DATA supporting the importance of ionizing radiation (IR) as ahuman carcinogen have come from studies of two populations. First,individuals who have been exposed to IR subsequently show an in-creased cancer incidence; examples include carcinoma of the skin ofthe hand in radiologists, carcinoma of the lung in radon and uraniumminers, and leukaemia and other cancers in the atomic bomb sur-vivors and among patients irradiated for ankylosing spondylitis. Theevidence also shows a linear relationship between dose and cancerrisk1. The second group of susceptible individuals are those with rarecancer-prone syndromes, such as ataxia telangiectasia (AT) andNijmegen breakage syndrome (NBS), in which radiation sensitivityis a characteristic phenotype. These two distinct, but closely related,diseases are characterized by an extreme sensitivity to IR; homozy-gotes are predisposed to developing cancers at a young age, in particu-lar lymphoreticular cancers, and show an acute radiation reactionwhen treated with conventional radiotherapeutic doses for cancer.Heterozygotes also have an increased cancer risk2,3.

Clinical observations of normal tissue damage are observed in asubset of patients following radiotherapy. This finding supports the

notion that genetic differences among individuals might account formuch of the unpredictable severity of normal tissue damage. Severalstudies have reported that ~5–10% of all breast cancer patients showsevere early or late tissue reactions following radiotherapy. Similarestimates for the proportion of the total population that is radiosensi-tive have come from cellular radiobiology studies of normal individ-uals4. Enhanced sensitivity to the chromosome-damaging effects ofIR is also a feature of many cancer-predisposing conditions, andmeasurements of chromosomal radiosensitivity in cancer populationshave found that quite a high proportion of such individuals have el-evated radiation sensitivity. Using a micronucleus induction assay,Scott et al. identified 31% of breast cancer patients, compared to 5%of healthy controls, as having elevated radiation sensitivity5.

From the studies carried out to date, it is clear that there are agrowing number of genetic loci that might influence radiation sensitivity and cancer predisposition; these studies suggest that theproducts of these genes are involved in some aspect of the normal response to IR. Recent studies2,3 on the ataxia telangiectasia mutated(ATM) and Nijmegen breakage syndrome (NBS1) gene products haveprovided valuable insights into the molecular mechanisms of the cel-lular response to IR-induced DNA damage and in the assessment ofcancer risk as a result of exposure to radiation.

Clinical features of ATThe incidence of AT is estimated to be between 1:40 000 and1:300 000, depending on the population studied6,7. The disease ischaracterized by progressive cerebellar ataxia, ocular telangiectasia,immunodeficiency, chromosomal instability, hypersensitivity to IRand predisposition to cancer. Reports have suggested that ~10% ofall AT patients develop a malignancy and have a high risk of devel-oping leukaemias and lymphomas (reviewed in Ref. 8). The frequencyof ATM heterozygotes in the population, based on both epidemiologi-cal studies and, more recently, on direct mutation analysis of theATM gene in control populations, is ~1%. Although heterozygous

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Radiation, DNA damage andcancer

Janet Hall and Sandra Angèle

The characterization of the rare, radiation-sensitive and cancer-prone syndromes, ataxia telangiectasiaand Nijmegen breakage syndrome, has demonstrated that genetic predisposition increases the risk ofdeveloping cancer after exposure to ionizing radiation (IR). Molecular analyses of these disorders providevaluable insights into the normal function of these two gene products in the cellular response to IR-induced DNA damage. Their contribution to a cellular radiosensitive phenotype and their role insporadic cancers can now be fully assessed. For example, the gene ataxia telangiectasia mutated (ATM)has recently been shown to be a tumour suppressor gene in T-cell prolymphocytic leukaemia, and there isincreasing evidence that individuals with one mutated ATM or Nijmegen breakage syndrome (NBS1) allelehave an increased predisposition to cancer.

R e v i e w s

Janet Hall* PhDScientist

Sandra Angèle PhDPostdoctoral Fellow

Unit of Mechanisms of Carcinogenesis, International Agency forResearch on Cancer, 150 cours Albert Thomas, 69372 Lyon

Cedex 08, France.Tel: 133 472 738596

Fax: 133 472 738322*e-mail: hall@iarc.fr

1357-4310/99/$ - see front matter © 1999 Elsevier Science. All rights reserved. PII: S1357-4310(99)01435-5

carriers of the ATM gene show none of theclinical features of AT, there is consistentevidence that they have a higher risk of can-cer (reviewed in Ref. 9). Whether these indi-viduals are at an increased risk of developingcancer as a result of routine therapeutic (andpossibly even diagnostic) procedures involv-ing radiation, as suggested by Swift et al.10

should be examined in detail. Several studieshave shown that the female relatives of ATpatients are at an increased risk of develop-ing breast cancer (Table 1). Based on theseestimates, the ATM gene might be respon-sible for up to 8% of all breast cancer cases. Insupport of the hypothesis that the ATM genemight be a susceptibility factor for breastcancer is the finding that loss of hetero-zygosity frequently occurs in the chromo-somal region 11q22–23 in breast tumours –the chromosomal location of the ATM gene16.However, molecular studies, reviewed in de-tail below, have yet to confirm the involve-ment of ATM in sporadic breast cancers.

Clinical features of NBSNBS was first described in 1981 in two pa-tients who showed microcephaly, immuno-globulin A deficiency and chromosomal instability. For many years, NBS was consid-ered to be a variant of AT (reviewed in

Ref. 2). Radiosensitivity and radioresistantDNA synthesis were observed in cells fromNBS patients, and a striking predispositionto hematopoietic malignancies, of which80% were lymphomas, was noted17. Thereare, however, distinct clinical differences be-tween the two syndromes (Fig. 1). NBS isvery rare in western Europe but is prevalentamong the eastern and central Europeanpopulation, in particular among the Czechand Polish people. NBS heterozygotes alsohave an increased risk for malignancies,suggesting that, as in the case of AT heterozy-gotes, gene carriers might face adverse effectsfrom routine procedures involving radiation18.

Function of the ATM gene productThe ATM gene was cloned in 1995, and mu-tation analysis in AT patients indicated that asingle locus on chromosome 11q23.1 was re-sponsible for the disease19. The gene com-prises 66 exons and encodes a 13-kb mRNA.The ATM protein has a molecular mass of350.6 kDa and is related to a family of yeastand mammalian proteins that contain a phos-phatidylinositol 39-kinase domain with pro-tein kinase activity (reviewed in Refs 2,3).These proteins are involved in cell-cyclecontrol and/or the detection of DNA damage.

The ATM gene plays an important role inthe cellular response to damage that is pro-duced by IR. Under normal physiologicalconditions, mammalian cells respond todamage by inhibiting cell-cycle progression,processing the DNA damage or, if the DNAis damaged beyond repair, inducing apop-tosis. The signalling cascades that are activatedby IR remain to be fully elucidated (Fig. 2),but both the ATM and p53 proteins have beenclearly shown to play a key role (reviewed in Refs 2,20). In response to IR, ATM-associated kinase activity is enhanced andp53 is phosphorylated at Ser15, resulting inan increase in the apparent intracellular p53protein concentration as a result of enhancedprotein stability21–23. The p53 protein is atranscriptional activator of many genes, in-cluding the WAF1/CIP1 (wild-type p53-activated fragment 1/CDK-interacting pro-tein 1) gene, which encodes p21 – a CDK(cyclin-dependent kinase) inhibitor thatplays an essential role in the p53-mediatedG1 cell-cycle block and in a decrease inDNA synthesis20. This activation of p53 issub-optimal in AT cells, resulting in a cell-cycle-checkpoint defect24. ATM also inter-acts with c-Abl, thus activating a separate radiation signal-transduction pathway thatoperates through stress-activated protein

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Table 1. Estimated relative risks of breast cancer inataxia telangiectasia (AT)

heterozygotes

Relative riska Ref.

6.8 (2.0–22.6) 10

5.1 (1.5–16.9) 11

1.3 (0.3–5.2) 12

3.9 (1.3–12.1) 13

3.9 (2.1–7.1)b 9

2.9 (1.1–7.6)c 14

6.4 (1.4–28.8)d

12.7 (0.2–11.8) 15

a95% confidence interval is shown in parentheses.bCombined relative risk from a review of Refs 10–13.cBased on 33 female relatives of AT patients from a

total of 775 blood relatives in the study; disease onset

before 60 years of age.dDisease onset at 60 years of age or older.

Figure 1. (a) Similarities and (b) differences between ataxia telangiectasia (AT) and Nijmegen breakagesyndrome (NBS).

a Similarities betweenAT and NBS

b Differences betweenAT NBS

• Elevatedα fetoprotein

• Normalα fetoprotein

Cerebellar ataxia;ocular

telangiectasia

Hypogonadism

11q23.1

ATM (350 kDa);protein kinase

Microcephaly;mild to moderate

mental retardation

Primary ovarianfailure

8q21.3

Nibrin (p95) (85 kDa);member of

double-strand DNArepair complex

• Developmental defects

• Fertility

• Chromosome location

• Protein

• Radiation sensitivity

• Growth retardation

• Chromosomal instabilityin cultured cells

• Immune deficiencies

• Cell-cycle response to radiation

• Increased cancer susceptibility:lymphoid

Frequently involving chromosomes7 and 14

Reduced humoral and cellular immunity;aberrant T-cell receptor rearrangement

Defective G1 – S and G2 arrest;inability to arrest DNA synthesis

ATT-cell lymphomasand leukaemias

NBSB-cell lymphomas

kinase (SAPK). This leads to the inductionof stress response genes via c-Jun and theregulation of cell-cycle checkpoint controlthrough p53-independent pathways (reviewedin Ref. 2).

ATM protein is localized in both the nucleus and cytoplasm where it appears to beassociated with vesicular structures25,26. Itsabsence from the nucleus in AT cells wouldblock the response to DNA damage, resultingin a defective p53 response. The presence ofATM in the cytoplasm might be part of thecellular response to reactive oxygen inter-mediates, which would be generated as by-products of energy metabolism or defencemechanisms. Indeed, it has been suggestedthat AT cells are in a constant state of oxidative stress27. Immunofluorescence and immunoprecipitation experiments have shownthat ATM binds to b-adaptin in the cytoplas-mic vesicles. b-adaptin is one of the compo-nents of the activator protein 2 (AP-2) adap-tor complex involved in clathrin-mediatedendocytosis of receptors. Therefore ATMmight also have roles in vesicle and/or proteintransport26.

The ATM protein clearly plays a role inmany physiological pathways, in addition toits role in response to DNA damage, as is notonly apparent from the phenotype of ATpatients but also from studies of ATM knock-out mice. These mice are growth retarded,sterile, radiation sensitive and have defectiveT-cell maturation and a striking tendency todevelop thymic lymphomas (reviewed inRef. 2). As recently reviewed by Taylor28, itis apparent that ATM has different functionsin different cell types and at different stagesof development, and it remains to be fully established how these contribute to the clinical features of AT.

Function of the NBS1 gene productThe cellular phenotype of NBS in response to IR is remarkably simi-lar to that observed in AT cells. Cultured cells from NBS patientsshow an increased frequency of chromosomal aberrations, reducedsurvival in colony-forming assays and radio-resistant DNA synthesis(reviewed in Ref. 2). Recently, abnormalities in the p53-inducible re-sponse to DNA damage and cell-cycle control after exposure to IRhave been documented in NBS cells29–32 and are similar to those pre-viously described in AT cells. This defect is present on a backgroundof apparently normal levels of ATM mRNA and protein29 and appearsto be specific to IR. These results suggest that the ATM and NBS1gene products function in the same pathway, which is involved in the detection and signalling of DNA damage. The lack of comple-mentation of radiation-induced chromosome aberrations in AT/NBS heterodikaryons supports this hypothesis33.

The NBS1 gene, which is located on chromosome 8q21.3, has re-cently been cloned by three different groups. A positional-cloningapproach was used by Varon et al.34 and Matsuura et al.35 to isolate a

gene encoding a novel protein, nibrin. The gene spans .50 kb ofgenomic DNA and has 16 exons. The NBS1 gene is alternativelyspliced and two mRNA transcripts of 2.4 and 4.4-kb are present in alltissues. The encoded protein of 754 amino acids has a predicted mol-ecular mass of 85 kDa. Nibrin contains two domains often found incell-cycle-checkpoint proteins, a forkhead associated (FHA) domainadjacent to a breast cancer carboxyl-terminal (BRCT) domain (re-viewed in Ref. 36). FHA domains are involved in mediating phospho-Ser/Thr-specific interactions and have been found in yeast proteinsthat are involved in S-phase checkpoints and damage repair37. BRCTdomains, recently described by Bork et al.38, are found in a variety of proteins, all of which appear to be involved in DNA-damage-responsive cell-cycle checkpoints. Carney et al.39 isolated the NBS1gene, whose protein product they called p95, in a search for membersof the double-strand break repair complex, which also contains theproteins hMRE11 and hRAD50 (human meiotic recombination 11and human RAD50, respectively). This complex becomes rapidly associated with double-strand breaks to form discrete nuclear foci in normal cells after exposure to IR and thus, like ATM, appears to function in close proximity to the sensors of DNA damage. The hMRE11–hRAD50–p95 complex has a manganese-dependent

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Figure 2. Role of ataxia telangiectasia mutated (ATM) protein in DNA double-strand break repair.Genotoxic agents, such as ionizing radiation (IR), induce double-strand breaks in DNA that can be recog-nized by a variety of cellular proteins. These include the ATM protein, the nibrin–hMRE11–hRAD50 com-plex, and the DNA-dependent protein kinase complex, which comprises a catalytic subunit (DNAPKcs) andthe DNA-binding proteins ku70 and ku80. In response to IR-induced DNA damage, ATM activates p53-mediated cell-cycle arrest, allowing DNA repair and control of the radiation-induced apoptotic response.ATM can also regulate the cell cycle by p53-independent pathways, involving c-Abl, replication protein A(RPA) and checkpoint 2 (Chk2). The growth arrest and DNA damage inducible (GADD45) gene, which isimplicated in DNA repair, contains a p53 responsive element. Its induction and the activation of stress acti-vated protein kinase (SAPK) are reduced in AT cells after exposure to IR. Likewise, the activation of the in-hibitor kB (IkB)/nuclear factor kB (NFkB) signalling pathway is also compromised in AT cells following exposure to IR. NFkB is maintained in the cytoplasm in a complex with IkB, a protein inhibitor of this transcription factor. IkB is an in vitro substrate for phosphorylation by the ATM kinase that in its phosphoryl-ated form undergoes proteolysis. The unbound NFkB can then translocate to the nucleus and activate the transcription of various genes, including anti-apoptotic genes. Abbreviations: hMRE11, human meioticrecombination 11; hRAD50, human RAD50.

IκB

NFκB p53 cAbl

SAPK

GADD45

Jun

Chk2 RPA

Cdc25C

p21WAF1/CIF1

Proteintransport

β-adaptin

Cell-cycleregulation

DNArepair

Ku80 Ku70

Apoptosis

DNAPKcs

ATMNucleus

DNA double-strand break

Nibrin–hRAD50–hMREIIcomplex

single-stranded DNA endonuclease and 39 to 59 exonuclease acti-vities40, which could be important for recombination, repair and genomic stability41.

The defect in the formation of nuclear foci after exposure to IRcannot fully account for the diminished p53 response that is seen inNBS cells. These aspects of the NBS phenotype indicate that p95(nibrin) and, by extension, the hMRE11–hRAD50 protein complex,is an integral part of the signal that activates the cellular response toIR-induced DNA damage. How the NBS1 and ATM gene productsinteract to bring about this dual activation of the pathways involvedin the processing of strand breaks and DNA damage-dependent cell-cycle checkpoints, and how these functions influence the clinicalfindings seen in affected individuals, remains to be elucidated.

ATM and intrinsic radiosensitivity inbreast cancerEpidemiological studies have shown that irradiation of the breast, especially amongyoung women, increases the risk of subse-quently developing breast cancer (reviewedin Refs 42,43). It might thus be expected thatgenes that are known to influence radiationsensitivity and that are associated with an in-creased breast cancer risk, such as the ATMgene, will be responsible for a proportion ofsuch cases. As previously mentioned, a sig-nificant proportion of breast cancer patientsshow an exaggerated acute or late (fibrosis,telangiectasia) adverse reaction of normaltissue following radiotherapy. Many reportsin the literature document a reduced cell sur-vival in cultured fibroblasts and lymphoblas-toid cell lines established from such individ-uals compared to the survival of normal cellsfollowing exposure to the same dose of IR,as seen in some AT heterozygotes. Therewould appear to be some correlation betweenthis in vitro radiosensitivity and the exagger-ated reaction to IR in patients, particularlythe occurrence of late normal tissue reactionsto radiotherapy44–46. These findings supportthe hypothesis that a germline mutation couldbe the basis of this radiation sensitivity, although many other factors must also influ-ence the in vivo response.

Mutation analysis of ATM and NBS1 –a direct link between radiationsensitivity and gene mutationMost of the mutations in AT patients areunique and uniformly distributed along thelength of the ATM gene. Most AT patientsare compound heterozygotes who have twodifferent ATM mutations; patients homozy-gous for the same ATM mutation are rare28.The predominant type of mutation found inthe ATM gene of AT patients with the ‘classi-cal’ form of the disease results in a truncatedand unstable ATM protein. However, a con-siderable percentage (29% in the UK) of in-

frame insertions/deletions or missense mutations have been found inthe ATM gene, many of which are associated with some degree of ex-pression of mutant ATM protein. The presence of such mutant ATMprotein with residual function, or the presence of a small amount ofnormal ATM protein, might lead to a milder clinical presentation anda reduced cancer predisposition15. In two British families, a T→Gtransversion at base pair 7271 was found, which would predict thereplacement of Val by Gly. This mutation was associated with a mildclinical phenotype, lower radiosensitivity and increased risk of breastcancer in both homozygotes and heterozygotes. It will be very im-portant to examine more closely genotype–phenotype correlations inall AT patients and heterozygotes because the possibility clearly exists that the risk of breast cancer might be increased by certain

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GlossaryAtaxia – A lack of balance.

Compound heterozygote – An individual who has both copies of a mutated gene but withdifferent mutations in each allele.

Founder effect – A unique mutation, found in a single family or in a small number of geo-graphically clustered families, that arose from a single founder.

Human meiotic recombination 11 (hMRE11) – A protein that associates with hRAD50 andnibrin/p95 and that is implicated in DNA double-strand break repair.

Human RAD50 (hRAD50) – A protein associated with hMRE11 and nibrin/p95 that is implicated in DNA double-strand break repair.

Positional cloning – A strategy by which a disease gene is identified by virtue of its map position on the chromosome rather than by its function.

Protein truncation test (PTT) – A test that can be used to detect gene mutations that resultin a premature truncation of the protein product. DNA fragments are amplified and tran-scribed in vitro using a forward primer containing the T7 promoter sequence and the initiatingATG codon and are then translated using rabbit reticulocytes lysate in the presence of [35S]-methionine. Translated radioactive proteins are separated by polyacrylamide gel electro-phoresis and are visualized by autoradiography. If a base substitution, leading to a stopcodon (TAA, TAG or TGA) has occurred in the DNA sequence, the translation of the proteinis stopped, resulting in a smaller size than would be expected.

Radiation sensitivity – An increased susceptibility to cellular damage by exposure to radi-ation. It can be measured by testing cultured cells for various end-points, including cyto-toxicity (survival assays), clastogenic responses (chromosome damage), mutagenicity andDNA repair.

Radioresistant DNA synthesis – When normal cells are exposed to ionizing radiation thereis a dose-dependent inhibition of the rate of DNA synthesis. By contrast, radioresistant DNAsynthesis occurs in ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS) cellsafter exposure to ionizing radiation.

Restriction endonuclease fingerprinting (REF) – A technique that can be used to detectgene mutations in large DNA fragments of up to 2 kb. The region of interest is amplified byPCR, and multiple restriction endonuclease digestions are performed prior to single-strandconformation polymorphism (SSCP) analysis. Sequence alterations will produce abnormalREF patterns, and the presence of mutations is confirmed by direct sequencing.

Telangiectasia – Dilated blood vessels.

Tumour suppressor gene – A gene that negatively regulates cell growth such that a mutation in it results in uncontrolled cell division and tumour progression.

mutations. Such findings would also suggest that other clinical phenotypes, not recognized as AT, might in fact be associated with an altered ATM gene product and manifested as radiation sensitivity.One such example is the recent finding of a homozygous ATMmutation in a radiosensitive melanoma cell line47.

The ATM gene has recently been shown to act as a tumour sup-pressor gene in sporadic cases of T-cell prolymphocytic leukaemia(T-PLL), a form of cancer often seen in young adult AT patients8,48–50.These results suggest that its inactivation is a key event in the devel-opment of this leukaemia. In future studies, it will be of interest tolook for mutations in other radiation-sensitive tumour lines and inother sporadic tumours of the types found in AT homozygotes andheterozygotes.

The role of ATM as a risk factor in sporadic breast cancer caseswithin the general population has been a consistent theme in the litera-ture since the first report by Swift et al.10 Several groups have tried to establish whether the ATM gene is indeed a major susceptibilitygene for breast cancer. As the ATM gene has been associated withboth in vivo and in vitro sensitivity to IR, several groups haveanalysed the ATM mutation status in radiation-sensitive breast cancerpatients (Table 2). Published studies have produced conflicting re-sults and have not revealed the magnitude of involvement of ATM insporadic breast cancer that would have been expected from the in-creased risk found in the AT family studies and from the frequency ofAT heterozygotes in the population. The largest study reported usedthe protein truncation test (PTT) for ATM mutation analysis. Thistechnique will not detect all mutations; in particular it will missamino-acid substitutions, such as the Val to Gly mutation discussedabove. In vitro radiation sensitivity also seems to show a better cor-relation with late, rather than with early, normal tissue reactions to

radiotherapy, and a large sample of breast cancer patients who showa late reaction to radiotherapy should be analysed for ATM mu-tations. The restriction endonuclease fingerprinting (REF) tech-nique, which efficiently detects sequence alterations in DNA frag-ments of up to 2 kb in length, offers a particularly suitable tool forthis purpose (Fig. 3). Functional studies on the cellular response toIR in cell lines established from radiation-sensitive breast cancer patients might also shed light on the involvement of other genes in breast cancer.

Future directionsThe identification of the ATM and NBS1 genes paves the way to eluci-dating their roles in the activation of cellular responses to IR. By manipulating their expression, it should be possible to alter the inher-ent radiosensitivity of tumour cells as a therapeutic tool3. The recentcloning of the NBS1 gene should allow its contribution to radiosensi-tivity and increased cancer risk to be fully assessed. The majority ofNBS patients of Slavic origin have a 5-bp deletion that would resultin a frameshift and thus premature protein truncation (Fig. 3). A simi-lar situation has been found in the ATM gene where significantfounder effects have been reported in North African Jewish57 andNorwegian, Costa Rican, Polish, Italian and Amish/Mennonite popu-lations58. Thus, in certain situations, it will be possible to assess thecontribution of these two genes in sporadic cancers using screeningtechniques that are designed to detect carriers of these common mutations.

It will be of great interest to assess the role of the NBS1 gene in B-cell lymphomas – the tumour type frequently found in young NBSpatients17. Owing to the similarities in the phenotype of NBS and ATcells, and the recent findings that the ATM gene acts as a tumour

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Table 2. Incidence of ataxia telangiectasia mutated (ATM) gene mutations in breast cancer patientsa

Cases Sample analysed Incidence of ATM mutations Age of onset of Reaction to Ref. (test system) breast cancer radiotherapy

Family history of cancer Blood DNA 3/88 53 6 12 Not indicated 51

(index case breast cancer) (PCR/SSCP)

Sporadicb Breast tumours and blood 0/38 55.8 6 13.8 Not indicated 52

DNA (PCR/SSCP)

Early onset Fibroblasts and LCLs 1/1 (PTT) 27 Minimal late effect 53

Early onset Lymphocytes 2/399 (PTT) <40 None 54

0/2 (PTT) Adverse

Selected for radiation sensitivity Lymphocytes 0/16 (REF) Not indicated Acutec 55

Family history of cancer Lymphocytes 1/18 (PTT) 48.3 (33–68) Not indicatedd 56

(index case breast cancer)

Selected for radiation sensitivity LCLs 0/15 (PTT) Not indicated Late 46

aAbbreviations: LCL, lymphoblastoid cell lines; PCR/SSCP, polymerase chain reaction/single-strand conformation polymorphism; PTT, protein truncation test; REF, restriction endonucleasefingerprinting.bUnselected.c3/16 showed some slight late effect.dAt time of blood sample, no radiotherapy had been given.

suppressor gene in T-PLL, it is tempting tospeculate that the NBS1 gene might also be atumour suppressor gene, the inactivation ofwhich is a key event in the development ofcertain classes of lymphomas.

Other gene products have also been impli-cated in the cellular response to IR, and mu-tations in these genes would be expected toconfer radiation sensitivity and/or a predis-position to cancer. Candidate genes includethose that encode the DNA-dependent pro-tein kinase, which depends on the presenceof strand breaks for its activity. This proteinkinase is a crucial component of the mam-malian DNA-double-strand break repair ap-paratus, and mammalian cells defective inthe components of this repair complex arehypersensitive to cell killing by IR59. Theprotein products of the breast cancer genes,BRCA1 and BRCA2 (Ref. 60), which are be-lieved to associate with RAD51, appear to beinvolved in some aspect of monitoringgenome integrity or the regulation of certainDNA-repair processes61. Other genes, whosecontribution to radiation sensitivity remainsto be determined, include those involved inDNA damage detection, the regulation ofcell-cycle control and apoptosis, such as themouse double minute 2 (mdm2) gene, TP53,c-Abl, the interferon regulatory factor 1(IRF-1) gene and WAF1/CIP1.

It is now 70 years since the first reportby H.J. Muller that radiation is mutagenic.Although considerable advances have beenmade in our knowledge of the genetic locithat influence radiation sensitivity and can-cer predisposition, the molecular mecha-nisms involved in the detection and repair ofIR-induced damage are still poorly under-stood in human cells. We are now muchcloser to elucidating how the many geneproducts identified to date, and the proteinsthat associate with them, will modify the cel-lular response to IR, the frequency of occur-rence of mutations in these genes and theirpenetrance at the population level and withinspecific groups of cancer patients. In the fu-ture, this will allow us to identify those at riskfrom diagnostic and/or therapeutic radiationand to tailor their treatment accordingly.

Acknowledgements. We gratefully acknowledge the contin-ued support and encouragement of R. Montesano and the tech-nical support of M. Vuillaume. We also thank G. Mollon forphotographic assistance. This work was supported in part bygrants from Association pour la Recherche sur le Cancer andLa Ligue Nationale Contre le Cancer, Comité Départementaldu Rhône. We apologize to those researchers whose work wasnot directly cited owing to space considerations.

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Figure 3. Techniques for the detection of ataxia telangiectasia mutated (ATM) and Nijmegen breakagesyndrome (NBS1) gene mutations. (a) Total RNA from cultured cells is reverse transcribed to produce afull-length cDNA of the ATM and NBS1 genes. The analysis of ATM cDNA (10 kb) by restriction endonu-clease fingerprinting (REF) involves PCR amplification of 8 partly overlapping fragments (A–H), usingprimers that amplify products of 1.0–1.6 kb. The analysis of the NBS cDNA (2.4 kb) using the proteintruncation test (PTT), in which 2 partly overlapping fragments are PCR amplified using defined primers.NBS1 contains two domains, a forkhead associated (FHA) domain adjacent to a breast cancer carboxyl-terminal (BRCT) domain. (b) The ATM REF fragments are digested with combinations of 7–10 restrictionendonucleases. The fragments are then pooled and end labelled with [33P]g-dATP and separated by elec-trophoresis on mutation detection enhancement (MDE) gels. The gels are dried and examined by auto-radiography. Sequence alterations that cause abnormal REF patterns can be detected by comparing theprofile of AT cells with those from individual mixes and from pooled mixes of normal (N) cell lines. The bluearrows show additional bands and the red arrows show the loss of bands in the REF pattern, demonstrat-ing that the AT cells in the asterixed (*) lane are mutated in this REF fragment. The NBS1 PTT fragmentsare in vitro transcribed and translated using [35S] methionine; the resulting protein fragments are separatedon a 12.5% polyacrylamide gel and are examined by autoradiography. The presence of a mutation in frag-ment 1, which would truncate the protein, is confirmed by the presence of a shorter protein product, as canbe seen in all the NBS cells examined. A normal cell line control (N) is shown. (c) Confirmation of the mu-tations shown in (b) by direct sequencing. In the AT cells (*), a CG→TA mutation has occurred at codon2714; all the NBS cells are homozygous for the 657del5 mutation (ACAAA), which would cause prematureprotein truncation and which is characteristic of Slavic NBS patients.

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References1 Doll, R. (1967) Prevention of cancer, in Pointers from Epidemiology, The

Nuffield Provincial Hospitals Trust2 Shiloh, Y. (1997) Ataxia telangiectasia and the Nijmegen breakage syndrome:

related disorders but genes apart, Annu. Rev. Genet. 31, 635–6623 Lavin, M.F. (1998) Radiosensitivity and oxidative signalling in ataxia

telangiectasia, Radiother. Oncol. 47, 113–1234 Mossman, K.L. (1997) Radiation protection of radiosensitive populations,

Health Phys. 72, 519–5235 Scott, D. et al. (1998) Radiation-induced micronucleus induction in lympho-

cytes identifies a high frequency of radiosensitive cases among breast cancerpatients: a test for predisposition, Br. J. Cancer 77, 614–620

6 Swift, M. et al. (1986) The incidence and gene frequency of ataxia telangiec-tasia in the United States, Am. J. Hum. Genet. 39, 573–583

7 Woods, C.G., Bundey, S.E. and Taylor, A.M.R. (1990) Unusual features in theinheritance of ataxia telangiectasia, Hum. Genet. 84, 555–562

8 Taylor, A.M.R., Metcalfe, J.A., Thick, J. and Mak, Y-F. (1996) Leukemia andlymphoma in ataxia telangiectasia, Blood 87, 423–438

9 Easton, D. (1994) Cancer risks in A-T heterozygotes, Int. J. Radiat. Biol. 66,S177–182

10 Swift, M., Reitnauer, P.J., Morrell, D. and Chase, C.L. (1987) Breast and othercancers in families with ataxia telangiectasia, New Engl. J. Med. 316,1289–1294

11 Swift, M., Morrell, D., Massey, R.B. and Chase, C.L. (1991) Incidence of cancerin 161 families affected by ataxia telangiectasia, New Engl. J. Med. 325,1831–1836

12 Pippard, E.C., Hall, A.J., Barker, D.J. and Bridges, B.A. (1988) Cancer in homo-zygotes and heterozygotes of ataxia-telangiectasia and xeroderma pigmento-sum in Britain, Cancer Res. 48, 2929–2932

13 Borresen, A.L. et al. (1990) Breast cancer and other cancers in Norwegian families with ataxia telangiectasia, Genes Chromosomes Cancer 2,339–402

14 Athma, P., Rappaport, R. and Swift, M. (1996) Molecular genotyping shows thatthe ataxia telangiectasia heterozygotes are predisposed to breast cancer,Cancer Genet. Cytogenet. 92, 130–134

15 Stankovic, T. et al. (1998) ATM mutations and phenotypes in ataxia-telangiec-tasia families in the British Isles: expression of mutant ATM and the risk ofleukemia, lymphoma and breast cancer, Am. J. Hum. Genet. 62, 334–345

16 Laake, K. et al. (1997) Loss of heterozygosity at 11q23.1 in breast carcinomas:indication of a gene distal and close to ATM, Genes Chromosomes Cancer 18,175–180

17 van der Burgt, I., Chrzanowska, K.H., Smeets, D. and Weemaes, C. (1996)Nijmegen breakage syndrome, J. Med. Genet. 33, 153–156

18 Seemanová, E. (1990) An increased risk for malignant neoplasms in hetero-zygotes for a syndrome of microcephaly, normal intelligence, growth retardation, remarkable facies, immunodeficiency and chromosomal instability, Mutat. Res. 238, 321–324

19 Savitsky, K. et al. (1995) A single ataxia telangiectasia gene with a productsimilar to PI-3 kinase, Science 268, 1749–1753

20 Ko, L.J. and Prives, C. (1996) p53:puzzle and paradigm, Genes Dev. 10,1054–1072

21 Banin, S. et al. (1998) Enhanced phosphorylation of p53 by ATM in responseto DNA damage, Science 281, 1674–1677

22 Canman, C.E. et al. (1998) Activation of the ATM kinase by ionising radiationand phosphorylation of p53, Science 281, 1677–1679

23 Khanna, K.K. et al. (1998) ATM associates with and phosphorylates p53: mapping the region of interaction, Nat. Genet. 20, 398–400

24 Kastan, M.B. et al. (1992) A mammalian cell cycle checkpoint pathway utiliz-ing p53 and GADD45 is defective in ataxia-telangiectasia, Cell 71, 587–597

25 Watters, D. et al. (1997) Cellular localisation of the ataxia-telangiectasia(ATM) gene product and discrimination between mutated and normal forms,Oncogene 14, 1911–1921

26 Lim, D-S. et al. (1998) ATM binds to b-adaptin in cytoplasmic vesicles, Proc.Natl. Acad. Sci. U. S. A. 95, 10146–10151

27 Rotman, G. and Shiloh, Y. (1997) Ataxia-telangiectasia: is ATM a sensor of oxidative damage and stress? BioEssays 19, 911–917

28 Taylor, A.M.R. (1998) What has the cloning of the ATM gene told us aboutataxia telangiectasia? Int. J. Radiat. Biol. 73, 365–371

29 Jongmans, W. et al. (1997) Nijmegen breakage syndrome cells fail to induce thep53-mediated DNA damage response following exposure to ionising radiation,Mol. Cell Biol. 17, 5016–5022

30 Sullivan, K.E., Veskler, E., Lederman, H. and Lees-Miller, S.P. (1997) Cell cycledefects and DNA repair in Nijmegen breakage syndrome, Clin. Immunol.Immunopathol. 82, 43–48

31 Matsuura, K. et al. (1998) Radiation induction of p53 in cells from Nijmegenbreakage syndrome is defective but not similar to ataxia telangiectasia,Biochem. Biophys. Res. Commun. 242, 602–607

32 Yamasaki, V., Wegner, R.D. and Kirchgessner, C.U. (1998) Characterisation ofcell cycle checkpoint responses in Nijmegen breakage syndrome cells, CancerRes. 58, 2316–2322

33 Stumm, M., Sperling, K. and Wegner, R-D. (1997) Non-complementation of radiation induced chromosome aberrations in ataxia telangiectasia/ataxiatelangiectasia variant heterodikaryons, Am. J. Hum. Genet. 60, 1246–1251

34 Varon, R. et al. (1998) Nibrin, a novel DNA double-strand break repair pro-tein, is mutated in Nijmegen breakage syndrome, Cell 93, 467–476

35 Matsuura, S. et al. (1998) Positional cloning of the gene for Nijmegen breakagesyndrome, Nat. Genet. 19, 179–181

36 Featherstone, C. and Jackson, S.P. (1998) DNA repair: The Nijmegen breakagesyndrome protein, Curr. Biol. 8, R622–R625

37 Hofmann, K. and Bucher, P. (1995) The FHA domain: a putative nuclear sig-nalling domain found in protein kinases and transcription factors, TrendsBiochem. Sci. 20, 347–349

38 Bork, P. et al. (1997) A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins, FASEB J. 11, 68–76

39 Carney, J.P. et al. (1998) The hMre11/hrad50 protein complex and Nijmegenbreakage syndrome: linkage of double-strand break repair to the cellularDNA damage response, Cell 93, 477–486

40 Trujillo, K.M., Yuan, S.S.F., Lee, E.Y.H.P. and Sung, P. (1998) Nuclease activitiesin a complex of human recombination and DNA repair factors Rad50, Mre11and p95, J. Biol. Chem. 273, 21447–21450

41 Lieber, M.R. (1998) Pathological and physiological double-strand breaks -roles in cancer, aging, and the immune system, Am. J. Pathol. 153, 1323–1332

The outstanding questions

• What are the genes involved in the early and late normaltissue responses to radiation?• How do the protein products of the ataxia telangiectasiamutated (ATM) and Nijmegen breakage syndrome (NBS1)genes interact to bring about the activation of the p53-dependentresponse to ionizing radiation and the initiation of the repair of double-strand breaks?• Is ATM a predisposing gene in sporadic breast cancers or isit only involved in a sub-group of breast cancers showing radiation sensitivity?• Is NBS1 a tumour suppressor gene in B-cell lymphomas?

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R e v i e w s MOLECULAR MEDICINE TODAY, APRIL 1999 (VOL. 5)

Focus on cancer

There’s something for cancer researchers in almost every issue of Molecular Medicine Today.As well as cutting-edge news, literature reports and meeting reports, we publish a wide varietyof reviews of relevance to cancer. Here’s a collection of recent reviews to whet your appetite...

McKenzie, K.E., Umbricht, C.B. and Sukumar, S. (1999) Applications of telomerase in the fightagainst cancer, Mol. Med. Today 5, 114–122

van Steeg, H. and Kraemer, K.H. (1999) Xeroderma pigmentosum and the role of UV-induced DNAdamage in skin cancer, Mol. Med. Today 5, 86–94

Colaco, C.A.L.S. (1999) Why are dendritic cells central to cancer immunotherapy? Mol. Med. Today 5, 14–17

Przepiorka, D. and Srivastava, P.K. (1998) Heat shock protein–peptide complexes as immunotherapy for humancancer, Mol. Med. Today 4, 478–484

Bustin, S. and Dorudi, S. (1998) Molecular assessment of tumour stage and disease recurrence using PCR-based assays, Mol. Med. Today 4, 389–396

Agrawal, B., Gendler, S.J. and Longenecker, B.M. (1998) The biological role of mucins in cellular interactions and immune regulation: prospects for cancer immunotherapy, Mol. Med. Today 4, 397–403

Ming, J.E., Roessler, E. and Muenke, M. (1998) Human developmental disorders and the sonic hedgehog pathway, Mol. Med. Today 4, 343–349

Romero, P., Cerottini, J-C. and Waanders, G.A. (1998) Novel methods to monitor antigen-specific cyto-toxic T-cell responses in cancer immunotherapy, Mol. Med. Today 4, 305–312

Kubbutat, M.H.G. and Vousden, K.H. (1998) Keeping an old friend under control: regulation of p53 stability, Mol. Med. Today 4, 250–256

Articles planned for future issues include: Papillomavirus-like particle vaccines for cervical cancer;Nucleoside transporters: molecular biology and implications for therapeutic development; Theputative role of cell adhesion molecules in endometriosis: can we learn from tumor metastasis?;Gene therapy for chronic myelogenous leukemia, and more.

Golgi

Dendritic cell

d

a Tumour cell

b Virally infected cell

e CTL precursor

CTL

c Microbe

42 Sankaranarayanan, K. and Chakraborty, R. (1995) Cancer predisposition, radiosensitivity and the risk of radiation-induced cancers. I. Background,Radiat. Res. 143, 121–143

43 Goss, P.E. and Sierra, S. (1998) Current perspectives in radiation-inducedbreast cancer, J. Clin. Oncol. 16, 338–347

44 Brock, W.A. et al. (1995) Fibroblast radiosensitivity versus acute and late normal skin responses in patients treated for breast cancer, Int. J. Radiat.Oncol. Biol. Phys. 32, 1371–1379

45 West, C. et al. (1995) A comparison of the radiosensitivity of lymphocytes fromnormal donors, cancer patients, individuals with ataxia telangiectasia (A-T)and A-T heterozygotes, Int. J. Radiat. Biol. 6, 175–184

46 Ramsay, J., Birrell, G. and Lavin, M. (1998) Testing for mutations of the ataxiatelangiectasia gene in radiosensitive breast cancer patients, Radiother. Oncol.47, 125–128

47 Ramsay, J. et al. (1998) Radiosensitive melanoma cell line with mutation of thegene for ataxia telangiectasia, Br. J. Cancer 77, 11–14

48 Vorechovsky, I. et al. (1997) Clustering of missense mutations in the ataxiatelangiectasia gene in sporadic T-cell leukaemia, Nat. Genet. 17, 96–99

49 Stoppa-Lyonnet, D. et al. (1998) Inactivation of the ATM gene in T-cell pro-lymphocytic leukemias, Blood 91, 3920–3926

50 Stilgenbauer, S. et al. (1997) Biallelic mutations in the ATM gene in T-pro-lymphocytic leukemia, Nat. Med. 3, 1155–1159

51 Vorechovsky, I. et al. (1996) ATM mutations in cancer families, Cancer Res. 56,4130–4133

52 Vorechovsky, I. et al. (1996) The ATM gene and susceptibility to breast cancer:analysis of 38 breast tumours reveals no evidence for mutation, Cancer Res.56, 2726–2732

53 Ramsay, J., Birrell, G. and Lavin, M. (1996) Breast cancer and radiotherapy inataxia telangiectasia heterozygote, Lancet 347, 1627

54 Fitzgerald, M.G. et al. (1997) Heterozygous ATM mutations do not contributeto early onset breast cancer, Nat. Genet. 15, 307–310

55 Appleby, J.M. et al. (1997) Absence of mutations in ATM gene in breast cancer patients with severe responses to radiotherapy, Br. J. Cancer 76,1546–1549

56 Bay, J-O. et al. (1998) No evidence for constitutional ATM mutation inbreast/gastric cancer families, Int. J. Oncol. 12, 1385–1390

57 Gilad, S. et al. (1996) Predominance of null mutations in ataxia-telangiectasia,Hum. Mol. Genet. 5, 433–439

58 Telatar, M. et al. (1998) Ataxia-telangiectasia: identification and detection offounder-mutations in the ATM gene in ethnic populations, Am. J. Hum. Genet.62, 86–97

59 Jackson, S.P. (1996) DNA damage detection by DNA dependent protein kinaseand related enzyme, Cancer Surv. 28, 261–279

60 Feunteun, J. (1998) Breast cancer and genetic instability: the molecules behindthe scenes, Mol. Med. Today 4, 263–267

61 Baumann, P. and West, S.C. (1998) Role of the human RAD51 protein in homologous recombination and double-strand break repair, Trends Biochem.Sci. 23, 247–251