We have developed novel, isogenic human mammary epithelial ATM knockdown cell lines that facilitated the discovery of a DNA-PK-dependent and CHK1-mediated component in the IR-induced G2 checkpoint in these cells. To our knowledge, this is the first report investigating the G2 DNA damage checkpoint in human mammary epithelial cells with reduced ATM levels.
Our focus on mammary epithelial cell lines was motivated by previous reports suggesting that mutations in the ATM
gene resulting in reduced ATM expression may contribute to breast cancer (33
). Thus, we sought to explore the mechanisms by which human mammary cells respond to DNA damage and how reduced ATM levels affect and alter these responses. With respect to clonogenic survival, we found that human mammary epithelial cells expressing reduced levels of ATM are sensitized to IR similar to the sensitization of fibroblasts from individuals with A-T (37
). In addition, these cells were also sensitized to bleomycin sulfate but not UV radiation. Much to our surprise, reduced expression of ATM in these mammary epithelial cells caused only a minor defect of the IR-induced G2
checkpoint after exposure to 2 Gy radiation, less than the attenuation generally observed following a comparable dose exposure of fibroblast or lymphoblast cells from individuals with A-T (26
Modulation of RNA interference pathways to “knockdown” gene expression in cells rarely results in complete loss of the protein of interest. To address the potentially confounding issue of residual ATM expression in our stable ATM knockdown cell lines, we transiently transfected the stable hTERT184-ATM5 and hTERT184-lacZ lines with siRNA targeting ATM, in an effort to reduce any residual amounts of ATM which could contribute to the G2 checkpoint observed after 2 Gy IR. Transient transfection of ATM siRNA did not attenuate the IR-induced G2 checkpoint in hTERT184-lacZ cells, probably because ATM levels were only reduced to approximately 25% of control levels. Transient transfection of ATM siRNA into the hTERT184-ATM5 stable knockdown line only slightly increased the attenuation of the IR-induced G2 checkpoint. While we cannot exclude the possibility that extremely low levels of ATM contributed to the arrest seen in ATM knockdown cells, we have reduced levels of ATM protein below 10% of control levels and it appears that an additional pathway exists that contributes to the G2 checkpoint in the presence of reduced levels of ATM in these human mammary epithelial cells.
Cellular responses to DNA damage include not only the activation of cell cycle checkpoints to prevent cell cycle progression but also activation of DNA repair pathways (3
). While DNA-PK plays a critical role in double-strand break repair, it also appears to play an active role in apoptosis induction after excessive DNA damage (39
). In addition, cells lacking DNA-PK have double strand break repair defects and are sensitized to radiation (5
). However, the role or roles DNA-PK may play in cell cycle checkpoint functions remain unresolved.
Our studies using wortmannin suggested that DNA-PK was involved in the IR-induced G2
checkpoint response in human mammary epithelial cells. Indeed, use of the DNA-PK inhibitor NU7026 compromised the IR-induced G2
checkpoint in the ATM knockdown cells after 2 Gy IR. A role for DNA-PK in the IR-induced G2
checkpoint was confirmed using RNAi to reduce the levels of DNA-PK, which resulted in a significant attenuation of the G2
checkpoint. Our data further show that this DNA-PK-dependent signaling is associated with phosphorylation of CHK1. Although CHK1 has been clearly shown to play an important role in the G2
) and to interact with DNA-PKcs (43
), this is the first demonstration of a DNA-PK-dependent, CHK1-mediated G2
The apparent redundancy of DNA-PK with regards to what was initially regarded as ATM-dependent IR-induced G2
checkpoint responses is further supported by several reports from in vivo
mouse studies that provide evidence for ATM and DNA-PK having complementary functions. Gurley and colleagues found that scid/scid
(mutated gene encoding DNA-PKcs) ATM+/-
embryos developed normally while scid/scid
embryos died early in embryogenesis, indicating redundant functions and/or synergistic interaction between mutations in these two related kinases (13
). Gladdy and colleagues further showed that, in fact, scid/scid
embryos failed to undergo normal organogenesis and this was due to increased p53-independent apoptosis (44
). In addition, Lee and colleagues reported that DNA-PK activity is regulated in a cell cycle-dependent manner and further showed that cells from scid/scid
mice underwent permanent G2
/M arrest after irradiation (45
). In line with these results, inhibition of DNA-PK by DNA-PK-specific inhibitors also caused G2
/M accumulation in response to irradiation and topoisomerase II poisons (46
) and DNA-PKcs deficient cells were found to accumulate in G2
/M after IR (48
). It is interesting that these studies showed that loss of DNA-PK led to G2
/M accumulation in response to DNA damage (45
). It was concluded that DNA-PKcs deficient cells had an intact IR-induced G2
). It is important to note, however, that in our work the DNA-PK-dependent component of the IR-induced G2
checkpoint was only apparent in cells containing reduced levels of ATM.
The mechanism of how loss of ATM and/or DNA-PK may contribute to breast cancer remains unclear. Inhibition or loss of DNA-PK activity would almost certainly cause the persistence of DNA double strand breaks that would activate ATM-dependent cell cycle checkpoint mechanisms. Loss of ATM may lead to continual cycling and further chromosome aberrations ultimately leading to genomic instability. On the other hand, DNA-PK, in addition to its known role in DNA repair, may be actively involved in DNA damage checkpoint signaling. Loss of both ATM and DNA-PK would compromise direct activation of signaling pathways leading to cell cycle arrest. In addition, it was recently shown that both DNA-PK and ATM can interact with and phosphorylate Artemis, which has been implicated in having a role in the cellular response to DNA damage (49
). These results are intriguing and it is possible that Artemis may play a role in DNA damage checkpoint responses in human mammary epithelial cells as well.
In summary, we have shown that human mammary epithelial cells have both ATM-dependent and DNA-PK-dependent pathways to activate checkpoint responses to DNA damage. Loss of both components may compromise genomic integrity, leading to chromosomal damage. Consistent with this notion, it is interesting that a recent report found an association between reduced DNA-PK activity in peripheral blood lymphocytes (PBL) from patients with sporadic cases of uterine, cervical, and breast cancer (50
). Thus, sporadic breast tumors may arise from multiple mutations affecting independent pathways. It will be interesting to determine if, in addition to loss of ATM (36
), the corresponding breast tumors also contain reduced DNA-PK activity. Alternatively, it will be interesting to determine whether those breast tumors that show no altered ATM levels or function might have alterations in DNA-PK function. A better understanding of the involvement of ATM and DNA-PK in breast cancer may provide better therapeutic strategies for treating mammary tumors. Clearly, the precise role DNA-PK plays in DNA damage responses in normal human mammary epithelial cells requires further investigation.