MAB3806 Sigma-AldrichAnti-ATM phosphoSer1981 Antibody, clone 10H11.E12

FOXM1 is implicated in genotoxic drug resistance but its mechanism of action remains elusive. We show here that FOXM1-depletion can sensitize breast cancer cells and mouse embryonic fibroblasts (MEFs) into entering epirubicin-induced senescence, with the loss of long-term cell proliferation ability, the accumulation of γH2AX foci, and the induction of senescence-associated β-galactosidase activity and cell morphology. Conversely, reconstitution of FOXM1 in FOXM1-deficient MEFs alleviates the accumulation of senescence-associated γH2AX foci. We also demonstrate that FOXM1 regulates NBS1 at the transcriptional level through an forkhead response element on its promoter. Like FOXM1, NBS1 is overexpressed in the epirubicin-resistant MCF-7Epi(R) cells and its expression level is low but inducible by epirubicin in MCF-7 cells. Consistently, overexpression of FOXM1 augmented and FOXM1 depletion reduced NBS1 expression and epirubicin-induced ataxia-telangiectasia mutated (ATM)phosphorylation in breast cancer cells. Together these findings suggest that FOXM1 increases NBS1 expression and ATM phosphorylation, possibly through increasing the levels of the MRN(MRE11/RAD50/NBS1) complex. Consistent with this idea, the loss of P-ATM induction by epirubicin in the NBS1-deficient NBS1-LBI fibroblasts can be rescued by NBS1 reconstitution. Resembling FOXM1, NBS1 depletion also rendered MCF-7 and MCF-7Epi(R) cells more sensitive to epirubicin-induced cellular senescence. In agreement, the DNA repair-defective and senescence phenotypes in FOXM1-deficent cells can be effectively rescued by overexpression of NBS1. Moreover, overexpression of NBS1 and FOXM1 similarly enhanced and their depletion downregulated homologous recombination (HR) DNA repair activity. Crucially, overexpression of FOXM1 failed to augment HR activity in the background of NBS1 depletion, demonstrating that NBS1 is indispensable for the HR function of FOXM1. The physiological relevance of the regulation of NBS1 expression by FOXM1 is further underscored by the strong and significant correlation between nuclear FOXM1 and total NBS1 expression in breast cancer patient samples, further suggesting that NBS1 as a key FOXM1 target gene involved in DNA damage response, genotoxic drug resistance and DNA damage-induced senescence.

Histone deacetylase (HDAC) inhibitors have been used to promote neuronal survival and ameliorate neurological dysfunction in a host of neurodegenerative disease models. The precise molecular mechanisms whereby HDAC inhibitors prevent neuronal death are currently the focus of intensive research. Here we demonstrate that HDAC inhibition prevents DNA damage-induced neurodegeneration by modifying the acetylation pattern of the tumor suppressor p53, which decreases its DNA-binding and transcriptional activation of target genes. Specifically, we identify that acetylation at K382 and K381 prevents p53 from associating with the pro-apoptotic PUMA gene promoter, activating transcription, and inducing apoptosis in mouse primary cortical neurons. Paradoxically, acetylation of p53 at the same lysines in various cancer cell lines leads to the induction of PUMA expression and death. Together, our data provide a molecular understanding of the specific outcomes of HDAC inhibition and suggest that strategies aimed at enhancing p53 acetylation at K381 and K382 might be therapeutically viable for capturing the beneficial effects in the CNS, without compromising tumor suppression.

The genome is constantly exposed to DNA damage agents, leading up to as many as 1 million individual lesions per cell per day. Cells have developed a variety of DNA damage repair (DDR) mechanisms to respond to harmful effects of DNA damage. Failure to repair the damaged DNA causes genomic instability and, as a result, leads to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers, thus providing a great opportunity for cancer therapy by developing anticancer agents that work by synthetic lethality-based mechanisms or enhancing the clinical efficacy of radiotherapy and existing chemotherapies. Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to DNA double-strand breaks. Ionizing radiation causes double-strand breaks and induces rapid ATM autophosphorylation on serine 1981 that initiates ATM kinase activity. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage-response pathways, most notably cell-cycle checkpoints. We describe here the development and validation of a high-throughput imaging assay measuring levels of phospho-ATM Ser1981 in HT29 cells after exposure to ionizing radiation. We also examined activation of downstream ATM effectors and checked specificity of the endpoint using known inhibitors of DNA repair pathways.

Protein phosphatase 2A (PP2A) is a family of heterotrimeric protein phosphatases that has a multitude of functions inside the cell, acting through various substrate targets in cell-signaling pathways. Recent evidence suggests that a subset of PP2A holoenzymes function as tumor suppressors and one particular family of B subunits, B56, are implicated in this function. However, the regulatory mechanisms that govern activation of B56-PP2A tumor-suppressive function have not been elucidated. In the present study, we demonstrate that ataxia-telangiectasia mutated (ATM) directly phosphorylates and specifically regulates B56?3, B56?2 and B56?, after DNA damage. We further show that phosphorylation of B56?3 at Ser510 leads to an increase in B56?3-PP2A complexes, and direction of PP2A phosphatase activity toward the substrate p53, activating its tumor-suppressive functions. In addition, we found that under cell growth conditions B56?3 is kept at low levels through the actions of the E3 ubiquitin ligase MDM2, and, importantly, phosphorylation of B56?3 by ATM leads to upregulation of the protein by blocking MDM2-mediated B56?3 ubiquitination. Finally, we show that Ser510 phosphorylation significantly enhances the ability of B56?3 to inhibit cell proliferation and anchorage-independent growth. These results provide mechanistic insight into the regulation of PP2A tumor-suppressive function, and suggest a model for parallel regulation of p53 and B56?3.

The ATM protein kinase, mutations of which are associated with the human disease ataxia-telangiectasia, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer or higher-order multimer, with the kinase domain bound to a region surrounding serine 1981 that is contained within the previously described 'FAT' domain. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. Most ATM molecules in the cell are rapidly phosphorylated on this site after doses of radiation as low as 0.5 Gy, and binding of a phosphospecific antibody is detectable after the introduction of only a few DNA double-strand breaks in the cell. Activation of the ATM kinase seems to be an initiating event in cellular responses to irradiation, and our data indicate that ATM activation is not dependent on direct binding to DNA strand breaks, but may result from changes in the structure of chromatin.