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    • The cycling and quiescence of MaSCs has

    2018-10-24

    The cycling and quiescence of MaSCs has been especially difficult to study (Visvader and Stingl, 2014). It has been shown that long-lived, label-retaining cells are enriched in the MaSC/basal compartment, indicating they are slowly dividing (Shackleton et al., 2006). However, it is still unclear how DNA damage affects the cycling and quiescence properties of MaSCs. In our study, we observed a slightly lower proliferation rate in wild-type MaSCs before IR as compared to other subpopulations. Interestingly, after IR, there was an increase in Ki67-positive cells in MaSCs and TICs, indicating more cells were entering the cell cycle from G0 into interphase. These data suggest that stem cells enter the cell cycle to initiate wound repair in the mammary gland and tumors to maintain tissue homeostasis, similar to what has been observed previously in stem cells in epidermis and agomelatine cancers (Kurtova et al., 2015; Mascré et al., 2012). In addition, we observed that non-stem cells in p53-null MECs and tumors failed to induce quiescence after IR, indicating that p53 regulates, at least partially, quiescence in the mammary gland. This result is supported by previous studies in the hematopoietic system (Asai et al., 2012; Liu et al., 2009). Moreover, previously it has been shown that a higher percentage of wild-type MaSCs reside in G2/M phase as compared to other compartments (Prater et al., 2014). In this study, we also observed this phenomenon not only in wild-type MaSCs but also in p53-null MaSCs and TICs, indicating that stem cells may be primed for rapid cell division to maintain tissue homeostasis. DSB repair in hair follicle bulge stem cells, hematopoietic stem cells, and glioma stem cells has been shown to be more rapid than in other cell types in their respective tissues (Bao et al., 2006; Mohrin et al., 2010; Sotiropoulou et al., 2010). In the present study, however, we didn’t observe increased efficiency of DSB repair in MaSCs. It’s possible that, by 12 hr after IR, all three subpopulations have already repaired most of the IR-induced damage, as we observed similar levels of γH2AX positivity at the 12 and 24 hr post-IR time points. However, it is technically difficult to investigate shorter time points in these in vivo experiments. Nevertheless, we observed increased NHEJ activity in basal/MaSC compartment, which may help facilitate repair immediately after DNA damage, thereby ensuring genome integrity. On the other hand, p53 has been linked directly to DSB repair activity by regulating the HR or NHEJ pathways in in vitro studies (Marmorstein et al., 1998; Stürzbecher et al., 1996; Tang et al., 1999). Although in this study we didn’t observe a decrease in NHEJ activity in p53-null MECs, we showed that the repair of DSB was significantly slower in p53-null MECs as compared to wild-type MECs. This result suggests that p53-null cells may accumulate mutations more easily after DNA damage in vivo. With respect to specific DSB repair mechanisms in the T7 p53-null tumor model, we demonstrated that TICs exhibited significantly higher NHEJ activity, contributing to their faster repair, as compared to non-TICs. However, the repair kinetics of TICs appeared much slower than normal MaSCs, which repaired most of the DSBs by 12 hr post-IR. In addition, a high percentage of p53-null tumor cells were actively cycling even following DNA damage. These observations suggest that, once damage occurs, p53-null tumor cells have an increase probability to accumulate mutations as compared to MECs. Finally, the increased activity of the NHEJ repair pathway in MaSCs and TICs might be a double-edged sword because NHEJ is an error-prone repair mechanism (Khanna and Jackson, 2001) and might, therefore, allow more mutations agomelatine to accumulate over time (Mohrin et al., 2010). In this study, we used three models to provide insight into the variations in the DDR from the normal mammary gland to breast cancer. First, we studied the DDR in normal and p53-null mammary glands from pubertal mice, which is the age when the mammary gland has a higher susceptibility to tumorigenesis after IR (Castiglioni et al., 2007; Land et al., 1980; Preston et al., 2002; Tang et al., 2014). We showed that loss of p53 in MECs interrupts DSB repair and cell-cycle regulation after IR, which potentially may increase the accumulation of mutations in mammary epithelium. Second, we used the T7 p53-null tumor model, which is histologically similar to human basal-like breast cancer (Zhang et al., 2008) and demonstrated that T7 TICs repaired DSB more rapidly with increased NHEJ activity and exhibited decreased apoptotic cell death, most likely contributing to their resistance to IR. These results may have important implications for designing targeting therapies to selectively sensitize TICs to radiation or chemotherapy by treating basal-like tumors with inhibitors of cell survival or NHEJ pathways.