after two X-ray shots (Figure 1B) did not entail a significant increase of 53BP1 foci in late passage HMECs (Mann Whitney p>0.05; Figure 4A).

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