Ataxia..Telangiectasia and Cellular Responses to DNA Damage'

(CANCERRESEARCH55. 5991-6001. December 15, 19951 Review

M. Stephen Meyn2

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

Abstract elevated frequencies of spontaneous and induced chromosome aber rations, high spontaneous rates of intrachromosomal recombination, Ataxia-telangiectasia (A-T) is a human disease characterized by high aberrant immune gene rearrangements, and inability to arrest the cell cancer risk, immune defects, radiation sensitivity, and genetic instability. Although A-T homozygotes are rare, the A-T gene may play a role in cycle in response to DNA damage (3â€”6)]. sporadic breast cancer and other common cancers. Abnormalities of DNA The nature of the A-T defect has been the subject of much repair, genetic recombination, chromatin structure, and cell cycle check speculation; most hypotheses focus on the radiation sensitivity of point control have been proposed as the underlying defect in A-T; how A-T cells. Early reports that A-T fibroblasts were unable to excise ever, previous models cannot satisfactorily explain the plelotropic A-T radiation-induced DNA adducts prompted suggestions that the phenotype. radiation sensitivity of A-T cells was due to an intrinsic defect in Two recent observations help clarify the molecular pathology of A-T: DNA repair (7). However, subsequent work indicated that not all (a) inappropriate p53-mediated apoptosis is the major cause of death in A-T fibroblasts have a defect in DNA adduct excision (8), and that A-T cells irradiated in culture; and (b) ATM, the putative gene for A-T, has extensive homology to several celi cycle checkpoint genes from other the kinetics of repair of DNA breaks and chromosome aberrations organisms. Building on these new observations, a comprehensive model is is grossly normal (reviewed in Ref. 3). Functional abnormalities of presented in which the ATM gene plays a crucial role in a signal trans specific repair enzymes have been proposed [e.g. , topoisomerase II duction network that activates multiple cellular functions In response to (9) and poly(ADP-ribosylase) (10)], but conclusive evidence of DNA damage. In this Damage Survefflance Network model, there is no repair enzyme defects in A-T is lacking. Structural abnormalities intrinsic defect in the machinery of DNA repair in A-T homozygotes, but of chromatin, subtle defects in DNA repair that affect repair their lack of a functional ATM gene results In an Inabifity to: (a) halt at multiple cell cycle checkpoints in response to DNA damage; (b) activate quality, and abnormalities of differentiation also have been offered damage-inducible DNA repair; and (c) prevent the triggering of pro as explanations for the A-T phenotype (11â€”15). grammed cell death by spontaneous and induced DNA damage. Absence Several investigators have suggested that a defect in genetic recom of damage-sensitive cell cycle checkpoints and damage-induced repair bination, resulting in an inability to productively rearrange and repair disrupts immune gene rearrangements and leads to genetic instability and genes, would provide a unifying explanation for radiation sensitivity, cancer. Triggering of apoptosis by otherwise nonlethal DNA damage is immune defects, and karyotypic abnormalities in A-T (5, 6, 16â€”18). primarily responsible for the radiation sensitivity ofA-T homozygotes and However, a defect in genetic recombination is difficult to resolve with results in an ongoing loss of cells, leading to cerebellar ataxia and neuro reports of near-normal frequencies of extrachromosomal recombina logical deterioration, as well as thymic atrophy, lymphocytopenla, and a paucity of germ cells. lion (4, 19, 20) and the observation that spontaneous rates of chro Experimental evidence supporting the Damage Surveffiance Network mosomal recombination in A-T fibroblast lines are 30- to 200-fold model is summarized, followed by a discussion of how defects in the higher than normal (4). Other investigators, struck by the inability of ATM-dependent signal transduction network might account for the A-T irradiated A-T cells to temporarily halt DNA replication and cell cycle phenotype and what insights this new understanding of A-T can offer progression, have proposed that A-T cells cannot recognize or respond regarding DNA damage response networks, genomic instability, and can to DNA damage (1 1, 12, 21â€”23).In these models, the radiation cer. sensitivity of A-I cells generally is assumed to result from an inability Previous Models for A-T3 to delay the cell cycle to allow sufficient time to repair DNA damage. These models could explain why A-T cells are radiation sensitive A-T is an autosomal recessive disease with a pleiotropic phenotype despite grossly normal DNA repair, but they cannot readily account that includes progressive cerebellar ataxia, cellular and humoral im for several studies in which experimental conditions that prolonged or mune defects, progenc changes of the skin, endocrine disorders, temporarily halted the cell cycle did not improve the survival of gonadal abnormalities, and a high incidence of cancer; the relative risk irradiated A-I cells (24â€”26). Neither do these models explain another of developing some tumors (e.g. , lymphoma) is several hundredfold puzzling feature of A-I: unlike most normal mammalian cells, fatally higher than normal ( 1, 2). Heterozygote carriers are also at increased irradiated A-I cells typically die before they can complete the first risk for cancer, particularly breast carcinoma (2). A-T cells are sen postirradiation mitosis (113â€”115). sitive to the killing effects of ionizing radiation, and they exhibit in The finding that the radiosensitivity of A-I cells in culture is vivo and in vitro abnormalities consistent with a defect involving primarily the result of inappropriate apoptosis, together with recent DNA metabolism and/or maintenance of genomic integrity [e.g., observations regarding the role that the p53 tumor suppressor protein plays in mediating cellular responses to DNA damage, has led to the Received 8/21/95; accepted 10/16/95. development of a new model regarding the nature of the A-I defect. 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 with Initial analysis of the A-T disease gene supports this model, which is 18 U.S.C. Section 1734 solely to indicate this fact. related to previous proposals in which the A-T gene product normally I This work has been supported by grants from the NIH and the A-I Children's Project. mediates cellular responses to DNA damage (e.g., see Ref. 22), but 2 lo whom requests for reprints should be addressed, at Yale University School of overcomes the objections to those models, cited above, while provid 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; ing a unifying explanation for the pleiotropic manifestations of the ICR, I-cell receptor;P13-kinase.phosphatidylinositol3-kinase. disease. 5991

Ihe Damage Surveillance Network Model a critical role. The detection of certain types of spontaneous or induced DNA damage triggers this signal transduction network, resulting in the Biological organisms are not passive targets ofDNA-damaging agents; activation of a group of cellular functions that promote genetic stability they actively respond to DNA damage [e.g., the SOS system in Esche by temporarily arresting the cell cycle and enhancing DNA repair. At the richia coli (30)]. A growing body of evidence indicates that the response of mammalian cellstoDNA damage iscomplex,perhapsinvolving same time, the A-I-dependent network promotes cellular survival by several interrelated signal-transductionnetworks that detect DNA dam inhibiting execution of damage-induced programmed cell death. In ad age, activate DNA repair, and alter the cell cycle (22, 27â€”29).Fig. IA dition to the five responses illustrated in Fig. 1A, there also may be other, depicts a damage response network in which the A-T gene product plays asyetundefined,A-I-dependentfunctions.

A. NormalIndividuals Activated DNARepair @ @GADD45 ,t p21 â€˜t'@@ GuS p53 Checkpoint Spontaneous and f Induced DNADamage @. ssandds ______S phase @ ImmuneGene DNAbreaks ATM Checkpoint @ Rearrangments \I ShortTelomeres p53 GRIM Checkpoint

B. A-T Homozygotes No enhanced reactIvation â€˜V d2 of Irradiated virus D air No enhanced mutagenesis @ @GADD45 of irradiated virus

@ /@ p21 increased genomic instability chromosomal translocations p53 epithelial cells with micronudel aneuploid cells in muftipletissues allelelossin erythrocytes Spontaneousand I ICR transrearrangements Induced recombination between repeated genes induced chromosome aberrations DNADamage solid tumors @ 55 and ds @. *- Disruption of Immune gene rearrangement ImmuneGene DNAbreaks 9@PQk@t Lowlevelsof lgA, IgE, g02 and lgG4 @ Rearrangments %4@ Lowproportionof I cells expressingW@3TCRs Frequent translocatlons near immune genes ShortTelomeres Increased risk of lymphoma and leukemia p53 Abnormal cell cycle kinetics following DNAdamage C ck nt RadioresistantDNAsynthesis

Programmed Cell Death

Increased spontaneous cell death progressive loss of neurons thymic hypoplasia and lymphocytopenia depletion or absence of germ cells hypoplastic thyroid and adrenals progeric changes in skin and hair cirrhosis and elevated serum AFP Increased mutagen-lnduced cell death Marked sensitivity to killingby: Ionizing radiation radiomimetic drugs

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 detection of 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 functions in response to DNA damage. resulting in the pleiotropic A-I phenotype. 5992

The primary abnormality in A-I homozygotes presumably creates in S phase, resulting in the phenomenon of radioresistant DNA syn a defect in this network that prevents the activation of these cellular thesis (11). The G2-M checkpoint appears to be defective in A-T cells functions in response to strand breaks, shortened telomeres, and other as well, in that both A-T fibroblasts and lymphocytes irradiated in 02 DNA lesions (Fig. 1B). This inability to respond to spontaneous and fail to undergo the initial radiation-induced 02-M delay seen in induced DNA damage results in increased genomic instability, as well normal cells (46, 47, 113). as in an unusually low threshold for the triggering of p53-mediated Lack of Damage-activated DNA Repair. Exposing humancells apoptosis by otherwise nonlethal DNA damage. These abnormalities to low doses of radiation before infection of irradiated virus improves lead, in turn, to the multiple in vivo and in vitro abnormalities seen in viral survival and increases the number of mutant viruses recovered A-I homozygotes (Fig. 1B). Although they are not as severely af (48). These effects, termed â€œenhancedsurvivalâ€•and â€œenhancedmu fected as homozygotes, A-I heterozygotes may not have a fully tagenesis,â€•have been demonstrated using both single-stranded and functional damage response network, because they express subtle double-stranded DNA viruses (48â€”50),but they are not as striking as abnormalities in their cellular responses to ionizing radiation (3) and the analogous phenomena in bacteria, Weigle reactivation and Weigle have an increased relative risk of cancer (2). mutagenesis (30). Weigle reactivation results from induction of an error-prone DNA A-I Homozygotes Lack DNA Damage-activated Functions repair pathway in bacteria; similarly, one or more damage-activated DNA repair processes could be responsible for enhanced survival and Absence of Cell Cycle Checkpoints. In normal eukaryotic cells, enhanced mutagenesis in mammalian cells. At least some form of the cell cycle halts temporarily after the induction of strand breaks in damage-activated repair may be p53 dependent in human cells. Smith cellular DNA by a variety of agents, including ionizing radiation, et aL (51) recently demonstrated that loss of p53 function in human radiomimetic drugs, restriction enzymes, and topoisomerase inhibitors cells was associated with modest decreases in clonogenic survival, as (reviewed in Refs. 23 and 29). There are at least three damage well as reduced repair of UV-damaged reporter plasmids. Activation sensitive cell cycle â€œcheckpointsâ€•inmammalian cells: one at the G1-S of excision DNA repair, as measured by an in vitro assay, also was border, one in S phase, and one at the G2-M boundary. These check points also may restrain the cell cycle in response to the generation of decreased in p53@ cells (52). The p53 protein role in damage strand breaks, shortened telomeres, and other DNA damage that activated repair might occur either by direct interaction with repair occurs spontaneously during the course of normal DNA metabolism enzyme complexes (52) and/or by activation ofGADD45, which itself (e.g. , site-specific gene rearrangements, genetic recombination, and has been shown to bind to proliferating cell nuclear antigen and repair of replication errors). Given the viability of yeast and mamma stimulate DNA excision repair in vitro (53). Although not extensively han mutants that lack functional damage-sensitive cell cycle check studied, damage-activated DNA repair appears to be impaired in A-I points (31â€”33),these checkpoints probably are not invoked during homozygotes because A-T fibroblasts have been shown to lack the most cell cycles. The relative timing of DNA replication, DNA repair, enhanced survival and enhanced mutagenesis expressed by control genetic recombination, and cell division ensures that genomic integ human cells for irradiated H-l parvovirus and adenovirus 2 (49, 50, rity is restored before the cell cycle reaches a checkpoint (23, 29). 54). The tumor suppressor protein p53 plays a key role in activating the Low Threshold for Triggering Programmed Cell Death after G1-s checkpoint after certain types of DNA damage [reviewed in Ref. DNA Damage. A growing body of evidence documents that low 29; e.g., mammalian cells increase intracellular levels of p53 protein doses of ionizing radiation kill some types of mammalian cells in vivo shortly after exposure to many agents that induce DNA strand breaks, and in vitro by activating apoptosis, a well-characterized form of and functional p53 protein typically is required for activation of the programmed cell death (reviewed in Ref. 55) that can be carried out Gl-s checkpoint after ionizing radiation exposure (22, 34, 35)]. G1-S in both cycling (56) and noncycing cells (57). cell cycle arrest occurs via p53-mediated transcriptional activation of Although researchers have only recently begun to focus on the the p21 (WAF-JICIPJISDIJ) gene, which codes for a protein that possible role of programmed cell death in the A-I phenotype, several binds to cyclin-dependent kinase-cyclin complexes and inhibits their in vivo and in vitro findings support such a possibility. Histological kinase activities (22, 36, 37). The p21 protein may be involved in the analyses of cerebella taken from A-T homozygotes at autopsy docu S-phase checkpoint as well because it can bind to proliferating cell ment a high frequency of abnormal Purkinje and granule cells that nuclear antigen/replication factor C/pol 6 complexes in vitro and exhibit the highly condensed, pyknotic nuclei expected from pro block the elongation step of DNA polymerization (38, 39). The grammed cell death in neurons (58â€”60).Anin vitro flow cytometric S-phase checkpoint may be p53 independent because cells lacking study that examined the effects of the radiomimetic drug streptomgrin functional p53 can express a radiation-induced S-phase checkpoint, as on A-T fibroblasts described changes in the DNA content of treated indicated by normal suppression of DNA synthesis after irradiation A-I cells that are consistent with apoptosis (61). (40). In mammaliancells,theG2-Mdamage-activatedcheckpoint Following up on these observations, we recently demonstrated that probably is also p53 independent (e.g., see Ref. 41); however, rela fibroblasts and lymphoblasts representing all A-I complementation lively little is known about its genetics and biochemistry. In contrast, groups undergo apoptotic death in culture after exposure to low at least 10 yeast genes are involved in controlling the G2-M check radiation and streptomgrin doses that do not induce appreciable ap point (31, 32, 42, 43). optosis in control cells (62).@This inappropriate apoptosis appears to A-I homozygotes lack the p53-mediated G1-S damage-sensitive be mediated by p53, in that it is suppressed in A-T fibroblasts, the p53 checkpoint, and the kinetics of p53, p21, and GADD45 induction by protein of which has been functionally inactivated by transfection ionizing radiation are abnormal in the cells of A-I homozygotes (22, with either a dominant-negative p.53 gene or a human papilloma virus 44, 45). These observations led Kastan et aL (22) to propose that the E6 gene. Iransfection-induced loss of p53 function did not affect A-T gene product(s) functions upstream of p53 in a damage-respon survival of control fibroblasts, but transfected A-I cells acquired sive pathway that leads to Ge-S arrest. This pathway is incorporated near-normal resistance to ionizing radiation, suggesting that p53- in the Damage Surveillance Network model as one branch of the ATM-dependent network (Fig. 1A). A-I cells do not have the S-phase 4 M. S. Meyn, L Strasfeld, and C. Allen. p53-mediated apoptosis is the primazy cause checkpoint, because they fail to arrest DNA synthesis when irradiated of radiosensitivity in ataxia-telangiectasia, manuscript in preparation. 5993

mediated apoptosis is the major cause of radiosensitivity in A-I cells also have marked elevations in the frequency of I lymphocytes in culture (62).@ expressing y/13, â€˜SIP,aTh,or y/6 heavy-chain ICRs as a result of aberrant interlocus gene rearrangements (5, 6) and a high frequency of Initial Analysis of the ATM Gene Supports the Damage B and I lymphocytes carrying chromosome translocations involving Surveillance Network Model sites on chromosomes 2, 7, and 14 near the immunoglobulin super Earlier this year, Savitsky et a!. (63) positionally cloned a gene gene family genes (reviewed in Ref. 17). from the 1lq23.l A-I locus that is mutated in A-I patients from all The Damage Surveillance Network model for A-I explains these complementation groups. The gene, termed ATM (A-I, mutated), is clinical findings by assuming that the gene rearrangements necessary conserved in vertebrates and codes for a I2-kb transcript that is for immunoglobulin switch recombination and ICR heavy-chain re abundantly expressed in multiple tissues in vivo. As shown in Fig. 2, arrangements frequently trigger the A-I damage surveillance network the carboxy terminus of the putative AIM protein is homologous to and its cell cycle checkpoints, perhaps in response to the creation of that of at least four checkpoint proteins from other organisms: Dro double-strand breaks during immune gene recombination. Normal sophila melanogaster MEI-41, Schizosaccharomyces pombe Rad3, cells halt the cell cycle temporarily to allow completion of these Saccharomyces cerevisiae MEC1p, and S. cerevisiae TEL1p (63â€”65). immune gene rearrangements (69), but cells of A-I homozygotes The region of strongest homology between these five proteins contains cannot activate cell cycle checkpoints. Hence, A-I cells that are a phosphatidylinositol 3-kinase domain, suggesting that proteins are undergoing switch recombination or ICR heavy-chain rearrangement involved in signal transduction. may attempt to replicate their DNA and/or proceed past the G2-M The ATM gene shares phenotypic similarities with these genes as checkpoint into mitosis before resolving these recombination com well. Like A-I homozygotes, mei-41, rad3, and med mutants are plexes. Manipulation of the immune recombination complexes during X-ray sensitive and lack damage-induced cell cycle checkpoints (42, DNA replication, chromosome condensation, and/or mitosis then dis 43, 65). In addition, A-I, mei-41, rad3, and tell homozygotes express rupts the complexes, creating chromosomes that contain free DNA increased chromosomal instability and have high spontaneous rates of ends generated during abortive immune gene rearrangement. These mitotic recombination (1, 4, 64â€”66).Taken together, the physical and free ends are highly recombinogenic and could serve as foci for phenotypic similarities between these checkpoint genes and ATM illegitimate recombinational events that lead to interlocus gene rear suggest that the ATM gene activates multiple cellular functions in rangements and chromosome translocations. The final result is an response to spontaneous and induced DNA damage. abnormally high frequency of lymphocytes carrying translocations near immune genes or expressing aberrantly recombinant ICRs. DNA Lack of Damage-activated Functions Explains the Phenotype of damage resulting from disrupted immune gene rearrangements also A-T Homozygotes may trigger apoptosis in A-I lymphocytes (see below), which, by Lack of damage-activated functions in A-I homozygotes due to a eliminating cells undergoing immunoglobulin switch or ICR recom defect in a signal transduction network that monitors genomic integ bination, would contribute to the selective immunoglobulin deficien rity, as proposed in the Damage Surveillance Network model de cies and paucity of a/@3ICR-expressing I cells seen in A-I homozy scribed above, could explain the pleiotropic nature of the clinical and gotes. laboratory abnormalities associated with the disease. Fig. lB summa Unlike A-I homozygotes, p53-null mice do not exhibit aberrant rizes the consequences of lack of damage-activated functions in A-T immune gene rearrangements, nor do their peripheral lymphocytes homozygotes, along with the resulting phenotypic effects. How the express the type of chromosome translocations characteristic of A-I Damage Surveillance Network model accounts for each aspect of the lymphocytes (33, 70). This suggests that loss of the p53-mediated A-I phenotype when previous models cannot is discussed below. G1-S checkpoint is not as disruptive to immune gene recombination as Cell Cycle Checkpoint Defects Disrupt Immune Gene Rear loss of the S-phase and G2-M checkpoints. rangements. The immunoglobulinheavy-chaingene andICR genes Cell Cycle Checkpoint Defects Cause Spontaneous Genetic In undergo site-specific gene rearrangements during normal develop stability. Cells that lack functional p53 protein fail to halt at the G1-S ment, resulting in production of IgA, IgE, IgG2, and IgG4 by B border after irradiation, but will arrest the cell cycle in S phase and at lymphocytes and in expression of a/j3 heavy-chain ICRs by I lym the G2-M border (34, 35, 40). Hence, cells that are defective for p53 phocytes (67, 68). A-I homozygotes have deficiencies in these spe function have a specific defect in the G1-S damage-sensitive check cific immunoglobulin classes, as well as a relative lack of I lympho point but not the S-phase or G2-M checkpoints. Several studies have cytes expressing a/@3heavy-chain TCRs (reviewed in Ref. 3). They demonstrated spontaneous genetic instability in mammalian cells that

Protein Length(a.a.)

@ ATM 3056 DNA-PK@5 4096 MEI-41 2357 TEL1p 2787 RAD3 2386 MEC1p 2368 kinase domain

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 domains that show 60â€”70%similaritybetween all family members; (I]), regions of 40â€”50%similarity.Alignments were generated using BLAST@analysis (148). a.a, amino acids. 5994

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1995 American Association for Cancer Research. A COMPREHENSIVE MODEL FOR ATAXIA-TELANGIECFASIA lack the G1-S checkpoint as a result of mutations in the p53 gene. increased incidence of solid tumors in A-I homozygotes by increas Fibroblasts from p53-null mice and patients with Li-Fraumeni syn ing the occurrence of spontaneous and induced chromosome aberra drome, as well as cell lines that have lost wild-type p53 alleles or tions, mitotic recombination, and LOH. harbor dominant-negative p53 mutations, lack functional p53 and A-I homozygotes face a 250- to 700-fold increased risk of devel show increased frequencies of chromosome loss, chromosome aber oping leukemia and non-Hodgkin's lymphoma (2, 85), tumors that rations, intrachromosomal genetic recombination, and/or gene ampli frequently harbor chromosome rearrangements involving immuno fication events (33, 62, 71â€”73).Given the genetic instability of cells globulin supergene family genes ( 17). Kastan et a!. (22) noted that that lack a -S cell cycle checkpoint because of loss of p53 function, both A-I homozygotes and p53-null mice have a predilection for the G1-S cell cycle checkpoint defect is likely to contribute to the high immune system tumors and suggested that an abnormal response to spontaneous frequencies of chromosome aberrations, genetic recom DNA strand breaks may be responsible for the high incidence of bination. aneuploid cells, and allele loss seen in different A-I cell lymphoid tumors in both cases. In the Damage Surveillance Network types in Vit'() and in vitro ( I 7. 74â€”77). Inability to arrest at the S-phase model, this abnormal response would be an inability to trigger the A-I and G2-M DNA damage checkpoints also may contribute to sponta damage surveillance network and activate its cell cycle checkpoints. neous chromosome instability in A-T cells, in that S. cerevisiae rad9 A-I lymphoid cells would become malignant as a consequence of the mutants that have lost the G,-M damage-sensitive checkpoint show activation of cellular oncogenes by chromosome translocations result marked increases in the frequency of spontaneous chromosome loss at ing from disruption of the normal rearrangement and repair of im mitosis (78). mune gene DNA. The specific increase in lymphoid tumors seen in The spontaneous genetic instability seen in nonimmune A-I cells A-I homozygotes suggests that the initial production of strand breaks may be due to a mechanism similar to that outlined above for the and other DNA damage is a rate-limiting step in oncogenesis, even in immune system@an inability to activate cell cycle checkpoints in the cells that are genetically unstable because of a lack of DNA damage face of spontaneous DNA damage allows A-T cells to attempt to sensitive cell cycle checkpoints. replicate DNA (G1-S and S-phase checkpoints) or enter mitosis Lack of Damage-activated DNA Repair Prevents Enhanced (G,-M checkpoint) before completion of repair of the damage. Rep Survival and Mutagenesis. Heightened ability to repair DNA dam lication or mitosis then results in breaks and gaps in chromosomal age resulting from radiation exposure to host cells is the putative DNA that promote the formation of acentric chromosome fragments, cause of enhanced survival and enhanced mutagenesis of irradiated facilitate generation of aneuploid daughter cells through missegrega viruses in mammalian cells. The contribution of damage-activated tion, and serve as substrates for chromosome translocations and mi repair to cellular survival after DNA damage is less certain. Loss of totic recombination. damage-activated repair is associated with a modest increase in UV The AIM-dependent damage surveillance network may monitor sensitivity but does not appreciably affect the clonogenic survival of telomeres as well, preventing the propagation of cells containing X-irradiated cells (5 1, 7 1, 86, 87). This differential effect on UV chromosomes with short telomeres by triggering cell cycle check damage may be due to the fact that damage-activated repair is pri points when telomeres fall below a critical size. In this scenario, short manly a form of enhanced excision repair (5 1). The Damage Surveil telomeres would be unable to arrest cell division in cells from A-I lance Network model assumes that, whatever its mechanism, this homozygotes, leading to the gradual accumulation of cells with short enhanced ability to repair DNA damage can be activated by normal ened telomeres. This would explain why A-I cells in culture have cells but not by A-I cells or cells without functional p53. shorter telomeres and fewer telomeric hybridization signals than do Lack of damage-activated repair does not appear to be a major control cells (79). Aberrant telomeres, in turn, may cause telomere factor in the X-ray sensitivity of A-I cells (see below), but it may be telomere associations, which are a characteristic karyotypic feature of partially responsible for the mild UV sensitivity seen in some A-I both A-I cells and senescing cells from normal individuals (17, 80). cells (3). Lack of damage-activated repair also could contribute to the Short telomeres also may contribute to the overall genomic instability higher than normal residual level of double-strand breaks seen in of A-I cells through their tendency to provoke chromosome X-irradiated A-I cells (82, 88). Alternatively, the residual double rearrangements. strand breaks in A-I cells could be due to initiation of apoptosis Cell Cycle Checkpoint Defects Contribute to Induced Chromo mediated nucleolytic degradation of genomic DNA. some Aberrations. The S. cerevisiae rad9 and S. pombe rad3 mu Dysfunctional Programmed Cell Death Leads to Spontaneous tants exhibit high levels of chromosome aberrations after irradiation, Cell Loss. Autopsies of A-I homozygotes have documented chronic presumably as a result of defective G1-S and G2-M damage check spontaneous loss of Purkinje cells and granule cells in the cerebellum, points (43, 8 1). Defective cell cycle checkpoints could contribute to as well as depletion of other neurons in the central nervous system of the high residual frequency of radiation-induced chromosome and older patients (reviewed in Ref. I). Other tissues reported to be chromatid aberrations seen in A-I cells when assayed at the first atrophic and/or hypoplastic in A-I homozygotes include the thymus, postirradiation mitosis (82, 83). Although the kinetics of DNA break gonads, thyroid, and adrenals ( 1). Cirrhotic changes have been seen in repair are grossly normal in A-I cells, the lack of checkpoint restraint the livers of A-I homozygotes, together with patches of regenerating on forward progress of the cell cycle into DNA synthesis and mitosis hepatocytes, which may be the cause of their high serum levels of effectively gives A-I cells less time in which to remove DNA breaks a-fetoprotein (1, 89). Taken together, these autopsy findings indicate before they give rise to chromosome aberrations (irradiation in that chronic spontaneous cell death occurs throughout the tissues of and chromatid aberrations (irradiation in G,). A-I homozygotes. Cell Cycle Checkpoint Defects Lead to Cancer. A growing body Cell cycle checkpoint defects cannot easily account for this ongoing of evidence indicates that multiple genetic changes resulting in loss of in vivo cell loss. Homozygous p53-null mice do not have detectable function at tumor suppressor genes and gain of function at oncogenes neurological abnormalities, immune defects, or sterility (33, 70). are critical to the development of cancer (29, 84). Accumulation of Hence, the G1-S checkpoint defect does not appear to be the cause of these changes is more likely to occur in cells from individuals with the cerebellar, immunological, and gonadal defects in A-I. Defective constitutional genetic instability. Hence, it is not surprising that can S-phase and G2-M checkpoints could cause a small part of the loss of cers occur at high frequencies in A-I homozygotes (85). G@-S, S dividing cells in vivo, as well as contribute to the high frequency of phase, and G,-M cell cycle checkpoint defects may contribute to the aneuploid giant cells found in A-I homozygotes (60, 76), a prediction 5995

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1995 American Association for Cancer Research. A COMPREHENSIVE MODEL FOR ATAXIA-TELANGIECTASIA based, in part, on the high frequency of spontaneous aneuploidy restoration of normal survival after X-irradiation but still express associated with the G2-M checkpoint defect in rad9 mutant yeast (78). radioresistant DNA synthesis, indicating that their S-phase check However, although defective S-phase and G2-M checkpoints may kill points remain defective. Fusion of HeLa cells to an A-I fibroblast line some dividing cells, they cannot cause the death ofcells arrested in G0 resulted in heterodikaryons that exhibited normal survival after irra (e.g., Purkinje cells and other neurons) in A-I homozygotes. In this diation, despite the lack of an S-phase checkpoint, as measured by regard, it seems significant that the cell types most affected by in vivo radioresistant DNA synthesis (40). Further evidence for the lack of degeneration in A-I are those that, in normal individuals, are most concordance between S-phase checkpoint abnormalities and increased sensitive to radiation-induced apoptotic cell death [e.g., lymphocytes, sensitivity to the lethal effects of radiomimetic agents is provided by spermatogonia, and neurons in the developing cerebellum (reviewed Mirzayans and Paterson (2 1), who found that fibroblasts from one in Ref. 90)]. A-I patient were hypersensitive to the killing effects of the mutagen The Damage Surveillance Network model predicts that the primary 4NQO but exhibited a normal S-phase checkpoint after 4NQO expo cause of spontaneous loss of both dividing and nondividing cells in sure, whereas fibroblasts from another A-I patient had normal sur tissues of A-I homozygotes is inappropriate activation of pro vival after 4NQO treatment but demonstrated complete lack of a grammed cell death by DNA damage that occurs spontaneously 4NQO-induced S-phase checkpoint. during normal DNA metabolism and as the result of ordinary envi Yeasts that lack a functional G2-M damage-sensitive checkpoint ronmental insults. In the Damage Surveillance Network model, because of mutations in the RAD9, RAD1, or RAD24 genes are chronic cell loss due to programmed cell death depletes Purkinje cells sensitive to the killing effects of ionizing radiation (102), presumably and other neurons from the central nervous system, I-cell and B-cell because these mutations effectively shorten the postirradiation cell precursors from the immune system, and germ cells from the gonads. cycle, thereby giving cells less time to complete repair before mitosis. These losses account for the progressive cerebellar ataxia and intel The behavior of the yeast G2-M checkpoint mutants suggests that the lectual arrest seen in A-I homozygotes, as well as their thymic radiation sensitivity of A-I cells could be due to unchecked progres atrophy, reduced numbers of circulating lymphocytes, and paucity of sion into mitosis before repair of potentially lethal DNA damage. germ cells (1). However, a defective G2-M damage checkpoint cannot explain an Dysfunctional Activation of Programmed Cell Death Causes unusual aspect of radiation sensitivity in A-I, the lack of â€œliquid Mutagen Sensitivity. The striking sensitivity of A-I cells to killing holdingâ€•recovery. In both prokaryotes and eukaryotes, experimental by ionizing radiation and radiomimetic drugs has been a long-standing manipulations that delay entry into the cell cycle or slow progression puzzle that has not been adequately explained in the past. of the cell cycle normally enhance the survival of irradiated cells (e.g., Multiple biochemical studies have failed to detect gross abnormal see Ref. 103), a phenomenon that usually is demonstrated in mam ities in the kinetics of single-strand and double-strand break repair in malian cells by their recovery of colony-forming ability after a po A-I cells (e.g., see Ref. 91, 92). Other reports have found no evidence stirradiation period of growth inhibition. As might be expected, treat that A-I cells are functionally defective in DNA repair (93â€”95).On ments that prolong or temporarily halt the postirradiation cell cycle the other hand, several studies found slight increases in the fraction of are especially effective in increasing the survival of mutants that lack breaks left unrepaired in irradiated A-I cells (82, 88), as well as a functional G2-M damage checkpoint [e.g., the S. cerevisiae rad9 abnormalities in the rejoining of restriction enzyme breaks in plasmids mutant (104) and the S. pombe radl mutant (105)]. In marked con transfected into A-I fibroblasts (13, 14). To account for the seeming trast, several studies have found that holding A-I fibroblasts in G0 for disparity between the various functional and biochemical studies of up to 7 days after irradiation does not significantly improve their irradiated A-I cells, it has been suggested that the repair defect in A-I survival (24â€”26).This lack of liquid holding recovery in A-I fibro is subtle, perhaps the result of impaired accuracy in strand rejoining blasts argues against G2-M damage-sensitive checkpoint defects play (13, 14) or an inability to repaira smallbut critical fractionof ing a major role in determining the survival of A-I cells after double-strand breaks (82, 96â€”98). irradiation. Subtle but critical defects in DNA repair might contribute to the Another aspect of the A-I phenotype that is not explained by radiation sensitivity of A-I cells and help to explain the increased previous A-I models is the circumstances under which irradiated A-I number of persistent chromosome aberrations observed in irradiated cells die. It is well established that the lethal effects of ionizing A-I cells at the first postirradiation mitosis (82, 83). However, DNA radiation are associated with the production of double-strand breaks in repair defects alone cannot readily account for the cell cycle abnor chromosomal DNA (reviewed in Ref. 106), and it has been estimated malities observed in A-I cells. To explain both radiosensitivity and that an average of 40 unrepaired double-strand breaks is sufficient to cell cycle abnormalities, several investigators have proposed that the kill a normal diploid human cell (107). It is unlikely that so few breaks enzymatic machinery for DNA repair and recombination is essentially introduced at random would directly damage a gene necessary for cell intact in A-I but that the in vivo and in vitro radiosensitivity of A-I survival. Instead, unrepaired double-strand breaks are thought to kill cells is due to inability to activate cell cycle checkpoints in response dividing mammalian cells because they give rise to acentric frag to DNA damage (1 1, 12). Experimental evidence suggests, however, ments, dicentrics, and other chromosome aberrations (108, 109). that the effects of checkpoint defects on the survival of irradiated A-I These chromosome aberrations then can undergo missegregation at cells are minor. mitosis, resulting in daughter cells with partial monosomies and It is unlikely that the G1-S checkpoint defect plays a significant role tnsomies, the unbalanced karyotypes of which eventually prove fatal in the sensitivity of A-I cells to the lethal effects of induced DNA (1 10, 11 1). This conclusion is supported by observations that, for most damage. Cells lacking the G1-S checkpoint as a result of defects in mammalian cells, radiation-induced death is not immediate but typi p53 expression or function are not radiosensitive (e.g. , see Refs. 40 cally occurs in the first- and second-generation offspring of irradiated and 71), and drug treatments that delay DNA replication in irradiated cells (summarized in Ref. 112). A-I cells (e.g., see Ref. 99) do not enhance survival. The S-phase Previous models for the A-I defect have assumed that, like normal checkpoint defect also has been dissociated from cell survival in A-I cells, irradiated A-I cells die as a result of genomic imbalance caused cells by gene transfer and cell fusion studies. After transfection of the by persistent radiation-induced chromosome aberrations (82, 83). A-I fibroblast line AT5BIVA with normal human genomic DNA, However, there is in vitro and in vivo evidence to the contrary. Unlike clones have been isolated that have partial (100) or complete (101) other mammalian cells, fatally irradiated A-I fibroblasts do not divide 5996

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1995 American Association for Cancer Research. A COMPREHENSIVE MODEL FOR ATAXIA-TELANGIECTASIA several times before death. Instead, the majority arrest in G2 before transduction network is not certain. However, consideration of a their first postirradiation mitosis (1 13â€”115).While arrested in G2, related human protein, DNA-PKâ‚¬@5,maybe instructive in this regard. they undergo apoptosis (62)@before any radiation-induced chromo DNA-PK@5 is the catalytic subunit of DNA-PK, a DNA-dependent some aberrations can missegregate. Several case reports document the protein kinase (122). Ku70 and Ku80, the other subunits of DNA-PK, extreme sensitivity of A-I homozygotes to the neurotoxic effects of form a heterodimer that binds without sequence specificity to double central nervous system irradiation given as treatment for cancer (116, strand DNA breaks, gaps, and short hairpins (123). Once bound to 117). Central nervous system neurons in children are nondividing DNA ends by the Ku polypeptides, DNA-PK activates its DNA-PK@5 cells, suggesting that at least part of the neurotoxicity of radiation subunit, which then can phosphorylate a variety of proteins in vitro, therapy in A-I homozygotes is the result of neurons being killed including p53 (122). This ability of the DNA-PK holoenzyme to while in G0. Because irradiated A-I fibroblasts and neurons die before phosphorylate proteins when bound to damaged DNA suggests that any induced chromosome aberrations can lead to genomic imbalance, DNA-PK not only may have a direct role in promoting the repair of other causes must be sought to explain their radiation sensitivity. certain types of DNA damage but also may serve as the front end of The Damage Surveillance Network model predicts that, because a signal transduction pathway that activates cellular responses to DNA A-I cells lack a functional ATM gene, they cannot prevent the damage. Further experimental support for a role in cellular damage initiation of programmed cell death by radiation-induced DNA lesions responses for DNA-PKâ‚¬@5isprovided by recent evidence that a (Fig. 1B). As a result, radiation damage that would be nonlethal in mutation in the DNA-PK@5 gene is responsible for the immune normal cells triggers programmed cell death in A-I cells, which then deficient SCID mouse (124, 125), and that mutant DNA-PK@@,ku7O, is carried out in G0 in noncycling cells and at the first postirradiation and ku8O genes are associated with radiosensitivity, defects in double mitosis in actively dividing cells. In this way, the Damage Surveil strand break repair, and abnormalities of VDJ recombination (126â€” lance Network model overcomes objections to previous cell cycle 129). Although ATM and DNA-PKâ‚¬.@5proteinsshare strong sequence based A-I models and accounts for why (a) both cycling and non homology in their P1-3 kinase domains (130), their mutant phenotypes cycling A-I cells are radiosensitive despite having only minor defects differ (e.g., see Ref. 126). In addition, A-I fibroblasts have normal in DNA repair; (b) irradiated A-I fibroblasts do not undergo liquid intracellular amounts of the kulO, ku8O, and DNA-PKâ‚¬.@5polypep holding recovery; and (c) irradiated A-I cells die before the first tides, and the DNA-PK enzymatic activity of A-I cell extracts is postirradiation cell division. normal (129). Taken together, these observations suggest that ATM and DNA-PKâ‚¬.@5actearly and independently in separate signal trans Discussion and Predictions duction pathways that respond to DNA damage. The similarities between ATM and DNA-PIQ5 suggest that, like DNA-PIQ5 the The Damage Surveillance Network model for A-I proposes that the ATM protein may be directly involved in the recognition of DNA A-I defect results in an inability to activate a group of diverse cellular damage, perhaps serving as the protein kinase subunit of a functional functions in response to DNA damage. It offers a unifying explanation complex that also includes ku7O- and ku8O-like polypeptides. of how a single-gene defect can cause the pleiotropic phenotype seen The Damage Surveillance Network model assumes that a major in A-I homozygotes and explains several puzzling aspects of the function of ATM protein is signal transduction. It is not yet clear how disease. The model assumes that the enzymatic machinery for DNA this occurs. However, initial sequence analysis of the ATM protein repair and genetic recombination is essentially intact, and it empha indicates that it has a P13-kinase domain (63). Similar PI3-kinase sizes the contribution of defective cell cycle checkpoints to genetic domains are found in the DNA-PK@5, MEI-4l, MEC1, RAD3, and instability and immune defects, two cardinal features of A-I. By TELl proteins, as well as TOR1 and IOR2, two yeast proteins that ascribing the disruption of immunoglobulin switch recombination and help to regulate normal progression of the cell cycle from G@into S ICR rearrangements to cell cycle checkpoint abnormalities, and pos (63â€”65, 130â€”132).These cell cycle control genes also share their tulating that disruptions of immune gene rearrangements and of repair P13-kinase domains with the mammalian PI3-kinase, which mediates of spontaneous DNA damage lead to generation of recombinogenic signal transduction pathways that control growth factor-dependent breaks and gaps in DNA, the Damage Surveillance Network model mitogenesis, membrane ruffling, and glucose uptake (reviewed in Ref. explains why immunoglobulin switch recombination and ICR rear 133). The existence of a PI3-kinase domain in the ATM protein raises rangement appear to be defective in A-I homozygotes, whereas the possibility that a phosphoinositide might serve as a secondary spontaneous rates of recombination between directly repeated nonim messenger for the ATM signal transduction network. However, al mune genes in A-I fibroblasts are markedly higher than normal (4). though the ATM protein has a PI3-kinase domain, its enzymatic The model can also account for the observation that chromosomal activity is unknown, and it is far from certain that phosphoinositols are translocations in A-I lymphocytes cluster near immune genes, the biologically relevant targets for its putative phosphotransferase whereas translocations in A-I fibroblasts appear to involve random activity. The most closely related mammalian protein, DNA-PIQ@, sites throughout the genome (1 18). By assuming that the damage has no detectable phosphinositol kinase activity in vitro but can surveillance network normally monitors telomere integrity, the model phosphorylate many proteins (130). The mammalian PI3-kinase and explains telomenc abnormalities seen in A-I cells. the yeast Vps34p P13-kinase also phosphorylate proteins (134, 135), The range of DNA lesions that might trigger the ATM network is reinforcing the possibility that the true targets of the phosphotrans uncertain, although strand breaks and gaps containing modified 3' ferase activity of the ATM protein may be proteins. termini are likely to play a major role, given the sensitivity of A-I Phenotypic and sequence similarities between the ATM gene and homozygotes to physical and chemical agents that induce strand cell cycle checkpoint genes from Drosophila (mei-41) and yeast breaks and small gaps containing 3' phosphoglycolates (119â€”121). (MECJ and radfl support the central assumption of the Damage Short telomeres also may activate the ATM network, suggesting that Surveillance Network model that the ATM gene controls a signal the ends of abnormally short telomeres may be structurally similar to transduction network that activates cell cycle checkpoints and other DNA breaks generated by these agents. As indicated in Fig. 1, the cellular functions in response to certain types of DNA damage. same lesions that activate A-I dependent cellular functions may also However, there are phenotypic differences between the genes [e.g., trigger p53-mediated programmed cell death. unlike A-I homozygotes, MECJ and rad3 mutants do not have How far upstream of p53 the ATM protein functions in the signal telomere abnormalities (64)]. Sequence analysis carried out by Green 5997

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1995 American Association for Cancer Research. A COMPREHENSIVE MODEL FOR ATAXIA-TELANGIECFASIA well et al. (64) indicates that TELl, another S. cerevisiae gene, is more functions that prevent DNA damage from causing genetic instability. closely related to ATM than either MEC] or rad3@. Like A-I homozy Acquisition of a genetic instability phenotype is a frequent event in gotes, TEL] mutants have shortened telomeres, elevated rates of tumorogenesis (136, 140, 141). Therefore, one might expect that the recombination between repeated genes, and increased frequencies of ATM gene, like p53 and p21, would function as a tumor suppressor aberrant chromosomal segregation (64). However, the TELl pheno gene. If so, inactivation of the ATM gene might occur during tumor type does not completely overlap with the A-I phenotype. TELl progression and be detectable as LOH of markers near the ATM gene mutants are not X-ray sensitive, nor do they have obvious defects in on 1lq23. This prediction is supported by multiple studies that found DNA damage-induced cell cycle checkpoints (64).@As suggested by that LOH at llq22â€”23 loci is a frequent event in sporadic breast, Greenwell et al. (64), this may be due to the presence of redundant ovarian, colon, and cervical cancers (142â€”145).Forexample, in an signal transduction systems in yeast that can partially mask the effect analysis of 62 sporadic breast cancers, 39% of the tumors had lost of a defective TELl protein. Alternatively, the ATM gene and its llq23 markers, with the common overlapping region ofLOH defining signal transduction network may be the functional equivalent of an â€”@20-cmregionthat includes the A-Tlocus (143). The LOH studies several different yeast damage response pathways. suggest that loss of ATM function is not just the cause of a rare A novel feature of the Damage Surveillance Network model is that, genetic disease but that it also plays a role in the development of many in response to DNA damage, the ATM protein activates cellular common tumors. With the recent isolation of the ATM gene, confir functions that promote genetic stability and survival, whereas it sup mation of these LOH studies should be forthcoming in the near future, presses any tendencies to commit cellular suicide via apoptosis. Ap and a more accurate picture of the role of ATM gene inactivation in Optosis is a normal process that is widely used by multicellular tumorogenesis will emerge. organisms to regulate the growth, development and maintenance of The A-I-dependent DNA damage-sensitive signal transduction net individual organs (55). This innate ability to commit cellular suicide work appears to be only one of an overlapping and partially redundant may have been incorporated into the repertoire of DNA damage web of intracellular networks that are involved in genetic homeostasis responses because it offers a means by which mammals and other in mammalian cells. We are now entering a period of intense study of multicellular organisms can eliminate cells that have sustained genetic the roles played by the ATM, p53, p21, and DNA-PK@5 genes in cell damage that threatens the survival of the organism. In this context, the cycle checkpoints, genetic instability, and programmed cell death. These studies should test the predictions of the Damage Surveillance increased spontaneous and DNA damage-induced cell loss in A-I Network model, further our understanding of these basic biological homozygotes can be seen as the result of lowering the threshold for processes, and shed light on the origin and development of cancer. triggering an otherwise normal mammalian response to DNA damage. Recently, p53 was shown to be required for the induction of apoptotic cell death in peripheral mouse lymphocytes by ionizing Acknowledgments radiation (86, 87). This finding suggests that, in addition to its â€œguard ian of the genomeâ€• role in activating the G1-S damage-sensitive I thank Drs. C. F. Arlett, R. Gatti, M. F. Lavin, M. C. Paterson, Y. Shiloh, checkpoint (136), p53 also acts as a â€œguardianof the organismâ€•by and C. M. R. Taylor for helpful discussions; Drs. D. E. Brash, L. B. K. mediating cellular decisions to trigger programmed cell death in the Herzing, and R. J. Monnat, Jr. for critical review of the manuscript; and Drs. face of DNA damage. If this is true, then a normal function of the C. W. Anderson, R. S. Hawley, T. Petes, and T. Pandita for sharing their unpublished results. This paper is dedicated to A-I patients and their families. wild-type ATM protein may be to promote survival in cells that have sustained DNA damage by physically interacting with the p53 protein in a way that inhibits p53-mediated activation of apoptosis. Alterna References tively, the ATM protein may counteract p53-mediated apoptosis in 1. Sedgwick, R. P., and Boder, E. Ataxia-telangiectasia. Handb. Clin. Neurol., 16: directly, perhaps by inhibiting a step in apoptosis that is downstream 347â€”423,1991. of p53. PI3-kinase is required for prevention of apoptosis in rat 2. Swift, M., Reitnauer, P. J., Morrell, D., and Chase, C. L. Breast and other cancers in families with ataxia-telangiectasia. N. EngI. J. 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