DNA damage of exposed tumour tissue leading to cell death is one of the detrimental effects of ionising radiation that is exploited, with beneficial consequences, for radiotherapy. The pattern of the discrete energy depositions during passage of the ionising track of radiation defines the spatial distribution of lesions induced in DNA with a fraction of the DNA damage sites containing clusters of lesions, formed over a few nanometres, against a background of endogenously induced individual lesions. These clustered DNA damage sites, which may be considered as a signature of ionising radiation, underlie the deleterious biological consequences of ionising radiation. The concepts developed rely in part on the fact that ionising radiation creates significant levels of clustered DNA damage, including complex double-strand breaks (DSB), to kill tumour cells as clustered damage sites are difficult to repair. This reduced repairability of clustered DNA damage using specific repair pathways is exploitable in radiotherapy for the treatment of cancer. We discuss some potential strategies to enhance radiosensitivity by targeting the repair pathways of radiation-induced clustered damage and complex DNA DSB, through inhibition of specific proteins that are not required in the repair pathways for endogenous damage. The variety and severity of DNA damage from ionising radiation is also influenced by the tumour microenvironment, being especially sensitive to the oxygen status of the cells. For instance, nitric oxide is known to influence the types of damage induced by radiation under hypoxic conditions. A potential strategy based on bioreductive activation of pro-drugs to release nitric oxide is discussed as an approach to deliver nitric oxide to hypoxic tumours during radiotherapy. The ultimate aim of this review is to stimulate thinking on how knowledge of the complexity of radiation-induced DNA damage may contribute to the development of adjuncts to radiotherapy.

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16 INK4a and p14 ARF , both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary ®broblasts from a member of a melanomaprone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16 INK4a de®cient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14 ARF . Although they have a ®nite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We ®nd that in human ®broblasts, ARF is not induced demonstrably by RAS, pointing to signi®cant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.

A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with associated base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will concentrate on the experimental findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addition certain non-DSB clustered damaged sites are processed within the cell to form additional DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a “friend”, leading to cell killing in tumour cells or as a “foe”, resulting in the formation of mutations and genetic instability in normal tissue.

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Ionizing radiation induces clustered DNA damage sites which have been shown to challenge the repair mechanism(s) of the cell. Evidence demonstrating that base excision repair is compromised during the repair of an abasic (AP) site present within a clustered damage site is presented. Simple bistranded clustered damage sites, comprised of either an AP-site and 8-oxoG or two AP-sites, one or five bases 3' or 5' to each other, were synthesized in oligonucleotides, and repair was carried out in xrs5 nuclear extracts. The rate of repair of an AP-site when present opposite 8-oxoG is reduced by up to 2-fold relative to that when an AP-site is present as an isolated lesion. The mechanism of repair of the AP-site shows asymmetry, depending on its position relative to 8-oxoG on the opposite strand. The AP-site is rejoined by short-patch base excision repair when the lesions are 5' to each other, whereas when the lesions are 3' to one another, rejoining of the AP-site occurs by both long-patch and short-patch repair processes. The major stalling of repair occurs at the DNA ligase step. 8-OxoG and an AP-site present within a cluster are processed sequentially, limiting the formation of double-strand breaks to <4%. In contrast, when two AP-sites are contained within the clustered DNA damage site, both AP-sites are incised simultaneously, giving rise to double-strand breaks. This study provides new insight into understanding the processes that lead to the biological consequences of radiation-induced DNA damage and ultimately tumorigenesis.

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Ionising radiation induces clustered DNA damage sites which pose a severe challenge to the cell’s repair machinery, particularly base excision repair. To date, most studies have focussed on two-lesion clusters. We have designed synthetic oligonucleotides to give a variety of three-lesion clusters containing abasic sites and 8-oxo-7, 8-dihydroguanine to investigate if the hierarchy of lesion processing dictates whether the cluster is cytotoxic or mutagenic. Clusters containing two tandem 8-oxoG lesions opposing an AP site showed retardation of repair of the AP site with nuclear extract and an elevated mutation frequency after transformation into wild-type or mutY Escherichia coli. Clusters containing bistranded AP sites with a vicinal 8-oxoG form DSBs with nuclear extract, as confirmed in vivo by transformation into wild-type E. coli. Using ung1 E. coli, we propose that DSBs arise via lesion processing rather than stalled replication in cycling cells. This study provides evidence that it is not only the prompt formation of DSBs that has implications on cell survival but also the conversion of non-DSB clusters into DSBs during processing and attempted repair. The inaccurate repair of such clusters has biological significance due to the ultimate risk of tumourigenesis or as potential cytotoxic lesions in tumour cells.

Previous investigations of a Li ± Fraumeni like family (Barnes et al., 1992) demonstrated that both the proband and her mother had elevated p53 protein levels in both tumour tissue and normal tissue at sites distant from the tumour, although no mutation was found in the p53 gene. In the present study two recently described functional assays for p53, an apoptotic assay and the functional assay for the separation of alleles in yeast (FASAY), have been employed to study the functional activity of p53 from this patient. The results of the apoptotic assay demonstrated that this patient had a p53 functional defect and the FASAY result suggested that this defect was in fact a germline mutation of the p53 gene. A point mutation of codon 337, which results in an amino acid substitution of a cysteine for an arginine, was demonstrated initially in cDNA and was con®rmed by sequencing of genomic DNA. This is an unusual mutation as it is in the oligomerisation domain of p53, in contrast to the majority of p53 mutations which are in the core DNA binding domain. This mutation results in a protein which still retains partial transactivational activity in the FASAY. The mutation of codon 337 is only the second reported case of a germline missense mutation occurring in the oligomerisation domain of p53.Keywords: p53; Li ± Fraumeni syndrome; oligomerisation domain; functional assaysThe tumour suppressor gene, p53, was ®rst discovered in 1979 (Lane and Crawford, 1979;Linzer and Levine, 1979). p53 is a 393 amino acid nuclear phosphoprotein which plays a central role in the cellular processes involved in the response to DNA damage (reviewed Kastan et al., 1995). It is made up of at least four domains, a transactivation domain, a central DNA binding domain, an oligomerisation domain and a regulatory domain, the latter two residing at the carboxy end of the protein.Following initial studies by Malkin et al. (1990) it is now established that germline p53 mutations are associated with Li ± Fraumeni syndrome (LFS), a dominantly inherited syndrome ®rst proposed by Fraumeni in 1969 (Li andFraumeni, 1969). However mutations in the coding region of p53 have only been con®rmed in about 50% of families with LFS (Birch et al., 1994). Families with this syndrome exhibit multiple primary neoplasms in childhood and early adulthood and the spectrum of cancers which commonly occurs in LFS families includes brain tumours, breast tumours, sarcomas, leukaemia and adrenal cortical tumours (Li et al., 1988). Families have now been recognised in which there is a similar spectrum of malignancies to LFS but the families do not ®t exactly into the criteria of LFS. These families have been named Li ± Fraumeni like (LFL) (Birch et al., 1994) and p53 germline mutations have been found in 7 ± 20% of these families (Eeles, 1995; Varley et al. manuscript in preparation). The patient who is the subject of this study is a member of a LFL family and meets the criteria for LFL families as set out by Birch et al. (1994).In 1992 Barnes et al. described a member of a LFL ...