Although TP status has been shown to play a
Although TP53 status has been shown to play a role in ATR inhibition and platinum resistance , , , in inhibition of ATM and sensitivity to IR , and in the activation of Chk1 and Chk2 , , ATM and ATR signaling can occur through the p38MAPK/MK2 pathway in p53-deficient ripasudil receptor . Our findings showing enhanced response of either platinum-based chemotherapy when combined with ATRi or IR when combined with inhibitors of ATR and ATM in GYN cancer cells harboring wild type (A2780, OVCAR3, HELA, SiHa) or mutant (A2780-CP20, KLE, HEC1B) TP53,  suggests the broad applicability of these combinations in the management of ovarian, endometrial, and cervical cancer.
Funding This study was funded by an award from the United States Army Medical Research and Materiel Command (W81XWH-11-2-0131, all authors but CJB) and CA148644 (CJB) from the National Cancer Institute (CJB).
Conflict of interest statement
Introduction Exposure of cells to IR triggers rapid autophosphorylation of serine-1981 that causes dimer dissociation and initiates monomer formation of ATM . It is recruited to sites of damage by the Mre11-Rad50-Nbs1 (MRN) complex , . The ATM kinase phosphorylates a large number of downstream molecules such as γ-H2AX, pATF2, NBS1, Chk2, p53, SMC1 and BRCA1, the deficiency of some of which leads to defective G1/S, G2/M and intra-S checkpoints , , , , , , . In the case of ATR, RPA-coated single-stranded DNA (ssDNA) generated as a result of stalled DNA replication forks or during processing of chromosomal lesions is crucial for the localization of ATR to sites of DNA damage in association with ATR-interacting protein (ATRIP) , , , . Although unlike ATR, ATM is not an essential gene, in humans, ATM deficiencies result in the disease ataxia-telangiectasia (AT) . Cells from AT patients are sensitive to IR but not to UV exposure. These observations suggested that ATM and ATR function in parallel pathways. However, several reports indicate that ATR also responds to DSBs including those produced at stalled replication forks, although the response may be delayed as compared to ATM . The ATR-ATRIP complex is required to maintain the G2/M checkpoint in response to IR and form intra-nuclear foci at IR induced damage sites. It has also been shown that in AT cells, phosphorylation of p53 in response to IR is delayed but not completely abrogated. It has also been shown that ATM activation triggers subsequent ATR activation upon DSB induction , . Recent successes in designing biochemical assays has allowed for the study of the substrate preference of ATM and provided insights of how blunt DNA ends or ends with short overhangs at resected DSBs activate ATM and how the single-strand/double-strand junctions of their substrates are crucial for ATM activation. The processing of these ends by various protein complexes belonging to family of nucleases or helicases like MRN , , Exo1 , , , CtIP ,  and BLM  leads to increased overhang length, which suppresses ATM and leads to subsequent ATR activation . Compared to x-rays or γ-rays, high linear energy transfer (LET) radiation leads to increased clustering and complexity of DSBs , providing an important tool to study altered DNA damage processing. In this study, we investigated the ATM to ATR switch in hTERT-immortalized human skin fibroblasts (82-6) after 1 or 2Gy γ-rays, high LET 600MeV/u 56Fe and 170MeV/u 28Si and moderate LET 250MeV/u 16O particles with LETs of approximately 180, 99 and 25keV/μm, respectively, utilizing Western blotting and immunofluorescence techniques. Live cell imaging experiments were performed with HT22-mouse hippocampal neuronal cell line stably expressing mCherry-53BP1.
Materials and methods
Discussion Both ATM and ATR proteins along with DNA-PKcs are critical molecules for the DNA damage response cascade. Previously, it was considered that ATM exclusively signals DNA DSBs and ATR signals SSBs (single strand breaks) and DNA replication. Although their individual roles have been studied extensively, their functioning in concert with each other has not been looked into systematically until recently . Studies have revealed that DSB-induced ATM activation is required for activation of ATR , , leading to homologous recombination repair pathway. In the absence of activation signal, ATM is known to exist as a dimer . It has been shown that Mre11-Rad50-Nbs1 (MRN complex) activates ATM kinase upon DNA damage induction . A number of studies had attempted to decipher the substrate specificity of these two major kinases. Biochemically, it has been shown previously that ATM and ATR can be activated by blunt-ended as well as resected linear plasmids . Utilizing an extract-based in vitro assay, the work was furthered by deciphering a structure-dependent regulation of the activation of ATM and ATR by double stranded DNA (dsDNA) , implying the results are valid only for DSBs. A two-step model of DNA resection was proposed showing that Mre11/CtIP stimulate end resection by promoting association of BLM or Exo1 with DNA ends , , , . Recently, it was also reported that after initial resection at DSB sites by the MRN complex conducive for ATM binding, ATM is involved in the resection initiation by CtIP which provides enough substrate for limited activation of ATR. ATR then phosphorylates CtIP (T859) promoting its chromatin association and robust resection leading to full blown ATR activation, thus providing evidence for the proposed switching of kinases at DSB sites .