The double-strand break (DSB) is believed to be one of the most severe types of DNA damage, and if left unrepaired is lethal to the cell. Several different types of repair act on the DSB. The most important in mammalian cells are nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR). NHEJ is the predominant type of DSB repair in mammalian cells, as opposed to lower eucaryotes, but HRR has recently been implicated in critical cell signaling and regulatory functions that are essential for cell viability. Whereas NHEJ repair appears constitutive, HRR is regulated by the cell cycle and inducible signal transduction pathways. More is known about the molecular details of NHEJ than HRR in mammalian cells. This review focuses on the mechanisms and regulation of DSB repair in mammalian cells, the signaling pathways that regulate these processes and the potential crosstalk between NHEJ and HRR, and between repair and other stress-induced pathways with emphasis on the regulatory circuitry associated with the ataxia telangiectasia mutated (ATM) protein.

Astrocyte elevated gene-1 (AEG-1) was initially identified as an HIV-1-and tumor necrosis factor A (TNF-A)-inducible transcript in primary human fetal astrocytes by a rapid subtraction hybridization approach. Interestingly, AEG-1 expression is elevated in subsets of breast cancer, glioblastoma multiforme and melanoma cells and AEG-1 cooperates with Ha-ras to promote transformation of immortalized melanocytes. Activation of the transcription factor nuclear factor KB (NF-KB), a TNF-A downstream signaling component, is associated with several human illnesses, including cancer, and NF-KB controls the expression of multiple genes involved in tumor progression and metastasis. We now document that AEG-1 is a significant positive regulator of NF-KB. Enhanced expression of AEG-1 via a replication-incompetent adenovirus (Ad.AEG-1) in HeLa cells markedly increased binding of the transcriptional activator p50/p65 complex of NF-KB. The NF-KB activation induced by AEG-1 corresponded with degradation of IKBA and nuclear translocation of p65 that resulted in the induction of NF-KB downstream genes. Infection with an adenovirus expressing the mt32IKBA superrepressor (Ad.IKBA-mt32), which prevents p65 nuclear translocation, inhibited AEG-1-induced enhanced agar cloning efficiency and increased matrigel invasion of HeLa cells. We also document that TNF-A treatment resulted in nuclear translocation of both AEG-1 and p65 wherein these two proteins physically interacted, suggesting a potential mechanism by which AEG-1 could activate NF-KB. Our findings suggest that activation of NF-KB by AEG-1 could represent a key molecular mechanism by which AEG-1 promotes anchorage-independent growth and invasion, two central features of the neoplastic phenotype.

Ataxia telangiectasia (A-T) mutated (ATM) is critical for cell cycle checkpoints and DNA repair. Thus, specific small molecule inhibitors targeting ATM could perhaps be developed into efficient radiosensitizers. Recently, a specific inhibitor of the ATM kinase, KU-55933, was shown to radiosensitize human cancer cells. Herein, we report on an improved analogue of KU-55933 (KU-60019) with K i and IC 50 values half of those of KU-55933. KU-60019 is 10-fold more effective than KU-55933 at blocking radiation-induced phosphorylation of key ATM targets in human glioma cells. As expected, KU-60019 is a highly effective radiosensitizer of human glioma cells. A-T fibroblasts were not radiosensitized by KU-60019, strongly suggesting that the ATM kinase is specifically targeted. Furthermore, KU-60019 reduced basal S473 AKT phosphorylation, suggesting that the ATM kinase might regulate a protein phosphatase acting on AKT. In line with this finding, the effect of KU-60019 on AKT phosphorylation was countered by low levels of okadaic acid, a phosphatase inhibitor, and A-T cells were impaired in S473 AKT phosphorylation in response to radiation and insulin and unresponsive to KU-60019. We also show that KU-60019 inhibits glioma cell migration and invasion in vitro, suggesting that glioma growth and motility might be controlled by ATM via AKT. Inhibitors of MEK and AKT did not further radiosensitize cells treated with KU-60019, supporting the idea that KU-60019 interferes with prosurvival signaling separate from its radiosensitizing properties. Altogether, KU-60019 inhibits the DNA damage response, reduces AKT phosphorylation and prosurvival signaling, inhibits migration and invasion, and effectively radiosensitizes human glioma cells. [Mol Cancer Ther 2009;8(10):2894-902]

Subtraction hybridization identified melanoma differentiationassociated gene-7 (mda-7) as a gene induced during terminal differentiation in human melanoma cells. On the basis of structure, chromosomal localization and cytokine-like properties, mda-7 is classified as IL-24. Administration of mda-7͞IL-24 by means of a replication-incompetent adenovirus (Ad.mda-7) induces apoptosis selectively in diverse human cancer cells without inducing harmful effects in normal fibroblast or epithelial cells. The present studies investigated the mechanism underlying this differential apoptotic effect. Infection of melanoma cells, but not normal immortal melanocytes, with Ad.mda-7 induced a time-and dose-dependent increase in expression, mRNA and protein, of a family of growth arrest and DNA damage (GADD)-inducible genes, which correlated with induction of apoptosis. Among the members of the GADD family of genes, GADD153, GADD45␣, and GADD34 displayed marked, and GADD45␥ showed minimal induction. Treatment of melanoma cells with SB203580, a selective inhibitor of the p38 mitogen-activated protein kinase (MAPK) pathway, effectively inhibited Ad.mda-7-induced apoptosis. Additional support for an involvement of the p38 MAPK pathway in Ad.mda-7-mediated apoptosis was documented by using an adenovirus expressing a dominant negative mutant of p38 MAPK. Infection with Ad.mda-7 increased the phosphorylation of p38 MAPK and heat shock protein 27 in melanoma cells but not in normal immortal melanocytes. In addition, SB203580 effectively inhibited Ad.mda-7-mediated induction of the GADD family of genes in a time-and dosedependent manner, and it effectively blocked Ad.mda-7-mediated down-regulation of the antiapoptotic protein BCL-2. Inhibition of GADD genes by an antisense approach either alone or in combination also effectively blocked Ad.mda-7-induced apoptosis in melanoma cells. These results support the hypothesis that Ad.mda-7 mediates induction of the GADD family of genes by means of the p38 MAPK pathway, thereby resulting in the selective induction of apoptosis in human melanoma cells. melanoma differentiation-associated gene-7 ͉ growth arrest DNA damage-inducible gene family ͉ programmed cell death

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Exposure of tumor cells to clinically relevant doses of ionizing radiation causes DNA damage as well as mitochondria-dependent generation of reactive oxygen species. DNA damage causes activation of ataxia telangiectasia mutated and ataxia telangiectasia mutated and Rad3-related protein, which induce cell cycle checkpoints and also modulate the activation of prosurvival and proapoptotic signaling pathways, such as extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH 2

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH 2 -terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0 -10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90 -240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor ␣ receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor ␣ (TGF␣) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGF␣ cleavage 120 -180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGF␣. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGF␣. Neutralization of TGF␣ function by an anti-TGF␣ antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGF␣-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.© 1999 by The American Society for Cell Biology 2493 INTRODUCTIONIonizing radiation has been shown to activate multiple signaling pathways within cells in vitro, which can lead to either increased cell death or increased proliferation depending on the cell type, the radiation dose, and the culture conditions (Xia et al., 1995;Rosette and Karin, 1996;Santana et al., 1996;Chmura et al., 1997;Schmidt-Ullrich et al., 1997;Carter et al., 1998;Haimovitz-Friedman, 1998;Kavanagh et al., 1998). Recently, a novel cellular target for ionizing radiation has been shown to be the ...