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The recognition and repair of DNA double-strand breaks (DSBs) is a complex process that draws upon a multitude of proteins. This is not surprising since this is a lethal lesion if left unrepaired and also contributes to genome instability and the consequential risk of cancer and other pathologies. Some of the key proteins that recognize these breaks in DNA are mutated in distinct genetic disorders that predispose to agent sensitivity, genome instability, cancer predisposition and/or neurodegeneration. These include members of the Mre11 complex (Mre11/Rad50/Nbs1) and ataxia-telangiectasia (A-T) mutated (ATM), mutated in the human genetic disorder A-T. The mre11 (MRN) complex appears to be the major sensor of the breaks and subsequently recruits ATM where it is activated to phosphorylate in turn members of that complex and a variety of other proteins involved in cell-cycle control and DNA repair. The MRN complex is also upstream of ATM and ATR (A-T-mutated and rad3-related) protein in responding to agents that block DNA replication. To date, more than 30 ATM-dependent substrates have been identified in multiple pathways that maintain genome stability and reduce the risk of disease. We focus here on the relationship between ATM and the MRN complex in recognizing and responding to DNA DSBs.

Bloom syndrome and ataxia-telangiectasia are autosomal recessive human disorders characterized by immunodeficiency, genome instability and predisposition to develop cancer. Recent data reveal that the products of these two genes, BLM and ATM, interact and function together in recognizing abnormal DNA structures. To investigate the function of these two molecules in DNA damage recognition, we generated double knockouts of ATM(-/-) BLM(-/-) in the DT40 chicken B-lymphocyte cell line. The double mutant cells were viable and exhibited a variety of characteristics of both ATM(-/-) and BLM(-/-) cells. There was no evidence for exacerbation of either phenotype; however, the more extreme radiosensitivity seen in ATM(-/-) and the elevated sister chromatid exchange seen in BLM(-/-) cells were retained in the double mutants. These results suggest that ATM and BLM have largely distinct roles in recognizing different forms of damage in DNA, but are also compatible with partially overlapping functions in recognizing breaks in radiation-damaged DNA.

Cells from patients with the genetic disorder ataxia-telangiectasia (A-T) are hypersensitive to ionizing radiation and radiomimetic agents, both of which generate reactive oxygen species capable of causing oxidative damage to DNA and other macromolecules. We describe in A-T cells constitutive activation of pathways that normally respond to genotoxic stress, Basal levels of p53 and p21(WAF1/CIP1), phosphorylation on serine 15 of p53, and the Tyr15-phosphorylated form of cdc2 are chronically elevated in these cells. Treatment of A-T cells with the antioxidant alpha -lipoic acid significantly reduced the levels of these proteins, pointing to the involvement of reactive oxygen species in their chronic activation. These findings suggest that the absence of functional ATM results in a mild but continuous state of oxidative stress, which could account for several features of the pleiotropic phenotype of A-T.

The signaling pathway through which ionizing radiation induces NF-kappaB activation is not fully understood. IkappaB-alpha, an inhibitory protein of NF-kappaB mediates the activation of NF-kappaB in response to various stimuli, including cytokines, mitogens, oxidants and other stresses. We have now identified an ionizing radiation-induced signaling pathway that is independent of TNF-alpha. IkappaB-alpha degradation is rapid in response to TNF-alpha induction, but it is absent in response to ionizing radiation exposure in cells from individuals with ataxia-telangiectasia (AT). Overexpression of wild-type ATM, the product of the gene defective in AT patients, restores radiation-induced degradation of IkappaB-alpha. Furthermore, phosphorylation of IkappaB-alpha by immunoprecipitated ATM kinase is increased in control fibroblasts and transfected AT cells following ionizing radiation exposure. These data provide support for a novel ionizing radiation-induced signaling pathway for activation of NF-kappaB and a molecular basis for the sensitivity of AT patients to oxidative stresses.

Loss of heterozygosity (LOH) involving the distal part of the short arm of chromosome 1 occurs frequently in ovarian adenocarcinomas but the tumour suppressor gene(s) targeted by this event is unknown. We have used five microsatellite markers in a panel of 56 ovarian adenocarcinomas to determine which part of 1p34 - 36 is the focus of this LOH. LOH was considerably more common at 1p36 (43%) than at 1p34 - 35 (18%), and 11 tumours showed LOH at 1p36 but not at 1p34 - 35. These data strongly suggest the presence of a tumour suppressor gene inactivated in ovarian adenocarcinoma at 1p36. The p53 homologue, p73, has recently been isolated and mapped to 1p36 and therefore is a candidate for this tumour suppressor gene. However, RT - PCR and Western analyses revealed strong expression of p73 in ovarian adenocarcinoma cell lines but very low or undetectable levels in normal ovarian surface epithelial cells. Immunohistochemical analysis of primary ovarian tumours showed that only 3/22 (14%) contained p73 expressing cells. There was no association between 1p36 LOH and p73 expression in ovarian tumours, nor between p73 and p53 expression. These findings strongly suggest that p73 is not the target of 1p36 LOH in ovarian adenocarcinomas but indicate the presence of an, as yet unidentified, tumour suppressor gene in this region that plays an important role in ovarian tumorigenesis.

There is evidence that ATM plays a wider role in intracellular signalling in addition to DNA damage recognition and cell cycle control. In this report we show that activation of the EGF receptor is defective in ataxia-telangiectasia (A-T) cells and that sustained stimulation of cells with EGF downregulates ATM protein in control cells but not in A-T cells expressing mutant protein. Concomitant with the downregulation of ATM, DNA-binding activity of the transcription factor Sp1 decreased in controls after EGF treatment but increased from a lower basal level in A-T cells to that in untreated control cells. Mutation in two Sp1 consensus sequences in the ATM promoter reduced markedly the capacity of the promoter to support luciferase activity in a reporter assay. Overexpression of anti-sense ATM cDNA in control cells decreased the basal level of Sp1, which in turn was increased by subsequent treatment of cells with EGF, similar to that observed in A-T cells. On the other hand full-length ATM cDNA increased the basal level of Sp1 binding in A-T cells, and in response to EGF Sp1 binding decreased, confirming that this is an ATM-dependent process. Contrary to that observed in control cells there was no radiation-induced change in ATM protein in EGF-treated A-T cells and likewise no alteration in Sp1 binding activity. The results demonstrate that EGF-induced downregulation of ATM (mutant) protein in A-T cells is defective and this appears to be due to less efficient EGFR activation and abnormal Sp1 regulation.

Cells from patients with the human genetic disorder ataxia-telangiectasia (A-T) are defective in the activation of cell cycle checkpoints in response to ionizing radiation damage. In order to understand the role of ATM in checkpoint control we investigated whether Schizosaccaromyces pombe chk1, a protein kinase implicated in controlling the G2 DNA damage checkpoint, might alter the radiosensitive phenotype in A-T cells. The fission yeast chk1 gene was cloned into an EBV-based vector under the control of a metallothionein promoter and transfected into A-T lymphoblastoid cells. Induction of chk1 enhanced the survival of an A-T cell line in response to radiation exposure as determined by cell viability and reduction of radiation-induced chromosome aberrations. This can be accounted for at least in part by the restoration of the G2 checkpoint to chk1 expressing cells. There was no evidence that chk1 expression corrected either the G1/S checkpoint or radioresistant DNA synthesis in S phase in these cells. These results suggest that chk1 when overexpressed acts downstream from ATM to restore the G2 checkpoint in these cells and correct the radiosensitive phenotype. These data allow us to dissociate individual checkpoint events and relate them to the radiosensitive phenotype in A-T cells.

The cloning of a full-length cDNA for the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) has been described recently. This cDNA, as well as a fragment representing a functional region from ATM, are capable of rescuing various aspects of the radiosensitive phenotype in A-T cells. We have subcloned full-length ATM cDNA in the opposite orientation in an EBV-based vector under the control of an inducible promoter to determine whether this anti-sense construct might sensitize control lymphoblastoid cells to ionizing radiation. The effectiveness of expression of this construct in control cells was monitored by loss of ATM protein which was evident over a period 6-12 h after induction. Under these conditions radiosensitivity was enhanced approximately threefold in control cells, approaching the degree of radiosensitivity observed in A-T cells. Expression of the anti-sense construct also increased the number of radiation-induced chromosomal breaks and led to the appearance of radioresistant DNA synthesis in these cells. Abrogation of the G1/S checkpoint was evident from the loss of the p53 response and that of its downstream effector, p21/WAF1, post-irradiation. The extent of accumulation of transfected cells in G2/M phase at 24 h post-irradiation was similar to that observed in A-T cells and the induction of stress-activated protein kinase by ionizing radiation was prevented by antisense ATM cDNA expression. These data demonstrate that full-length ATM anti-sense cDNA, by reducing the amount of ATM protein, is effective in imposing a series of known defects characteristic of the A-T phenotype. This inducible system provides an experimental model to further investigate mechanisms underlying radiosensitivity and cell cycle control.