No abstract

Aging involves a deterioration of cell functions and changes that may predispose the cell to undergo an oncogenic transformation. The carcinogenic risks following radiation exposure rise with age among adults. Increasing inflammatory response, loss of oxidant/antioxidant equilibrium, ongoing telomere attrition, decline in the DNA damage response efficiency, and deleterious nuclear organization are age-related cellular changes that trigger a serious threat to genomic integrity. In this review, we discuss the mechanistic interplay between all these factors, providing an integrated view of how they contribute to the observed age-related increase in radiation sensitivity. As life expectancy increases and so it does the medical intervention, it is important to highlight the benefits of radiation protection in the elderly. Thus, a deep understanding of the mechanistic processes confining the threat of aging-related radiosensitivity is currently of foremost relevance.

Phosphorylation of the H2AX protein is an early step in the double strand break (DSB) repair pathway; therefore, phosphorylated histone (γH2AX) foci scoring is widely used as a measure for DSBs. Foci scoring is performed either manually or semi-automatically using hand-operated capturing and image analysis software. In general, both techniques are laborious and prone to artifacts associated with manual scoring. While a few fully automated methods have been described in the literature, none of them have been used to quantify γH2AX foci in combination with a cell cycle phase analysis. Adding this feature to a rapid automated γH2AX foci quantification method would reduce the scoring uncertainty that arises from the variations in the background level of the γH2AX signal throughout the cell cycle. The method was set up to measure DNA damage induced in human mammary epithelial cells by irradiation under a mammogram device. We adapted a FISH (fluorescent in situ hybridization) Spot-counting system, which has a slide loader with automatic scanning and cell capture system throughout the thickness of each cell (z-stack), to meet our assay requirements. While scanning the sample, the system classifies the selected nuclei according to the signal patterns previously described by the user. For our purposes, a double staining immunofluorescence was carried out with antibodies to detect γH2AX and pericentrin, an integral component of the centrosome. We could thus distinguish both the number of γH2AX foci per cell and the cell cycle phase. Furthermore, restrictive settings of the program classifier reduced the “touching nuclei” problem described in other image analysis software. The automated scoring was faster than and as sensitive as its manually performed counterpart. This system is a reliable tool for γH2AX radio-induced foci counting and provides essential information about the cell cycle stage. It thus offers a more complete and rapid assessment of DNA damage.

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.

Abbreviations: AU, arbitrary units; DSB, DNA double-strand break; FISH, fluorescence in situ hybridization; IRIF, ionizing radiation induced foci; FI, fluorescence intensity; mFISH, multicolor fluorescence in situ hybridization; MRN complex, MRE11-Rad50-Nbs1 complex; SD, standard deviation; TIF, telomere-dysfunction induced foci.The presence of gH2AX foci on apparently intact mitotic chromosomes is controversial because they challenge the assumed relationship between gH2AX foci and DNA double-strand breaks (DSBs). In this work, we show that after irradiation during interphase, a variety of gH2AX foci are scored in mitotic cells. Surprisingly, approximately 80% of the gH2AX foci spread over apparently undamaged chromatin at Terminal or Interstitial positions and they can display variable sizes, thus being classified as Small, Medium and Big foci. Chromosome and chromatid breaks that reach mitosis are spotted with Big (60%) and Medium (30%) Terminal gH2AX foci, but very rarely are they signaled with Small gH2AX foci. To evaluate if Interstitial gH2AX foci might be signatures of misrejoining, an mFISH analysis was performed on the same slides. The results show that Interstitial gH2AX foci lying on apparently intact chromatin do not mark sites of misrejoining, and that misrejoined events were never signaled by a gH2AX foci during mitosis. Finally, when analyzing the presence of other DNA-damage response (DDR) factors we found that all gH2AX foci-regardless their coincidence with a visible break-always colocalized with MRE11, but not with 53BP1. This pattern suggests that these gH2AX foci may be hallmarks of both microscopically visible and invisible DNA damage, in which an active, although incomplete or halted DDR is taking place.