It is generally accepted that DNA damage checkpoint is the mechanism that allows time for DNA damage repair. However, several lines of evidence challenge this paradigm, especially, in the case of G1 checkpoint. The first evidence is the complete difference between the repair kinetics of DNA double-strand breaks (very rapid) and the timing of G1 checkpoint induction (very slow) after ionizing radiation. The second evidence is that inactivation of p53, which is a central player of G1 checkpoint, does not render cells radiosensitive, rather, such cells become radioresistant. Moreover, it was shown that G1 arrest persists almost permanently after irradiation, until the time when most of the initial damage should be repaired and disappear. Therefore, cells should have a mechanism to maintain G1 checkpoint signaling by amplifying the signal from a limited number of damage. In this review, we discuss what is the bona fide role of G1 arrest and how G1 checkpoint signal is maintained long after irradiation.

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The presence of DNA breaks has extensive biochemical implications for the integrity of the genome. It is well established that distinct DNA damage response proteins are recruited to, and accumulate at, sites of genomic lesions, including kinases that initiate multiple DNA damage signaling cascades. The repair of DNA breaks is facilitated by the phosphorylation of H2AX, which organizes DNA damage response factors in the vicinity of the lesion. Metabolism of the DNA breaks occurs in a chromatin environment and modulating chromatin structure is necessary for the fidelity of the DNA damage response. We set out to determine in living cells both how chromatin is remodeled in the presence of DNA breaks and whether the establishment of large sub-cellular DNA damage response domains influences other DNA metabolic processes, such as transcription. Using a photoactivatable histone H2B, we examined the mobility and structure of chromatin immediately after the introduction of DNA breaks. We find that chromatin-containing damaged DNA exhibits limited mobility but undergoes an initial energy-dependent local expansion that occurs independently of H2AX and ATM. We also took advantage of the large copy number, tandem gene arrangement, and spatial organization of ribosomal transcription units as a model system to measure the kinetics of transcription in real time in the presence of DNA breaks. We find that RNA polI inhibition is not the direct result of the physical DNA break but mediated by ATM kinase activity and surrogate DNA repair proteins. We propose that the localized opening of chromatin at DNA breaks establishes an accessible biochemically unique sub-nuclear environment that facilitates DNA damage signaling and repair.

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Eukaryotic chromatin is segregated into highly condensed heterochromatin and comparably relaxed euchromatin. Although heterochromatic gene expression is either transiently or permanently impeded, the integrity of heterochromatic DNA is critical for cell survival as it contributes to the regulation of nuclear architecture, gene expression, ribosome biogenesis, chromosome stability and mitosis. Formed by a plethora of proteins, structurally complex heterochromatin is generally inaccessible to DNA processing enzymes, including those repair factors required to rejoin DNA double strand breaks (DSBs). To be repaired, heterochromatic lesions require the Ataxia Telangiectasia Mutated (ATM) pathway to transiently modify heterochromatic factors surrounding the DSB, relaxing its structure and thereby allowing DNA non-homologous end-joining (NHEJ) to function. Cells deficient for ATM or proteins involved in its signalling cascade repair euchromatic DSBs normally but are unable to resolve lesions within heterochromatin. Depletion of key heterochromatic proteins, including the KAP-1 transcriptional co-repressor, Heterochromatin Protein 1 (HP1) or histone deacetylases 1&2 (HDAC1&2), relieves the requirement for ATM signalling in DSB repair. Importantly, KAP-1 is a highly dose dependent, transient and specific substrate of ATM and the manipulation of KAP-1 phosphorylation regulates heterochromatic DSB repair. We propose that KAP-1 is a critical heterochromatic factor that undergoes specific modifications following DSB formation to promote repair in a manner that allows localised and transient chromatin relaxation but precludes widespread dismantling of the heterochromatic superstructure.

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The G2/M checkpoint is important in preventing cells with unrepaired DNA double strand breaks (DSBs) entering mitosis, an event which is likely to result in genomic instability. We recently reported that checkpoint arrest is maintained until close to completion of DSB repair and that the duration of checkpoint arrest depends on the dose and DSB repair capacity rather than lasting for a fixed period of time. ATM leads to phosphorylation of Chk1/2 in G2 phase following exposure to ionizing radiation. These transducer kinases can phosphorylate and inhibit Cdc25 activity, which is the phosphatase regulating mitotic entry. In this study we dissect three processes that contribute to the maintenance of checkpoint arrest in irradiated G2 phase cells. First, the ATR-Chk1 pathway contributes to maintaining checkpoint arrest, although it is dispensable for the initial activation of checkpoint arrest. Second, ongoing ATM to Chk2 signalling from unrepaired DSBs contributes to checkpoint arrest. This process plays a greater role in a repair defective background. Finally, slow decay of the initially activated Chk2 also contributes to the maintenance of checkpoint arrest. 53BP1 and MDC1 defective cells show an initial checkpoint defect after low doses but are proficient in initial activation of arrest after high doses. After higher radiation doses, however, 53BP1^-/- and MDC1^-/- MEFs fail to maintain checkpoint arrest. Furthermore 53BP1^-/- and MDC1^-/- MEFs display elevated mitotic breakage even after high doses. We show that the defect in the maintenance of checkpoint arrest conferred by 53BP1 and MDC1 deficiency substantially enhances chromosome breakage.

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A multitude of clinically distinct human disorders exist whose underlying cause is a defect in the response to or repair of DNA damage. The clinical spectrum of these conditions provides evidence for the role of the DNA damage response (DDR) in mediating diverse processes such as genomic stability, immune system function and normal human development. Cell lines from these disorders provide a valuable resource to help dissect the consequences of compromised DDR at the molecular level. Ataxia telangiectasia and Rad3-related (ATR) and Ataxia telangiectasia Mutated (ATM) are apical protein kinases that play central roles in coordinating the cells response to DNA damage. Whilst ATM is activated by DNA double strand breaks (DSB's), ATR is activated by single stranded regions of DNA (ssDNA) which can occur, for example, during DNA replication fork stalling. There is significant functional overlap between these two kinases. In fact, they phosphorylate many of the same substrates, including p53 and Brca1. Nevertheless, ATR appears to be essential for embryonic development, unlike ATM. Mutations in ATM result in Ataxia telangiectasia (A-T) a progressive neurological disorder. Interestingly, a hypomorphic mutation in ATR is associated with Seckel syndrome, a clinically distinct disorder to that of A-T. Seckel syndrome is characterised by profound proportionate growth retardation with severe microcephaly. Why defects in these two related kinases should result in such distinct human disorders is unclear. Recently, mutations in Pericentrin/Kendrin (PCNT) have also been demonstrated in Seckel syndrome. PCNT encodes a core structural centrosomal protein. Interestingly, defective PCNT results in impaired ATR-dependent, but not ATM-dependent G2-M cell cycle checkpoint arrest. Using evidence from murine knockout studies and human cell-based work I will discuss the biological impact of compromised ATR-pathway function with the aim of trying to understand the link between genotype-phenotype in this context.

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Recently we identified β-galactoside α(2, 6)-sialyltransferase (ST6Gal I) as a candidate biomarker for ionizing radiation. The expression of ST6Gal I and the level of protein sialylation increased following radiation exposure in a dose-dependent manner. We also found that radiation induced ST6Gal I cleavage and the cleaved form of ST6Gal I was soluble and secreted. Sialylation of integrin β1, a glycosylated cell surface protein, was stimulated by irradiation and this increased its protein stability. Overexpression of ST6Gal I in SW480 colon cancer cells that initially showed a low enzyme activity of ST6Gal I increased the sialylation of integrin β1 and also increased the stability of the protein. Inhibition of sialylation by transfection with neuramidase or by treatment with short interfering (si) RNA targeting ST6Gal I (Si-ST6Gal I) reversed the effects of ST6Gal I expression. In addition, ST6Gal I overexpression increased clonogenic survival following radiation exposure and reduced radiation-induced cell death and caspase 3 activation. In conclusion, we suggest that exposure to ionizing radiation was found to increase sialylation of glycoproteins such as integrin β1 by inducing the expression of ST6 Gal I, and finally protein sialylation contributed to cellular radiation resistance.

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Genesis of new cells in the mammalian brain has previously been regarded as a negligible event; an assumption that long prevented our understanding in the development of neoplasias. The recent discovery of perpetual lineages derived from neural stem cells has resulted in a new approach to studying the cellular behaviour of potential cancer stem cells in the brain. Glioblastoma multiforme (GBM), the most aggressive and lethal brain tumour is derived from such a group of cancerous stem cells known as glioma stem cells. GBM cells are impervious to conventional therapies such as surgical resection and ionizing radiation because of their pluripotent and radioresistant properties. Thus in our study, we aim to investigate whether a combination of chemo- and radio- therapies is an effective treatment for glioma stem cells. The study utilizes a specific kinase inhibitor (ATMi) of the ATM (Ataxia-telangiectasia mutated) protein which is an essential protein in DNA-damage responses. In the presence of both low dose radiation and ATMi, glioma stem cells have rapid onset of cell death and reduction in growth. Since DNA damage can be inherited through cell division, accumulated DNA breaks in later generations may also lead to cell death. The limitation of conventional radiation therapy is that administration of fractionated (low) doses to reduce any potential harm to the surrounding healthy cells in the brain outweighs the benefits of high radiation doses to induce actual arrest in the propagation of malignant cells. Our study demonstrates a benefit in using low dose radiation combined with chemotherapy resulting in a reduction in malignancy of glioma stem cells.

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Ataxia Telangiectasia Mutated Protein (ATM) is one of the first DNA damage sensors and is involved in telomere repair. Telomeres help maintain the stability of our chromosomes by protecting their ends from degradation. AT patients lacking the gene ATM are susceptible to acquire chromosomal anomalies and show heightened susceptibility to cancer. Here we show that cells from AT patients display considerable telomere attrition. Further, induced DNA damage and genomic instability were found to be more in DNA repair deficient ATM^-/- cells (treated and untreated) than in normal cells. Results demonstrate that the cells ATM- deficient (heterozygous and homozygous) cells are sensitive to arsenite- and radiation-induced oxidative stress. Elevated numbers of chromosome alterations was seen in arsenic-treated and irradiated ATM^-/- cells. The results might help in understanding the extent of susceptibility of AT patients to oxidative stress.

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The microenvironment is consisted both of soluble factors involving growth factors and of insoluble factors. Stroma cells contribute to form the microenvironment through a secretion of these factors. Fibroblast, which is known as stroma cells, also secretes various soluble/ insoluble factors, but the secretion level is significantly up-regulated when they reach to a finite replicative lifespan. Recent accumulating studies not only in vitro but also in vivo provide us that secreted proteins from senescent cells promote pro-survival pathway in bystander cells, especially tumor cells rather than normal cells. Since various stresses including ionizing radiation (IR) prematurely induces cellular senescent stage, called Stress-Induced Premature Senescence (SIPS), there is the possibility that the secretion pathway in cells undergoing SIPS is also activated. Here, we propose that pro-survival factor is secreted from SIPS cells to provide the stress-resistant microenvironment.

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Emerging evidence suggests the existence of a new mode of epidermal growth factor receptor (EGFR) signalling in which activated EGFR undergoes nuclear translocation. We provide evidence that the nuclear EGFR transport is a stress specific cellular reaction, which is linked to src dependent EGFR internalization into caveolae. Internalized EGFR is sorted into a peri-nuclear localization. This peri-nuclear EGFR may serve as a reservoir for nuclear transport which is regulated by PKC ε. Nuclear EGFR induces transcription of genes essential for cell proliferation and cell cycle regulation. In addition, nuclear EGFR has physical contact with compounds of the DNA repair machinery and is involved in removal of DNA-damage. The exact role of nuclear EGFR has to be elucidated in future experiments.

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Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. We have shown that irradiating human mammary epithelial cells (HMEC) cultured with that transforming growth factor β1 (TGFβ) can generate a persistent phenotype in daughter cells characterized by spindle cell morphology, increased mesenchymal markers, decreased epithelial markers and increased cellular motility and invasion, which are hallmarks of epithelial to mesenchymal transition (EMT). Neither radiation nor TGFβ alone elicited EMT, although IR increased chronic TGFβ signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/MAPK signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFβ-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFβ, and inhibition of Erk activation, blocked the EMT phenotype. Preliminary studies suggest that eqi-toxic doses of sparsely and densely ionizing radiation resulted in comparable EMT when cells were cultivated in the presence of TGFβ, Furthermore radiation dose response studies show that this effect has a very low threshold in that a single exposure of 3-200 cGy radiation elicits the ‘same’ phenotypic switch, which is consistent with non-targeted effects. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to radiation carcinogenesis.

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Radiation-induced bystander effect is defined as the induction of biological effects in cells that are not directly traversed by radiation, but merely in the presence of cells that are. Although radiation induced bystander effects have been well defined in a variety of in vitro models using a range of endpoints including clonogenic survival, mutations, neoplastic transformation, apoptosis, micronucleus, chromosomal aberrations and DNA double strand breaks, the mechanism(s) as well as the presence of such an effect in vivo are not well described. In this review, we summarize the evidence of radiation induced bystander effect in various in vivo systems including rodents, fish and plants. Many biological endpoints such as epigenetic changes, DNA damage, miRNA, apoptosis, cell proliferation, gene expression and tumorgenesis have been demonstrated in the non-targeted regions in vivo. Although the bystander effect is evolutionarily conserved in rodent systems, the bystander response depends on gender, tissue and strain. However, the studies about mechanism of radiation induced bystander effect in vivo are still limited.

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