Serah Choi

Ionizing Radiation Induces Ataxia Telangiectasia Mutated‐Dependent Checkpoint Signaling and G2 But Not G1 Cell Cycle Arrest in Pluripotent Human Embryonic Stem Cells

Human embryonic stem (ES) cells are highly sensitive to environmental insults including DNA damaging agents, responding with high levels of apoptosis. To understand the response of human ES cells to DNA damage, we investigated the function of the ataxia telangiectasia mutated (ATM) DNA damage signaling pathway in response to γ‐irradiation. Here, we demonstrate for the first time in human ES cells that ATM kinase is phosphorylated and properly localized to the sites of DNA double‐strand breaks within 15 minutes of irradiation. Activation of ATM kinase resulted in phosphorylation of its downstream targets: Chk2, p53, and Nbs1. In contrast to murine ES cells, Chk2 and p53 were localized to the nucleus of irradiated human ES cells. We further show that irradiation resulted in a temporary arrest of the cell cycle at the G2, but not G1, phase. Human ES cells resumed cycling approximately 16 hours after irradiation, but had a fourfold higher incidence of aberrant mitotic figures compared to nonirradiated cells. Finally, we demonstrate an essential role of ATM in establishing G2 arrest since inhibition with the ATM‐specific inhibitor KU55933 resulted in abolishment of G2 arrest, evidenced by an increase in the number of cycling cells 2 hours after irradiation. In summary, these results indicate that human ES cells activate the DNA damage checkpoint, resulting in an ATM‐dependent G2 arrest. However, these cells re‐enter the cell cycle with prominent mitotic spindle defects. STEM CELLS 2009;27:1822–1835

Adenoviral proteins mimic nutrient/growth signals to activate the mTOR pathway for viral replication

Like tumor cells, DNA viruses have had to evolve mechanisms that uncouple cellular replication from the many intra‐ and extracellular factors that normally control it. Here we show that adenovirus encodes two proteins that activate the mammalian target of rapamycin (mTOR) for viral replication, even under nutrient/growth factor‐limiting conditions. E4‐ORF1 mimics growth factor signaling by activating PI3‐kinase, resulting in increased Rheb.GTP loading and mTOR activation. E4‐ORF4 is redundant with glucose in stimulating mTOR, does not affect Rheb.GTP levels and is the major mechanism whereby adenovirus activates mTOR in quiescent primary cells. We demonstrate that mTOR is activated through a mechanism that is dependent on the E4‐ORF4 protein phosphatase 2A‐binding domain. We also show that mTOR activation is required for efficient S‐phase entry, independently of E2F activation, in adenovirus‐infected quiescent primary cells. These data reveal that adenovirus has evolved proteins that activate the mTOR pathway, irrespective of the cellular microenvironment, and which play a requisite role in viral replication.

Mitochondrial hyperfusion induced by loss of the fission protein Drp1 causes ATM-dependent G2/M arrest and aneuploidy through DNA replication stress

Summary Mitochondrial fission and fusion cycles are integrated with cell cycle progression. In this paper, we demonstrate that the inhibition of mitochondrial fission protein Drp1 causes an unexpected delay in G2/M cell cycle progression and aneuploidy. In investigating the underlying molecular mechanism, we revealed that inhibiting Drp1 triggers replication stress, which is mediated by a hyperfused mitochondrial structure and unscheduled expression of cyclin E in the G2 phase. This persistent replication stress then induces an ATM-dependent activation of the G2 to M transition cell cycle checkpoint. Knockdown of ATR, an essential kinase in preventing replication stress, significantly enhanced DNA damage and cell death of Drp1-deficienct cells. Persistent mitochondrial hyperfusion also induces centrosomal overamplification and chromosomal instability, which are causes of aneuploidy. Analysis using cells depleted of mitochondrial DNA revealed that these events are not mediated by the defects in mitochondrial ATP production and reactive oxygen species (ROS) generation. Thus dysfunctional mitochondrial fission directly induces genome instability by replication stress, which then initiates the DNA damage response. Our findings provide a novel mechanism that contributes to the cellular dysfunction and diseases associated with altered mitochondrial dynamics.

Irreversible chromosome damage accumulates rapidly in the absence of ATM kinase activity

Mutations in the ATM kinase cause the neurodegenerative disorder ataxia telangiectasia (A-T) and affected individuals are exquisitely radiation-sensitive and cancer-prone. Cells derived from A-T individuals contain chromosome aberrations and exhibit profound cellular radiosensitivity. ATM is an apical kinase critical for the activation of cell cycle checkpoints and the induction of apoptosis in irradiated cells. However, defects in these pathways are insufficient to account for the chromosomal instability seen in A-T cells. We show here that the small molecule KU55933 can be used as a “molecular switch” to selectively and transiently inhibit ATM kinase activity in cells. We subsequently show that the cellular radiosensitization seen when ATM kinase activity is inhibited for one hour following exposure to γ-rays, accounts for over 70% of the total cellular radiosensitization seen when ATM kinase activity is inhibited for 17h. Finally, we show that inhibition of ATM kinase activity for one hour following exposure to irradiation doubles the number of chromosome aberrations occurring in late-S- and G2-, but not M-phase, cells. These observations are unexpected and suggest that irreversible chromosome damage accumulates very rapidly when ATM kinase activity is transiently inhibited following irradiation. We propose that we have revealed an essential, yet previously undescribed, role for ATM kinase in suppressing chromosomal instability

Transient ATM Kinase Inhibition Disrupts DNA Damage-Induced Sister Chromatid Exchange

Radiation triggers distinct responses in cells in which the kinase ATM is transiently inhibited versus those that have adapted to its loss. Time and Tools for Repair ATM is a kinase activated in response to DNA damage, such as that caused by exposure of cells to ionizing radiation. ATM triggers cell cycle checkpoints that allow cells time to repair the damage before proceeding to replicate their DNA. With reversible inhibitors, White et al. show that ATM and DNA-PK, another kinase induced by DNA damage, initiated distinct mechanisms of DNA repair in cells exposed to ionizing radiation. Despite activation of the cell cycle checkpoint, cells in which either of these two kinases was inhibited for as little as 1 hour 15 min after exposure to ionizing radiation exhibited reduced survival and accumulated persistent chromosomal damage. Furthermore, the authors show that ATM-deficient cells adapt and can initiate repair by sister chromatid exchange through an ATM-independent mechanism. Cells derived from ataxia telangiectasia (A-T) patients exhibit defective cell cycle checkpoints because of mutations in the gene encoding ATM (ataxia telangiectasia mutated). After exposure to ionizing radiation (IR), A-T cells exhibit sensitivity to IR-induced cellular damage that results in increased chromosome aberrations and cell death (radiosensitivity). ATM is a member of a family of kinases that become activated in response to DNA damage. We showed that even transient inhibition of ATM kinase for 1 hour, initiated 15 minutes after cellular irradiation, resulted in an accumulation of persistent chromosome aberrations and increased cell death. Using reversible inhibitors of DNA-PK (DNA-dependent protein kinase), another kinase involved in responding to DNA damage, and ATM, we showed that these two kinases acted through distinct DNA repair mechanisms: ATM resolved DNA damage through a mechanism involving sister chromatid exchange (SCE), whereas DNA-PK acted through nonhomologous end joining. Furthermore, because DNA damage–induced SCE occurred in A-T fibroblasts that lack functional ATM protein, and the inhibitors of ATM kinase had no effect on DNA damage–induced SCE in A-T fibroblasts, we showed that the consequences of short-term inhibition of the kinase activity of ATM and adaptation to ATM protein disruption were distinct. This suggests that A-T fibroblasts have adapted to the loss of ATM and have alternative mechanisms to initiate SCE.

Lack of DNA Polymerase θ (POLQ) Radiosensitizes Bone Marrow Stromal Cells In Vitro and Increases Reticulocyte Micronuclei after Total-Body Irradiation

Abstract Mammalian POLQ (pol θ) is a specialized DNA polymerase with an unknown function in vivo. Roles have been proposed in chromosome stability, as a backup enzyme in DNA base excision repair, and in somatic hypermutation of immunoglobulin genes. The purified enzyme can bypass AP sites and thymine glycol. Mice defective in POLQ are viable and have been reported to have elevated spontaneous and radiation-induced frequencies of micronuclei in circulating red blood cells. To examine the potential roles of POLQ in hematopoiesis and in responses to oxidative stress responses, including ionizing radiation, bone marrow cultures and marrow stromal cell lines were established from Polq+/+ and Polq−/− mice. Aging of bone marrow cultures was not altered, but Polq−/− cells were more sensitive to γ radiation than were Polq+/+ cells. The D0 was 1.38 ± 0.06 Gy for Polq+/+ cells compared to 1.27 ± 0.16 and 0.98 ± 0.10 Gy (P  =  0.032) for two Polq−/− clones. Polq−/− cells were moderately more sensitive to bleomycin than Polq+/+ cells and were not hypersensitive to paraquat or hydrogen peroxide. ATM kinase activation appeared to be normal in γ-irradiated Polq−/− cells. Inhibition of ATM kinase activity increased the radiosensitivity of Polq+/+ cells slightly but did not affect Polq−/− cells. Polq−/− mice had more spontaneous and radiation-induced micronucleated reticulocytes than Polq+/+ and +/− mice. The sensitivity of POLQ-defective bone marrow stromal cells to ionizing radiation and bleomycin and the increase in micronuclei in red blood cells support a role for this DNA polymerase in cellular tolerance of DNA damage that can lead to double-strand DNA breaks.

Adenovirus Overrides Cellular Checkpoints for Protein Translation

mTOR is a critical regulator of protein translation, and plays an important role in controlling cellular replication. Recent studies indicate that nutrient and growth factor mediated activation of mTOR is deregulated in human cancer, and therefore represents an attractive tumor target. However, activation of mTOR is a complex process that is not yet fully understood. DNA viruses and tumor cells often perturb similar cellular pathways to facilitate their replication. In a recent study, we used adenovirus as a novel tool to probe the mechanisms underlying the inappropriate activation of mTOR in quiescent primary cells. These studies revealed that adenovirus encodes two viral proteins, E4-ORF1 and E4-ORF4, which activate mTOR, even in the absence of nutrient/growth factor signals, and which play a role in promoting viral replication. E4-ORF1 mimics growth factor signaling to mTOR by activating PI3-kinase, whereas E4-ORF4, which binds and relocalizes PP2A, can substitute for glucose mediated activation of mTOR. We discuss insights from this study, together with the similarities that may exist between viruses and tumor cells with respect to the mechanistic and functional requirements for mTOR activation in driving their aberrant DNA replication.

Inhibition of ATM kinase activity does not phenocopy ATM protein disruption: implications for the clinical utility of ATM kinase inhibitors.

Biallelic mutations in ataxia-telangiectasia mutated (ATM), which encodes for a protein kinase, cause ataxia telangiectasia (A-T). A-T is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation (IR). A-T patients typically lack both detectable ATM protein and ATM kinase activity, and small molecule inhibitors of ATM kinase activity have been developed as strategies to improve radiotherapy for the treatment of cancers. As predicted, inhibition of ATM kinase activity is sufficient to radiosensitize cells. However, we recently showed that inhibition of ATM kinase activity disrupts DNA damage-induced sister chromatid exchange (SCE). This result was unanticipated since SCE is normal in A-T cells that lack detectable ATM protein. In these studies, we showed, for the first time, that the consequences of inhibition of ATM kinase activity and adaptation to ATM protein disruption are distinct. Here, we discuss the mechanistic implications of this finding for the function of ATM at the replication fork and the clinical utility of ATM kinase inhibitors.

Quantification of nanoscale nuclear refractive index changes during the cell cycle

Intrigued by our recent finding that the nuclear refractive index is significantly increased in malignant cells and histologically normal cells in clinical histology specimens derived from cancer patients, we sought to identify potential biological mechanisms underlying the observed phenomena. The cell cycle is an ordered series of events that describes the intervals of cell growth, DNA replication, and mitosis that precede cell division. Since abnormal cell cycles and increased proliferation are characteristic of many human cancer cells, we hypothesized that the observed increase in nuclear refractive index could be related to an abundance or accumulation of cells derived from cancer patients at a specific point or phase(s) of the cell cycle. Here we show that changes in nuclear refractive index of fixed cells are seen as synchronized populations of cells that proceed through the cell cycle, and that increased nuclear refractive index is strongly correlated with increased DNA content. We therefore propose that an abundance of cells undergoing DNA replication and mitosis may explain the increase in nuclear refractive index observed in both malignant and histologically normal cells from cancer patients. Our findings suggest that nuclear refractive index may be a novel physical parameter for early cancer detection and risk stratification.

Intraesophageal manganese superoxide dismutase-plasmid liposomes ameliorates novel total-body and thoracic radiation sensitivity of NOS1-/- mice.

Abstract The effect of deletion of the nitric oxide synthase 1 gene (NOS1−/−) on radiosensitivity was determined. In vitro, long-term cultures of bone marrow stromal cells derived from NOS1−/− were more radioresistant than cells from C57BL/6NHsd (wild-type), NOS2−/− or NOS3−/− mice. Mice from each strain received 20 Gy thoracic irradiation or 9.5 Gy total-body irradiation (TBI), and NOS1−/− mice were more sensitive to both. To determine the etiology of radiosensitivity, studies of histopathology, lower esophageal contractility, gastrointestinal transit, blood counts, electrolytes and inflammatory markers were performed; no significant differences between irradiated NOS1−/− and control mice were found. Video camera surveillance revealed the cause of death in NOS1−/− mice to be grand mal seizures; control mice died with fatigue and listlessness associated with low blood counts after TBI. NOS1−/− mice were not sensitive to brain-only irradiation. MnSOD-PL therapy delivered to the esophagus of wild-type and NOS1−/− mice resulted in equivalent biochemical levels in both; however, in NOS1−/− mice, MnSOD-PL significantly increased survival after both thoracic and total-body irradiation. The mechanism of radiosensitivity of NOS1−/− mice and its reversal by MnSOD-PL may be related to the developmental esophageal enteric neuronal innervation abnormalities described in these mice.