Ataxia-Telangiectasia and Cellular Responses to DNA Damage...Ataxia-telangiectasia (A-T) is a human disease characterized by high cancer risk, immune defects, radiation sensitivity,

1995;55:5991-6001. Published online December 1, 1995.Cancer Res M. Stephen Meyn Ataxia-Telangiectasia and Cellular Responses to DNA Damage

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(CANCERRESEARCH55. 5991-6001. December 15, 19951

Review

Ataxia..Telangiectasia and Cellular Responses to DNA Damage'

M. Stephen Meyn2

Departments of Genetics and Pediatrics, Yale University School of Medicine, New Haven, connecticut 06510

Abstract

Ataxia-telangiectasia (A-T) is a human disease characterized by highcancer risk, immune defects, radiation sensitivity, and genetic instability.Although A-T homozygotes are rare, the A-T gene may play a role insporadic breast cancer and other common cancers. Abnormalities of DNArepair, genetic recombination, chromatin structure, and cell cycle checkpoint control have been proposed as the underlying defect in A-T; however, previous models cannot satisfactorily explain the plelotropic A-Tphenotype.

Two recent observations help clarify the molecular pathology of A-T:(a) inappropriate p53-mediated apoptosis is the major cause of death inA-T cells irradiated in culture; and (b) ATM, the putative gene for A-T,has extensive homology to several celi cycle checkpoint genes from otherorganisms. Building on these new observations, a comprehensive model ispresented in which the ATM gene plays a crucial role in a signal transduction network that activates multiple cellular functions In response toDNA damage. In this Damage Survefflance Network model, there is nointrinsic defect in the machinery of DNA repair in A-T homozygotes, buttheir lack of a functional ATM gene results In an Inabifity to: (a) halt atmultiple cell cycle checkpoints in response to DNA damage; (b) activatedamage-inducible DNA repair; and (c) prevent the triggering of programmed cell death by spontaneous and induced DNA damage. Absenceof damage-sensitive cell cycle checkpoints and damage-induced repairdisrupts immune gene rearrangements and leads to genetic instability andcancer. Triggering of apoptosis by otherwise nonlethal DNA damage isprimarily responsible for the radiation sensitivity ofA-T homozygotes andresults in an ongoing loss of cells, leading to cerebellar ataxia and neurological deterioration, as well as thymic atrophy, lymphocytopenla, and apaucity of germ cells.

Experimental evidence supporting the Damage Surveffiance Networkmodel is summarized, followed by a discussion of how defects in the

ATM-dependent signal transduction network might account for the A-Tphenotype and what insights this new understanding of A-T can offerregarding DNA damage response networks, genomic instability, and cancer.

Previous Models for A-T3

A-T is an autosomal recessive disease with a pleiotropic phenotypethat includes progressive cerebellar ataxia, cellular and humoral immune defects, progenc changes of the skin, endocrine disorders,gonadal abnormalities, and a high incidence of cancer; the relative riskof developing some tumors (e.g. , lymphoma) is several hundredfoldhigher than normal ( 1, 2). Heterozygote carriers are also at increasedrisk for cancer, particularly breast carcinoma (2). A-T cells are sensitive to the killing effects of ionizing radiation, and they exhibit in

vivo and in vitro abnormalities consistent with a defect involvingDNA metabolism and/or maintenance of genomic integrity [e.g.,

Received 8/21/95; accepted 10/16/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I This work has been supported by grants from the NIH and the A-I Children's

Project.2 lo whom requests for reprints should be addressed, at Yale University School of

Medicine, 333 Cedar Street, P.O. Box 208005, New Haven. CI 06520-8005.3 The abbreviations used are: A-I, ataxia-telangiectasia; LOH, loss of heterozygosity;

ICR, I-cell receptor;P13-kinase.phosphatidylinositol3-kinase.

elevated frequencies of spontaneous and induced chromosome aberrations, high spontaneous rates of intrachromosomal recombination,aberrant immune gene rearrangements, and inability to arrest the cellcycle in response to DNA damage (3â€”6)].

The nature of the A-T defect has been the subject of much

speculation; most hypotheses focus on the radiation sensitivity ofA-T cells. Early reports that A-T fibroblasts were unable to exciseradiation-induced DNA adducts prompted suggestions that theradiation sensitivity of A-T cells was due to an intrinsic defect inDNA repair (7). However, subsequent work indicated that not allA-T fibroblasts have a defect in DNA adduct excision (8), and that

the kinetics of repair of DNA breaks and chromosome aberrationsis grossly normal (reviewed in Ref. 3). Functional abnormalities ofspecific repair enzymes have been proposed [e.g. , topoisomerase II(9) and poly(ADP-ribosylase) (10)], but conclusive evidence ofrepair enzyme defects in A-T is lacking. Structural abnormalitiesof chromatin, subtle defects in DNA repair that affect repairquality, and abnormalities of differentiation also have been offeredas explanations for the A-T phenotype (11â€”15).

Several investigators have suggested that a defect in genetic recombination, resulting in an inability to productively rearrange and repairgenes, would provide a unifying explanation for radiation sensitivity,immune defects, and karyotypic abnormalities in A-T (5, 6, 16â€”18).However, a defect in genetic recombination is difficult to resolve withreports of near-normal frequencies of extrachromosomal recombinalion (4, 19, 20) and the observation that spontaneous rates of chro

mosomal recombination in A-T fibroblast lines are 30- to 200-foldhigher than normal (4). Other investigators, struck by the inability ofirradiated A-T cells to temporarily halt DNA replication and cell cycleprogression, have proposed that A-T cells cannot recognize or respondto DNA damage (1 1, 12, 21â€”23).In these models, the radiationsensitivity of A-I cells generally is assumed to result from an inabilityto delay the cell cycle to allow sufficient time to repair DNA damage.These models could explain why A-T cells are radiation sensitivedespite grossly normal DNA repair, but they cannot readily accountfor several studies in which experimental conditions that prolonged or

temporarily halted the cell cycle did not improve the survival ofirradiated A-I cells (24â€”26). Neither do these models explain another

puzzling feature of A-I: unlike most normal mammalian cells, fatallyirradiated A-I cells typically die before they can complete the firstpostirradiation mitosis (113â€”115).

The finding that the radiosensitivity of A-I cells in culture isprimarily the result of inappropriate apoptosis, together with recentobservations regarding the role that the p53 tumor suppressor proteinplays in mediating cellular responses to DNA damage, has led to thedevelopment of a new model regarding the nature of the A-I defect.Initial analysis of the A-T disease gene supports this model, which isrelated to previous proposals in which the A-T gene product normallymediates cellular responses to DNA damage (e.g., see Ref. 22), butovercomes the objections to those models, cited above, while providing a unifying explanation for the pleiotropic manifestations of thedisease.

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A COMPREHENSIVE MODEL FOR ATAXIA-TELANGIECTASIA

Ihe Damage Surveillance Network Model

Biological organisms are not passive targets ofDNA-damaging agents;they actively respond to DNA damage [e.g., the SOS system in Escherichia coli (30)]. A growing body of evidence indicates that the responseof mammaliancellstoDNA damage iscomplex,perhapsinvolvingseveral interrelated signal-transductionnetworks that detect DNA damage, activate DNA repair, and alter the cell cycle (22, 27â€”29).Fig. IAdepicts a damage response network in which the A-T gene product plays

a critical role. The detection of certain types of spontaneous or inducedDNA damage triggers this signal transduction network, resulting in theactivation of a group of cellular functions that promote genetic stability

by temporarily arresting the cell cycle and enhancing DNA repair. At the

same time, the A-I-dependent network promotes cellular survival byinhibiting execution of damage-induced programmed cell death. In addition to the five responses illustrated in Fig. 1A, there also may be other,

asyetundefined,A-I-dependentfunctions.

No enhanced reactIvationof Irradiated virus

No enhanced mutagenesisof irradiated virus

increased genomic instabilitychromosomal translocationsepithelial cells with micronudelaneuploid cells in muftipletissuesalleleloss in erythrocytesICR transrearrangementsrecombination between repeated genesinduced chromosome aberrationssolid tumors

Disruption of Immune gene rearrangementLowlevelsof lgA, IgE, g02 and lgG4Lowproportionof I cells expressingW@3TCRsFrequent translocatlons near immune genesIncreased risk of lymphoma and leukemia

Abnormal cell cycle kinetics following DNAdamageC ck nt RadioresistantDNAsynthesis

Fig. I . A, a DNA Damage Surveillance Network. As part of this signal transduction network, the ATM protein activates at least five cellular functions in response to the detectionof spontaneous or induced DNA damage. In B, the DNA damage surveillance network is defective in A-I homozygotes. A-I homozygotes cannot activate ATM-dependent functionsin response to DNA damage. resulting in the pleiotropic A-I phenotype.

ActivatedDNARepair

GuSCheckpoint

S phaseCheckpoint

GRIMCheckpoint

â€˜V d2

D air

9@PQk@t

A. NormalIndividuals

@GADD45@,t p21 â€˜t'@@

p53

Spontaneous and fInduced

DNADamage@. ssandds ___________

ImmuneGene DNAbreaks@ ATMRearrangments \ I@

ShortTelomeresp53

B. A-T Homozygotes

@GADD45@ /@ p21@p53

Spontaneousand IInduced

DNADamage@ 55 and ds @.@ *-

ImmuneGene DNAbreaksRearrangments %4@@

ShortTelomeresp53

ProgrammedCell Death

Increased spontaneous cell deathprogressive loss of neuronsthymic hypoplasia and lymphocytopeniadepletion or absence of germ cellshypoplastic thyroid and adrenalsprogeric changes in skin and haircirrhosis and elevated serum AFP

Increased mutagen-lnduced cell deathMarked sensitivity to killingby:

Ionizing radiationradiomimetic drugs

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The primary abnormality in A-I homozygotes presumably createsa defect in this network that prevents the activation of these cellularfunctions in response to strand breaks, shortened telomeres, and otherDNA lesions (Fig. 1B). This inability to respond to spontaneous andinduced DNA damage results in increased genomic instability, as wellas in an unusually low threshold for the triggering of p53-mediatedapoptosis by otherwise nonlethal DNA damage. These abnormalitieslead, in turn, to the multiple in vivo and in vitro abnormalities seen inA-I homozygotes (Fig. 1B). Although they are not as severely affected as homozygotes, A-I heterozygotes may not have a fullyfunctional damage response network, because they express subtleabnormalities in their cellular responses to ionizing radiation (3) andhave an increased relative risk of cancer (2).

A-I Homozygotes Lack DNA Damage-activated Functions

Absence of Cell Cycle Checkpoints. In normal eukaryotic cells,the cell cycle halts temporarily after the induction of strand breaks incellular DNA by a variety of agents, including ionizing radiation,radiomimetic drugs, restriction enzymes, and topoisomerase inhibitors(reviewed in Refs. 23 and 29). There are at least three damagesensitive cell cycle â€œcheckpointsâ€•in mammalian cells: one at the G1-Sborder, one in S phase, and one at the G2-M boundary. These checkpoints also may restrain the cell cycle in response to the generation ofstrand breaks, shortened telomeres, and other DNA damage thatoccurs spontaneously during the course of normal DNA metabolism(e.g. , site-specific gene rearrangements, genetic recombination, andrepair of replication errors). Given the viability of yeast and mammahan mutants that lack functional damage-sensitive cell cycle checkpoints (31â€”33),these checkpoints probably are not invoked duringmost cell cycles. The relative timing of DNA replication, DNA repair,genetic recombination, and cell division ensures that genomic integrity is restored before the cell cycle reaches a checkpoint (23, 29).

The tumor suppressor protein p53 plays a key role in activating theG1-s checkpoint after certain types of DNA damage [reviewed in Ref.29; e.g., mammalian cells increase intracellular levels of p53 proteinshortly after exposure to many agents that induce DNA strand breaks,

and functional p53 protein typically is required for activation of theGl-s checkpoint after ionizing radiation exposure (22, 34, 35)]. G1-Scell cycle arrest occurs via p53-mediated transcriptional activation ofthe p21 (WAF-JICIPJISDIJ) gene, which codes for a protein thatbinds to cyclin-dependent kinase-cyclin complexes and inhibits theirkinase activities (22, 36, 37). The p21 protein may be involved in theS-phase checkpoint as well because it can bind to proliferating cellnuclear antigen/replication factor C/pol 6 complexes in vitro andblock the elongation step of DNA polymerization (38, 39). TheS-phase checkpoint may be p53 independent because cells lackingfunctional p53 can express a radiation-induced S-phase checkpoint, asindicated by normal suppression of DNA synthesis after irradiation(40). In mammaliancells,theG2-Mdamage-activatedcheckpointprobably is also p53 independent (e.g., see Ref. 41); however, relalively little is known about its genetics and biochemistry. In contrast,at least 10 yeast genes are involved in controlling the G2-M checkpoint (31, 32, 42, 43).

A-I homozygotes lack the p53-mediated G1-S damage-sensitivecheckpoint, and the kinetics of p53, p21, and GADD45 induction byionizing radiation are abnormal in the cells of A-I homozygotes (22,44, 45). These observations led Kastan et aL (22) to propose that theA-T gene product(s) functions upstream of p53 in a damage-responsive pathway that leads to Ge-S arrest. This pathway is incorporatedin the Damage Surveillance Network model as one branch of theATM-dependent network (Fig. 1A). A-I cells do not have the S-phasecheckpoint, because they fail to arrest DNA synthesis when irradiated

in S phase, resulting in the phenomenon of radioresistant DNA synthesis (11). The G2-M checkpoint appears to be defective in A-T cellsas well, in that both A-T fibroblasts and lymphocytes irradiated in 02fail to undergo the initial radiation-induced 02-M delay seen innormal cells (46, 47, 113).

Lack of Damage-activated DNA Repair. Exposing humancellsto low doses of radiation before infection of irradiated virus improvesviral survival and increases the number of mutant viruses recovered(48). These effects, termed â€œenhancedsurvivalâ€•and â€œenhancedmutagenesis,â€•have been demonstrated using both single-stranded anddouble-stranded DNA viruses (48â€”50),but they are not as striking asthe analogous phenomena in bacteria, Weigle reactivation and Weiglemutagenesis (30).

Weigle reactivation results from induction of an error-prone DNArepair pathway in bacteria; similarly, one or more damage-activatedDNA repair processes could be responsible for enhanced survival andenhanced mutagenesis in mammalian cells. At least some form ofdamage-activated repair may be p53 dependent in human cells. Smithet aL (51) recently demonstrated that loss of p53 function in humancells was associated with modest decreases in clonogenic survival, aswell as reduced repair of UV-damaged reporter plasmids. Activationof excision DNA repair, as measured by an in vitro assay, also wasdecreased in p53@ cells (52). The p53 protein role in damageactivated repair might occur either by direct interaction with repairenzyme complexes (52) and/or by activation ofGADD45, which itselfhas been shown to bind to proliferating cell nuclear antigen andstimulate DNA excision repair in vitro (53). Although not extensivelystudied, damage-activated DNA repair appears to be impaired in A-Ihomozygotes because A-T fibroblasts have been shown to lack theenhanced survival and enhanced mutagenesis expressed by controlhuman cells for irradiated H-l parvovirus and adenovirus 2 (49, 50,54).

Low Threshold for Triggering Programmed Cell Death afterDNA Damage. A growing body of evidence documents that lowdoses of ionizing radiation kill some types of mammalian cells in vivoand in vitro by activating apoptosis, a well-characterized form ofprogrammed cell death (reviewed in Ref. 55) that can be carried outin both cycling (56) and noncycing cells (57).

Although researchers have only recently begun to focus on thepossible role of programmed cell death in the A-I phenotype, severalin vivo and in vitro findings support such a possibility. Histologicalanalyses of cerebella taken from A-T homozygotes at autopsy document a high frequency of abnormal Purkinje and granule cells thatexhibit the highly condensed, pyknotic nuclei expected from programmed cell death in neurons (58â€”60).An in vitro flow cytometricstudy that examined the effects of the radiomimetic drug streptomgrinon A-T fibroblasts described changes in the DNA content of treatedA-I cells that are consistent with apoptosis (61).

Following up on these observations, we recently demonstrated thatfibroblasts and lymphoblasts representing all A-I complementationgroups undergo apoptotic death in culture after exposure to lowradiation and streptomgrin doses that do not induce appreciable apoptosis in control cells (62).@This inappropriate apoptosis appears tobe mediated by p53, in that it is suppressed in A-T fibroblasts, the p53protein of which has been functionally inactivated by transfectionwith either a dominant-negative p.53 gene or a human papilloma virusE6 gene. Iransfection-induced loss of p53 function did not affect

survival of control fibroblasts, but transfected A-I cells acquirednear-normal resistance to ionizing radiation, suggesting that p53-

4 M. S. Meyn, L Strasfeld, and C. Allen. p53-mediated apoptosis is the primazy cause

of radiosensitivity in ataxia-telangiectasia, manuscript in preparation.

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mediated apoptosis is the major cause of radiosensitivity in A-I cellsin culture (62).@

Initial Analysis of the ATM Gene Supports the DamageSurveillance Network Model

Earlier this year, Savitsky et a!. (63) positionally cloned a genefrom the 1lq23.l A-I locus that is mutated in A-I patients from allcomplementation groups. The gene, termed ATM (A-I, mutated), isconserved in vertebrates and codes for a I2-kb transcript that isabundantly expressed in multiple tissues in vivo. As shown in Fig. 2,the carboxy terminus of the putative AIM protein is homologous tothat of at least four checkpoint proteins from other organisms: Drosophila melanogaster MEI-41, Schizosaccharomyces pombe Rad3,Saccharomyces cerevisiae MEC1p, and S. cerevisiae TEL1p (63â€”65).The region of strongest homology between these five proteins containsa phosphatidylinositol 3-kinase domain, suggesting that proteins areinvolved in signal transduction.

The ATM gene shares phenotypic similarities with these genes aswell. Like A-I homozygotes, mei-41, rad3, and med mutants areX-ray sensitive and lack damage-induced cell cycle checkpoints (42,43, 65). In addition, A-I, mei-41, rad3, and tell homozygotes expressincreased chromosomal instability and have high spontaneous rates ofmitotic recombination (1, 4, 64â€”66).Taken together, the physical andphenotypic similarities between these checkpoint genes and ATMsuggest that the ATM gene activates multiple cellular functions inresponse to spontaneous and induced DNA damage.

Lack of Damage-activated Functions Explains the Phenotype ofA-T Homozygotes

Lack of damage-activated functions in A-I homozygotes due to adefect in a signal transduction network that monitors genomic integrity, as proposed in the Damage Surveillance Network model described above, could explain the pleiotropic nature of the clinical andlaboratory abnormalities associated with the disease. Fig. lB summarizes the consequences of lack of damage-activated functions in A-Thomozygotes, along with the resulting phenotypic effects. How theDamage Surveillance Network model accounts for each aspect of the

A-I phenotype when previous models cannot is discussed below.Cell Cycle Checkpoint Defects Disrupt Immune Gene Rear

rangements. The immunoglobulinheavy-chaingene andICR genesundergo site-specific gene rearrangements during normal development, resulting in production of IgA, IgE, IgG2, and IgG4 by Blymphocytes and in expression of a/j3 heavy-chain ICRs by I lymphocytes (67, 68). A-I homozygotes have deficiencies in these specific immunoglobulin classes, as well as a relative lack of I lymphocytes expressing a/@3heavy-chain TCRs (reviewed in Ref. 3). They

also have marked elevations in the frequency of I lymphocytesexpressing y/13, â€˜SIP,aTh, or y/6 heavy-chain ICRs as a result ofaberrant interlocus gene rearrangements (5, 6) and a high frequency ofB and I lymphocytes carrying chromosome translocations involvingsites on chromosomes 2, 7, and 14 near the immunoglobulin supergene family genes (reviewed in Ref. 17).

The Damage Surveillance Network model for A-I explains theseclinical findings by assuming that the gene rearrangements necessaryfor immunoglobulin switch recombination and ICR heavy-chain rearrangements frequently trigger the A-I damage surveillance networkand its cell cycle checkpoints, perhaps in response to the creation ofdouble-strand breaks during immune gene recombination. Normalcells halt the cell cycle temporarily to allow completion of theseimmune gene rearrangements (69), but cells of A-I homozygotescannot activate cell cycle checkpoints. Hence, A-I cells that areundergoing switch recombination or ICR heavy-chain rearrangementmay attempt to replicate their DNA and/or proceed past the G2-Mcheckpoint into mitosis before resolving these recombination complexes. Manipulation of the immune recombination complexes duringDNA replication, chromosome condensation, and/or mitosis then disrupts the complexes, creating chromosomes that contain free DNAends generated during abortive immune gene rearrangement. Thesefree ends are highly recombinogenic and could serve as foci forillegitimate recombinational events that lead to interlocus gene rearrangements and chromosome translocations. The final result is anabnormally high frequency of lymphocytes carrying translocationsnear immune genes or expressing aberrantly recombinant ICRs. DNAdamage resulting from disrupted immune gene rearrangements alsomay trigger apoptosis in A-I lymphocytes (see below), which, byeliminating cells undergoing immunoglobulin switch or ICR recombination, would contribute to the selective immunoglobulin deficiencies and paucity of a/@3ICR-expressing I cells seen in A-I homozygotes.

Unlike A-I homozygotes, p53-null mice do not exhibit aberrantimmune gene rearrangements, nor do their peripheral lymphocytesexpress the type of chromosome translocations characteristic of A-Ilymphocytes (33, 70). This suggests that loss of the p53-mediatedG1-S checkpoint is not as disruptive to immune gene recombination asloss of the S-phase and G2-M checkpoints.

Cell Cycle Checkpoint Defects Cause Spontaneous Genetic Instability. Cells that lack functional p53 protein fail to halt at the G1-Sborder after irradiation, but will arrest the cell cycle in S phase and atthe G2-M border (34, 35, 40). Hence, cells that are defective for p53function have a specific defect in the G1-S damage-sensitive checkpoint but not the S-phase or G2-M checkpoints. Several studies havedemonstrated spontaneous genetic instability in mammalian cells that

Protein Length(a.a.)@ ATM 3056

DNA-PK@5 4096

MEI-41

TEL1p

RAD3

2357

2787

2386

MEC1p 2368

Fig. 2. Block alignment of predicted protein products for the ATM (146), DNA-PK@@(130), mei-41 (65), TELl (64), radY (146), and MECI (147) genes. (U. P1-3 kinase domainsthat show 60â€”70%similarity between all family members; (I]), regions of 40â€”50%similarity. Alignments were generated using BLAST@analysis (148). a.a, amino acids.

kinasedomain

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A COMPREHENSIVE MODEL FOR ATAXIA-TELANGIECFASIA

lack the G1-S checkpoint as a result of mutations in the p53 gene.Fibroblasts from p53-null mice and patients with Li-Fraumeni syndrome, as well as cell lines that have lost wild-type p53 alleles orharbor dominant-negative p53 mutations, lack functional p53 and

show increased frequencies of chromosome loss, chromosome aberrations, intrachromosomal genetic recombination, and/or gene amplification events (33, 62, 71â€”73).Given the genetic instability of cellsthat lack a -S cell cycle checkpoint because of loss of p53 function,the G1-S cell cycle checkpoint defect is likely to contribute to the highspontaneous frequencies of chromosome aberrations, genetic recombination. aneuploid cells, and allele loss seen in different A-I celltypes in Vit'() and in vitro ( I 7. 74â€”77). Inability to arrest at the S-phase

and G2-M DNA damage checkpoints also may contribute to spontaneous chromosome instability in A-T cells, in that S. cerevisiae rad9mutants that have lost the G,-M damage-sensitive checkpoint showmarked increases in the frequency of spontaneous chromosome loss atmitosis (78).

The spontaneous genetic instability seen in nonimmune A-I cellsmay be due to a mechanism similar to that outlined above for theimmune system@an inability to activate cell cycle checkpoints in theface of spontaneous DNA damage allows A-T cells to attempt toreplicate DNA (G1-S and S-phase checkpoints) or enter mitosis(G,-M checkpoint) before completion of repair of the damage. Replication or mitosis then results in breaks and gaps in chromosomalDNA that promote the formation of acentric chromosome fragments,facilitate generation of aneuploid daughter cells through missegregation, and serve as substrates for chromosome translocations and mitotic recombination.

The AIM-dependent damage surveillance network may monitortelomeres as well, preventing the propagation of cells containingchromosomes with short telomeres by triggering cell cycle checkpoints when telomeres fall below a critical size. In this scenario, shorttelomeres would be unable to arrest cell division in cells from A-Ihomozygotes, leading to the gradual accumulation of cells with shortened telomeres. This would explain why A-I cells in culture haveshorter telomeres and fewer telomeric hybridization signals than do

control cells (79). Aberrant telomeres, in turn, may cause telomeretelomere associations, which are a characteristic karyotypic feature ofboth A-I cells and senescing cells from normal individuals (17, 80).Short telomeres also may contribute to the overall genomic instabilityof A-I cells through their tendency to provoke chromosomerearrangements.

Cell Cycle Checkpoint Defects Contribute to Induced Chromosome Aberrations. The S. cerevisiae rad9 and S. pombe rad3 mutants exhibit high levels of chromosome aberrations after irradiation,presumably as a result of defective G1-S and G2-M damage checkpoints (43, 8 1). Defective cell cycle checkpoints could contribute tothe high residual frequency of radiation-induced chromosome andchromatid aberrations seen in A-I cells when assayed at the firstpostirradiation mitosis (82, 83). Although the kinetics of DNA breakrepair are grossly normal in A-I cells, the lack of checkpoint restrainton forward progress of the cell cycle into DNA synthesis and mitosiseffectively gives A-I cells less time in which to remove DNA breaksbefore they give rise to chromosome aberrations (irradiation inand chromatid aberrations (irradiation in G,).

Cell Cycle Checkpoint Defects Lead to Cancer. A growing bodyof evidence indicates that multiple genetic changes resulting in loss offunction at tumor suppressor genes and gain of function at oncogenesare critical to the development of cancer (29, 84). Accumulation ofthese changes is more likely to occur in cells from individuals withconstitutional genetic instability. Hence, it is not surprising that cancers occur at high frequencies in A-I homozygotes (85). G@-S, Sphase, and G,-M cell cycle checkpoint defects may contribute to the

increased incidence of solid tumors in A-I homozygotes by increasing the occurrence of spontaneous and induced chromosome aberrations, mitotic recombination, and LOH.

A-I homozygotes face a 250- to 700-fold increased risk of developing leukemia and non-Hodgkin's lymphoma (2, 85), tumors thatfrequently harbor chromosome rearrangements involving immunoglobulin supergene family genes ( 17). Kastan et a!. (22) noted thatboth A-I homozygotes and p53-null mice have a predilection forimmune system tumors and suggested that an abnormal response toDNA strand breaks may be responsible for the high incidence oflymphoid tumors in both cases. In the Damage Surveillance Networkmodel, this abnormal response would be an inability to trigger the A-Idamage surveillance network and activate its cell cycle checkpoints.A-I lymphoid cells would become malignant as a consequence of theactivation of cellular oncogenes by chromosome translocations resulting from disruption of the normal rearrangement and repair of immune gene DNA. The specific increase in lymphoid tumors seen inA-I homozygotes suggests that the initial production of strand breaksand other DNA damage is a rate-limiting step in oncogenesis, even incells that are genetically unstable because of a lack of DNA damagesensitive cell cycle checkpoints.

Lack of Damage-activated DNA Repair Prevents EnhancedSurvival and Mutagenesis. Heightened ability to repair DNA damage resulting from radiation exposure to host cells is the putativecause of enhanced survival and enhanced mutagenesis of irradiatedviruses in mammalian cells. The contribution of damage-activatedrepair to cellular survival after DNA damage is less certain. Loss ofdamage-activated repair is associated with a modest increase in UVsensitivity but does not appreciably affect the clonogenic survival ofX-irradiated cells (5 1, 7 1, 86, 87). This differential effect on UVdamage may be due to the fact that damage-activated repair is primanly a form of enhanced excision repair (5 1). The Damage Surveillance Network model assumes that, whatever its mechanism, thisenhanced ability to repair DNA damage can be activated by normalcells but not by A-I cells or cells without functional p53.

Lack of damage-activated repair does not appear to be a majorfactor in the X-ray sensitivity of A-I cells (see below), but it may bepartially responsible for the mild UV sensitivity seen in some A-Icells (3). Lack of damage-activated repair also could contribute to thehigher than normal residual level of double-strand breaks seen inX-irradiated A-I cells (82, 88). Alternatively, the residual doublestrand breaks in A-I cells could be due to initiation of apoptosismediated nucleolytic degradation of genomic DNA.

Dysfunctional Programmed Cell Death Leads to SpontaneousCell Loss. Autopsies of A-I homozygotes have documented chronicspontaneous loss of Purkinje cells and granule cells in the cerebellum,as well as depletion of other neurons in the central nervous system ofolder patients (reviewed in Ref. I). Other tissues reported to beatrophic and/or hypoplastic in A-I homozygotes include the thymus,gonads, thyroid, and adrenals ( 1). Cirrhotic changes have been seen inthe livers of A-I homozygotes, together with patches of regeneratinghepatocytes, which may be the cause of their high serum levels ofa-fetoprotein (1, 89). Taken together, these autopsy findings indicatethat chronic spontaneous cell death occurs throughout the tissues ofA-I homozygotes.

Cell cycle checkpoint defects cannot easily account for this ongoingin vivo cell loss. Homozygous p53-null mice do not have detectableneurological abnormalities, immune defects, or sterility (33, 70).Hence, the G1-S checkpoint defect does not appear to be the cause ofthe cerebellar, immunological, and gonadal defects in A-I. DefectiveS-phase and G2-M checkpoints could cause a small part of the loss ofdividing cells in vivo, as well as contribute to the high frequency ofaneuploid giant cells found in A-I homozygotes (60, 76), a prediction

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based, in part, on the high frequency of spontaneous aneuploidyassociated with the G2-M checkpoint defect in rad9 mutant yeast (78).However, although defective S-phase and G2-M checkpoints may killsome dividing cells, they cannot cause the death ofcells arrested in G0(e.g., Purkinje cells and other neurons) in A-I homozygotes. In thisregard, it seems significant that the cell types most affected by in vivodegeneration in A-I are those that, in normal individuals, are mostsensitive to radiation-induced apoptotic cell death [e.g., lymphocytes,spermatogonia, and neurons in the developing cerebellum (reviewedin Ref. 90)].

The Damage Surveillance Network model predicts that the primarycause of spontaneous loss of both dividing and nondividing cells intissues of A-I homozygotes is inappropriate activation of programmed cell death by DNA damage that occurs spontaneouslyduring normal DNA metabolism and as the result of ordinary environmental insults. In the Damage Surveillance Network model,chronic cell loss due to programmed cell death depletes Purkinje cellsand other neurons from the central nervous system, I-cell and B-cell

precursors from the immune system, and germ cells from the gonads.These losses account for the progressive cerebellar ataxia and intellectual arrest seen in A-I homozygotes, as well as their thymicatrophy, reduced numbers of circulating lymphocytes, and paucity ofgerm cells (1).

Dysfunctional Activation of Programmed Cell Death CausesMutagen Sensitivity. The striking sensitivity of A-I cells to killingby ionizing radiation and radiomimetic drugs has been a long-standingpuzzle that has not been adequately explained in the past.

Multiple biochemical studies have failed to detect gross abnormalities in the kinetics of single-strand and double-strand break repair inA-I cells (e.g., see Ref. 91, 92). Other reports have found no evidence

that A-I cells are functionally defective in DNA repair (93â€”95).Onthe other hand, several studies found slight increases in the fraction ofbreaks left unrepaired in irradiated A-I cells (82, 88), as well asabnormalities in the rejoining of restriction enzyme breaks in plasmidstransfected into A-I fibroblasts (13, 14). To account for the seemingdisparity between the various functional and biochemical studies ofirradiated A-I cells, it has been suggested that the repair defect in A-Iis subtle, perhaps the result of impaired accuracy in strand rejoining(13, 14) or an inability to repaira smallbut critical fractionofdouble-strand breaks (82, 96â€”98).

Subtle but critical defects in DNA repair might contribute to theradiation sensitivity of A-I cells and help to explain the increasednumber of persistent chromosome aberrations observed in irradiatedA-I cells at the first postirradiation mitosis (82, 83). However, DNA

repair defects alone cannot readily account for the cell cycle abnormalities observed in A-I cells. To explain both radiosensitivity andcell cycle abnormalities, several investigators have proposed that theenzymatic machinery for DNA repair and recombination is essentiallyintact in A-I but that the in vivo and in vitro radiosensitivity of A-Icells is due to inability to activate cell cycle checkpoints in responseto DNA damage (1 1, 12). Experimental evidence suggests, however,that the effects of checkpoint defects on the survival of irradiated A-Icells are minor.

It is unlikely that the G1-S checkpoint defect plays a significant rolein the sensitivity of A-I cells to the lethal effects of induced DNAdamage. Cells lacking the G1-S checkpoint as a result of defects inp53 expression or function are not radiosensitive (e.g. , see Refs. 40and 71), and drug treatments that delay DNA replication in irradiatedA-I cells (e.g., see Ref. 99) do not enhance survival. The S-phase

checkpoint defect also has been dissociated from cell survival in A-Icells by gene transfer and cell fusion studies. After transfection of theA-I fibroblast line AT5BIVA with normal human genomic DNA,clones have been isolated that have partial (100) or complete (101)

restoration of normal survival after X-irradiation but still expressradioresistant DNA synthesis, indicating that their S-phase checkpoints remain defective. Fusion of HeLa cells to an A-I fibroblast lineresulted in heterodikaryons that exhibited normal survival after irradiation, despite the lack of an S-phase checkpoint, as measured byradioresistant DNA synthesis (40). Further evidence for the lack ofconcordance between S-phase checkpoint abnormalities and increasedsensitivity to the lethal effects of radiomimetic agents is provided byMirzayans and Paterson (2 1), who found that fibroblasts from oneA-I patient were hypersensitive to the killing effects of the mutagen

4NQO but exhibited a normal S-phase checkpoint after 4NQO exposure, whereas fibroblasts from another A-I patient had normal survival after 4NQO treatment but demonstrated complete lack of a4NQO-induced S-phase checkpoint.

Yeasts that lack a functional G2-M damage-sensitive checkpointbecause of mutations in the RAD9, RAD1, or RAD24 genes aresensitive to the killing effects of ionizing radiation (102), presumablybecause these mutations effectively shorten the postirradiation cellcycle, thereby giving cells less time to complete repair before mitosis.The behavior of the yeast G2-M checkpoint mutants suggests that theradiation sensitivity of A-I cells could be due to unchecked progression into mitosis before repair of potentially lethal DNA damage.However, a defective G2-M damage checkpoint cannot explain anunusual aspect of radiation sensitivity in A-I, the lack of â€œliquidholdingâ€•recovery. In both prokaryotes and eukaryotes, experimentalmanipulations that delay entry into the cell cycle or slow progressionof the cell cycle normally enhance the survival of irradiated cells (e.g.,see Ref. 103), a phenomenon that usually is demonstrated in mammalian cells by their recovery of colony-forming ability after a postirradiation period of growth inhibition. As might be expected, treatments that prolong or temporarily halt the postirradiation cell cycleare especially effective in increasing the survival of mutants that lack

a functional G2-M damage checkpoint [e.g., the S. cerevisiae rad9mutant (104) and the S. pombe radl mutant (105)]. In marked contrast, several studies have found that holding A-I fibroblasts in G0 forup to 7 days after irradiation does not significantly improve theirsurvival (24â€”26).This lack of liquid holding recovery in A-I fibroblasts argues against G2-M damage-sensitive checkpoint defects playing a major role in determining the survival of A-I cells afterirradiation.

Another aspect of the A-I phenotype that is not explained byprevious A-I models is the circumstances under which irradiated A-Icells die. It is well established that the lethal effects of ionizingradiation are associated with the production of double-strand breaks inchromosomal DNA (reviewed in Ref. 106), and it has been estimatedthat an average of 40 unrepaired double-strand breaks is sufficient tokill a normal diploid human cell (107). It is unlikely that so few breaksintroduced at random would directly damage a gene necessary for cellsurvival. Instead, unrepaired double-strand breaks are thought to kill

dividing mammalian cells because they give rise to acentric fragments, dicentrics, and other chromosome aberrations (108, 109).These chromosome aberrations then can undergo missegregation atmitosis, resulting in daughter cells with partial monosomies andtnsomies, the unbalanced karyotypes of which eventually prove fatal(1 10, 1 1 1). This conclusion is supported by observations that, for most

mammalian cells, radiation-induced death is not immediate but typically occurs in the first- and second-generation offspring of irradiatedcells (summarized in Ref. 112).

Previous models for the A-I defect have assumed that, like normalcells, irradiated A-I cells die as a result of genomic imbalance causedby persistent radiation-induced chromosome aberrations (82, 83).However, there is in vitro and in vivo evidence to the contrary. Unlikeother mammalian cells, fatally irradiated A-I fibroblasts do not divide

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several times before death. Instead, the majority arrest in G2 beforetheir first postirradiation mitosis (1 13â€”115). While arrested in G2,they undergo apoptosis (62)@ before any radiation-induced chromosome aberrations can missegregate. Several case reports document theextreme sensitivity of A-I homozygotes to the neurotoxic effects ofcentral nervous system irradiation given as treatment for cancer (116,117). Central nervous system neurons in children are nondividingcells, suggesting that at least part of the neurotoxicity of radiationtherapy in A-I homozygotes is the result of neurons being killedwhile in G0. Because irradiated A-I fibroblasts and neurons die beforeany induced chromosome aberrations can lead to genomic imbalance,other causes must be sought to explain their radiation sensitivity.

The Damage Surveillance Network model predicts that, becauseA-I cells lack a functional ATM gene, they cannot prevent theinitiation of programmed cell death by radiation-induced DNA lesions(Fig. 1B). As a result, radiation damage that would be nonlethal innormal cells triggers programmed cell death in A-I cells, which thenis carried out in G0 in noncycling cells and at the first postirradiationmitosis in actively dividing cells. In this way, the Damage Surveillance Network model overcomes objections to previous cell cyclebased A-I models and accounts for why (a) both cycling and noncycling A-I cells are radiosensitive despite having only minor defectsin DNA repair; (b) irradiated A-I fibroblasts do not undergo liquidholding recovery; and (c) irradiated A-I cells die before the firstpostirradiation cell division.

Discussion and Predictions

The Damage Surveillance Network model for A-I proposes that theA-I defect results in an inability to activate a group of diverse cellularfunctions in response to DNA damage. It offers a unifying explanationof how a single-gene defect can cause the pleiotropic phenotype seenin A-I homozygotes and explains several puzzling aspects of thedisease. The model assumes that the enzymatic machinery for DNArepair and genetic recombination is essentially intact, and it emphasizes the contribution of defective cell cycle checkpoints to geneticinstability and immune defects, two cardinal features of A-I. Byascribing the disruption of immunoglobulin switch recombination andICR rearrangements to cell cycle checkpoint abnormalities, and postulating that disruptions of immune gene rearrangements and of repairof spontaneous DNA damage lead to generation of recombinogenicbreaks and gaps in DNA, the Damage Surveillance Network modelexplains why immunoglobulin switch recombination and ICR rearrangement appear to be defective in A-I homozygotes, whereasspontaneous rates of recombination between directly repeated nonimmune genes in A-I fibroblasts are markedly higher than normal (4).The model can also account for the observation that chromosomaltranslocations in A-I lymphocytes cluster near immune genes,whereas translocations in A-I fibroblasts appear to involve randomsites throughout the genome (1 18). By assuming that the damagesurveillance network normally monitors telomere integrity, the modelexplains telomenc abnormalities seen in A-I cells.

The range of DNA lesions that might trigger the ATM network isuncertain, although strand breaks and gaps containing modified 3'termini are likely to play a major role, given the sensitivity of A-Ihomozygotes to physical and chemical agents that induce strandbreaks and small gaps containing 3' phosphoglycolates (119â€”121).Short telomeres also may activate the ATM network, suggesting thatthe ends of abnormally short telomeres may be structurally similar toDNA breaks generated by these agents. As indicated in Fig. 1, thesame lesions that activate A-I dependent cellular functions may alsotrigger p53-mediated programmed cell death.

How far upstream of p53 the ATM protein functions in the signal

transduction network is not certain. However, consideration of arelated human protein, DNA-PKâ‚¬@5,may be instructive in this regard.DNA-PK@5 is the catalytic subunit of DNA-PK, a DNA-dependentprotein kinase (122). Ku70 and Ku80, the other subunits of DNA-PK,form a heterodimer that binds without sequence specificity to doublestrand DNA breaks, gaps, and short hairpins (123). Once bound toDNA ends by the Ku polypeptides, DNA-PK activates its DNA-PK@5subunit, which then can phosphorylate a variety of proteins in vitro,including p53 (122). This ability of the DNA-PK holoenzyme tophosphorylate proteins when bound to damaged DNA suggests thatDNA-PK not only may have a direct role in promoting the repair ofcertain types of DNA damage but also may serve as the front end ofa signal transduction pathway that activates cellular responses to DNAdamage. Further experimental support for a role in cellular damageresponses for DNA-PKâ‚¬@5is provided by recent evidence that amutation in the DNA-PK@5 gene is responsible for the immunedeficient SCID mouse (124, 125), and that mutant DNA-PK@@,ku7O,and ku8O genes are associated with radiosensitivity, defects in doublestrand break repair, and abnormalities of VDJ recombination (126â€”129). Although ATM and DNA-PKâ‚¬.@5proteins share strong sequencehomology in their P1-3 kinase domains (130), their mutant phenotypesdiffer (e.g., see Ref. 126). In addition, A-I fibroblasts have normalintracellular amounts of the kulO, ku8O, and DNA-PKâ‚¬.@5polypeptides, and the DNA-PK enzymatic activity of A-I cell extracts isnormal (129). Taken together, these observations suggest that ATMand DNA-PKâ‚¬.@5act early and independently in separate signal transduction pathways that respond to DNA damage. The similaritiesbetween ATM and DNA-PIQ5 suggest that, like DNA-PIQ5 theATM protein may be directly involved in the recognition of DNAdamage, perhaps serving as the protein kinase subunit of a functionalcomplex that also includes ku7O- and ku8O-like polypeptides.

The Damage Surveillance Network model assumes that a majorfunction of ATM protein is signal transduction. It is not yet clear howthis occurs. However, initial sequence analysis of the ATM proteinindicates that it has a P13-kinase domain (63). Similar PI3-kinasedomains are found in the DNA-PK@5, MEI-4l, MEC1, RAD3, andTELl proteins, as well as TOR1 and IOR2, two yeast proteins thathelp to regulate normal progression of the cell cycle from G@into S(63â€”65, 130â€”132).These cell cycle control genes also share theirP13-kinase domains with the mammalian PI3-kinase, which mediatessignal transduction pathways that control growth factor-dependentmitogenesis, membrane ruffling, and glucose uptake (reviewed in Ref.133). The existence of a PI3-kinase domain in the ATM protein raisesthe possibility that a phosphoinositide might serve as a secondarymessenger for the ATM signal transduction network. However, although the ATM protein has a PI3-kinase domain, its enzymaticactivity is unknown, and it is far from certain that phosphoinositols arethe biologically relevant targets for its putative phosphotransferaseactivity. The most closely related mammalian protein, DNA-PIQ@,has no detectable phosphinositol kinase activity in vitro but canphosphorylate many proteins (130). The mammalian PI3-kinase andthe yeast Vps34p P13-kinase also phosphorylate proteins (134, 135),reinforcing the possibility that the true targets of the phosphotransferase activity of the ATM protein may be proteins.

Phenotypic and sequence similarities between the ATM gene andcell cycle checkpoint genes from Drosophila (mei-41) and yeast(MECJ and radfl support the central assumption of the DamageSurveillance Network model that the ATM gene controls a signaltransduction network that activates cell cycle checkpoints and othercellular functions in response to certain types of DNA damage.However, there are phenotypic differences between the genes [e.g.,unlike A-I homozygotes, MECJ and rad3 mutants do not havetelomere abnormalities (64)]. Sequence analysis carried out by Green

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A COMPREHENSIVE MODEL FOR ATAXIA-TELANGIECFASIA

well et al. (64) indicates that TELl, another S. cerevisiae gene, is moreclosely related to ATM than either MEC] or rad3@. Like A-I homozygotes, TEL] mutants have shortened telomeres, elevated rates ofrecombination between repeated genes, and increased frequencies ofaberrant chromosomal segregation (64). However, the TELl phenotype does not completely overlap with the A-I phenotype. TELlmutants are not X-ray sensitive, nor do they have obvious defects inDNA damage-induced cell cycle checkpoints (64).@As suggested byGreenwell et al. (64), this may be due to the presence of redundantsignal transduction systems in yeast that can partially mask the effectof a defective TELl protein. Alternatively, the ATM gene and itssignal transduction network may be the functional equivalent ofseveral different yeast damage response pathways.

A novel feature of the Damage Surveillance Network model is that,in response to DNA damage, the ATM protein activates cellularfunctions that promote genetic stability and survival, whereas it suppresses any tendencies to commit cellular suicide via apoptosis. ApOptosis is a normal process that is widely used by multicellularorganisms to regulate the growth, development and maintenance ofindividual organs (55). This innate ability to commit cellular suicidemay have been incorporated into the repertoire of DNA damageresponses because it offers a means by which mammals and othermulticellular organisms can eliminate cells that have sustained geneticdamage that threatens the survival of the organism. In this context, theincreased spontaneous and DNA damage-induced cell loss in A-Ihomozygotes can be seen as the result of lowering the threshold fortriggering an otherwise normal mammalian response to DNA damage.

Recently, p53 was shown to be required for the induction ofapoptotic cell death in peripheral mouse lymphocytes by ionizingradiation (86, 87). This finding suggests that, in addition to its â€œguardian of the genomeâ€• role in activating the G1-S damage-sensitivecheckpoint (136), p53 also acts as a â€œguardianof the organismâ€•bymediating cellular decisions to trigger programmed cell death in theface of DNA damage. If this is true, then a normal function of thewild-type ATM protein may be to promote survival in cells that havesustained DNA damage by physically interacting with the p53 proteinin a way that inhibits p53-mediated activation of apoptosis. Alternatively, the ATM protein may counteract p53-mediated apoptosis indirectly, perhaps by inhibiting a step in apoptosis that is downstreamof p53. PI3-kinase is required for prevention of apoptosis in ratpheochromocytoma cells deprived of nerve growth factor (137) andmay protect against apoptosis in B cells (138). Perhaps the ATMprotein, through its kinase domain, acts as a general inhibitor ofapoptosis, whether apoptosis is triggered by DNA damage or by lackof trophic factors. It is also possible that the ATM protein does notdirectly prevent DNA-damage induced apoptosis, but that functionalloss of the ATM signal transduction network in A-I homozygotesresults in the activation of a set of backup cellular damage responsesthat includes p53-dependent apoptosis.

Although inactivation of the ATM gene may be favored duringtumor progression because loss of ATM gene function could contribute to genetic instability, the Damage Surveillance Network modelpredicts that such a loss also may render a cell sensitive to being killedby DNA damage-induced apoptosis. Hence, inactivation of ATM orrelated genes may explain the sensitivity of many tumor cells toinduction of apoptosis by ionizing radiation, radiomimetic chemicals,and topoisomerase inhibitors (139), a sensitivity exploited by manycancer treatments.

The Damage Surveillance Network model predicts that a majorfunction of the ATM gene product is to activate multiple cellular

functions that prevent DNA damage from causing genetic instability.Acquisition of a genetic instability phenotype is a frequent event intumorogenesis (136, 140, 141). Therefore, one might expect that theATM gene, like p53 and p21, would function as a tumor suppressorgene. If so, inactivation of the ATM gene might occur during tumorprogression and be detectable as LOH of markers near the ATM geneon 1lq23. This prediction is supported by multiple studies that foundthat LOH at llq22â€”23 loci is a frequent event in sporadic breast,ovarian, colon, and cervical cancers (142â€”145).For example, in ananalysis of 62 sporadic breast cancers, 39% of the tumors had lostllq23 markers, with the common overlapping region ofLOH definingan â€”@20-cmregion that includes the A-Tlocus (143). The LOH studiessuggest that loss of ATM function is not just the cause of a raregenetic disease but that it also plays a role in the development of manycommon tumors. With the recent isolation of the ATM gene, confirmation of these LOH studies should be forthcoming in the near future,and a more accurate picture of the role of ATM gene inactivation intumorogenesis will emerge.

The A-I-dependent DNA damage-sensitive signal transduction network appears to be only one of an overlapping and partially redundantweb of intracellular networks that are involved in genetic homeostasisin mammalian cells. We are now entering a period of intense study ofthe roles played by the ATM, p53, p21, and DNA-PK@5 genes in cellcycle checkpoints, genetic instability, and programmed cell death.These studies should test the predictions of the Damage SurveillanceNetwork model, further our understanding of these basic biologicalprocesses, and shed light on the origin and development of cancer.

Acknowledgments

I thank Drs. C. F. Arlett, R. Gatti, M. F. Lavin, M. C. Paterson, Y. Shiloh,and C. M. R. Taylor for helpful discussions; Drs. D. E. Brash, L. B. K.Herzing, and R. J. Monnat, Jr. for critical review of the manuscript; and Drs.C. W. Anderson, R. S. Hawley, T. Petes, and T. Pandita for sharing theirunpublished results. This paper is dedicated to A-I patients and their families.

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Ataxia-Telangiectasia and Cellular Responses to DNA Damage...Ataxia-telangiectasia (A-T) is a human disease characterized by high cancer risk, immune defects, radiation sensitivity,