The downstream targets of ATR

The downstream targets of ATR involved in mediating human telomerase recruitment have not yet been identified. Under stalled fork conditions, activated ATR is able to phosphorylate and activate ATM (Stiff et al., 2006, and Figures 4C and 4D); whether this R 59-022 mg of ATM participates in a positive feedback loop in removing TRF1 to accelerate ATR-mediated telomere length regulation remains to be investigated. We also provide evidence that exposing telomeric single-stranded DNA in a different way, by overexpression of POT1 lacking its DNA-binding domain (Loayza and De Lange, 2003), provides a signal for rapid telomere lengthening that is also ATM dependent (Figure 5). Overexpression of POTΔOB does not perturb binding of TRF1 to the telomere (Loayza and De Lange, 2003); the ATM-dependence of telomere lengthening in this context reinforces the notion that in addition to mediating telomerase recruitment through a TRF1-dependent pathway, there must be other TRF1-independent mechanisms by which ATM mediates telomerase localization at telomeres. This is consistent with the observation that lack of TRF1 does not completely rescue the telomerase recruitment defect caused by ATM depletion (Figure 2G). We demonstrated that one TRF1-independent function of ATM is its impact upon the ability of hTR and hTERT to assemble into a functional enzyme complex (Figure 6), which is a prerequisite for localization of hTR to telomeres (Tomlinson et al., 2008). ATR also plays a role in assembly of human telomerase; we do not know if the substrates of these two kinases in this process are the same. This role is reflected in a substantial decrease in the amount of hTR recovered after hTERT immunoprecipitation and in the total immunoprecipitated telomerase activity following ATM and ATR knockdown. The specific activity of telomerase remains unchanged, demonstrating that both ATM and ATR have no effect on telomerase catalytic activity, consistent with results in yeast (Chan et al., 2001). No consensus PIKK phosphorylation motifs exist in the RNA-binding domain of hTERT, implying either that ATM or ATR can mediate telomerase assembly by targeting regions not in the RNA-binding domain or that they can regulate telomerase assembly by phosphorylating unknown substrates (Figure 7B). Our data support a model incorporating multiple roles for ATM and ATR in the presence of human telomerase at telomeres (Figure 7). One pathway involving both ATM and ATR is mediated by phosphorylation of TRF1 and its removal from the telomere, leading to replication fork stalling in telomeric DNA, which acts as a trigger for telomerase recruitment. A second pathway involves the role of ATM and ATR in facilitating telomerase assembly; additional phosphorylation targets of ATM, ATR, and other PIKKs in the telomerase recruitment process may remain to be identified. These data reveal that although it is important for telomeres to repress DNA damage signaling in order to avoid deleterious fusions, telomeres have also evolved the ability to carefully exploit aspects of DNA damage signaling pathways to regulate telomerase presence at the telomere. Increased understanding of regulation of telomerase assembly and access to the telomere may provide valuable insight in the process of developing highly specific cancer therapeutics.
Experimental Procedures
Author Contributions
Acknowledgments
Introductions Cancers are major public health problem of the world. Among them, lung cancer considers for more deaths than any other cancer in both women and men, occurred in approximately 1.8 million patients in 2012 and caused an estimated 1.6 million deaths in the world (Brambilla E, 2014). In Taiwan, it is also the leading cause of cancer death, the 5-year survival rate was only 15.9%, with a median survival of 13.2 months (Wang et al., 2013). Lung cancer commonly does not cause signs and symptoms in its earliest stages almost until the cancer is advanced. Clinical treatments for lung cancer include palliative care, surgery, radiation therapy, and chemotherapy (Gorelik et al., 2010), these therapies have improved 5-year survival rates only in early-stage, but not advanced or end-stage lung cancer patients (Crino et al., 2010). Targeted therapy of lung cancer is increasing in importance for advanced lung cancer; however, adverse side effects and drug resistant are limiting the response rate and usage of these target therapeutic drugs (Kobayashi et al., 2005). Therefore, the development of new drugs with safety and efficacy is an extremely important issue.