- Email: [email protected]
ATM: the product of the gene mutated in ataxia-telangiectasia
The International Journal of Biochemistry & Cell Biology 31 (1999) 735±740 www.elsevier.com/locate/ijbcb
Molecules in focus
ATM: the product of the gene mutated in ataxiatelangiectasia Martin F. Lavin a, b,* a
The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, Qld 4029, Australia Department of Surgery, The University of Queensland, PO Royal Brisbane Hospital, Brisbane, Qld 4029, Australia
b
Received 5 January 1999; accepted 12 April 1999
Abstract Ataxia-telangiectasia mutated (ATM) is the product of the gene mutated in the human genetic disorder ataxiatelangeictasia (A-T). It is a 370 kDa protein that is a member of the phosphatidyl inositol 3-kinases superfamily. AT cells and those derived from Atmÿ/ÿ mice are characterized by hypersensitivity to ionizing radiation and defective cell cycle checkpoints. Defects are observed at all cell cycle checkpoints in A-T cells post-irradiation including the G1/S interface where ATM plays an important role in the activation of the tumour suppressor gene product p53. Activation leads to the induction of p21/WAF1, inhibition of cyclin-dependent kinase activity, failure to phosphorylate key substrates such as the retinoblastoma protein and consequently G1 arrest. ATM also plays an important role in the regulation and surveillance of meiotic progression. Absence of ATM gives rise to a spectrum of defects including immunode®ciency, neurodegeneration, radiosensitivity and cancer predisposition. It is clear that a better de®nition of the role of ATM in DNA damage recognition, cell cycle control and cell signalling may assist in the treatment of the progressive neurodegeneration in this syndrome. # 1999 Published by Elsevier Science Ltd. All rights reserved.
1. Introduction Ataxia-telangiectasia (A-T) is a multisystem disorder characterized by cerebellar degeneration, immunode®ciency, defective recognition of damage to DNA, radiosensitivity, cell cycle checkpoint defects and cancer predisposition * Tel.: +61 (07) 3362 0341; fax: +61 (07) 3362 0106. E-mail address: [email protected] (Martin F. Lavin)
[1,2]. A-T cells in culture are also characterized by a number of hallmarks that include slow growth rate, extreme radiosensitivity, radio resistant DNA synthesis, chromosomal instability, speci®c translocations, defective repair of a subtype of DNA strand breaks and defective cell cycle checkpoint activation [1]. These abnormalities, that reveal a defect in DNA damage recognition and as a consequence genome instability, can be correlated with the increased incidence of
1357-2725/99/$ - see front matter # 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 2 8 - X
736
M. F. Lavin / The International Journal of Biochemistry & Cell Biology 31 (1999) 735±740
cancer in A-T patients [3]. It is also of interest that mutations in the ataxia-telangiectasia mutated (ATM) gene have been detected in sporadic leukaemia in non-A-T patients [4±6]. The basis of the radio sensitivity has not yet been determined but several reports point to a slower rate of repair of DNA double strand breaks or the continued existence of residual breaks [7]. The identity of the A-T gene, ATM [8], is now ®rmly established and interacting proteins have been identi®ed that further strengthens a role for ATM in DNA damage recognition and cell cycle checkpoint control [9,10]. The identi®cation of new partner proteins will assist in elucidating the many roles that apparently exist for ATM. 2. Structure Savitsky et al. [8] cloned a partial ATM cDNA, 5.9 kb in size, that encoded a protein containing a domain related to that in the phosphatidyl inositol 3-kinases (PI3-kinases) superfamily [11]. The ATM gene was subsequently shown to extend over 150 kb of genomic DNA and to code for an mRNA of approx. 13 kb in size [12]. No evidence for alternate transcripts in the open reading frame was obtained but complex alternate splicing was observed within the 5 ' and 3'-untranslated regions of the gene [13], Fulllength cDNA was eventually cloned and shown to correct aspects of the radiosensite phenotype in A-T cells as well as the defective cell cycle checkpoints [14,15]. Partial correction by a cDNA fragment containing the PI3-kinases region was also reported [16]. That a single gene mutation was responsible for A-T was con®rmed when it was shown that Atmÿ/ÿ mice had a similar phenotype [17±20]. These mice displayed some growth retardation, mild neurological dysfunction, male and female infertility, immunode®ciency, radio sensitivity, and a prediliction to thymic lymphomas [17±19]. None of these studies with Atmÿ/ÿ mice reproduced the neurodegenerative changes observed in A-T patients, but one study employing electron microscopy revealed evidence of degeneration of neurons in the cer-
ebellar cortex of 2 month old Atmÿ/ÿ mice [21]. Mice doubly null for Atm and p53 exhibit a rapid acceleration of tumourigenesis compared to the single knockouts [22]. On the other hand a requirement for Atm in radiation-induced apoptosis in the developing CNS is mediated by p53 [20]. 3. Synthesis and degradation ATM is predominantly found in the nucleus of proliferating cells but 10±20% of the protein is present in cytoplasmic vesicles including peroxisomes and endosomes [10,23]. An alteration in this distribution in favour of the cytoplasm is observed in Purkinje cells [24] which may be related to the dierentiation state of the cells. No agent has been identi®ed that can alter this distribution in proliferilnig cells. Expression of ATM and subcellular distribution varies greatly from tissue to tissue and cell type to cell type (SoonLee et al., unpublished). No obvious nuclear localization signals are detected in ATM and neither an ectopically expressed N-terminal fragment of the protein (Keating et al., unpublished) nor a C-terminal fragment (Brown et al., unpublished) are capable of entering the nucleus. This suggests that in its normal conformation ATM interacts with another protein that facilitates its entry into the nucleus. A sequence at the extreme C-terminus of ATM, related to that observed in human catalase (a peroxisomal enzyme), has the potential for localizing the protein to peroxisomes (Watters et al., submitted). The state of dierentiation of a speci®c cell type may determine how much ATM is tracked to the nucleus. The half-life of the protein is in excess of 8 h and there is no evidence for dierent splice forms. However, complex alternate splicing is observed within the 3 ' and 5 ' UTR regions of the transcript. These splice forms point to posttranscriptional regulation of ATM transcripts [13]. This form of regulation may explain the ability of PHA to increase markedly the amount of ATM protein and its kinase activity in peripheral blood mononuclear cells (Fukao et al., in press).
M. F. Lavin / The International Journal of Biochemistry & Cell Biology 31 (1999) 735±740
DEEP FIGURE Remove frame ref and adjust check for multiple ®gure
Fig. 1. (Caption overleaf)
737
738
M. F. Lavin / The International Journal of Biochemistry & Cell Biology 31 (1999) 735±740
4. Biological function A-T patients exposed to radiotherapy exhibit an adverse response and A-T cells in culture are 3±4 times more radiosensitive than controls [25]. Rejoining of radiation-induced double strand breaks is less ecient in A-T ®broblasts than in controls, with approx. 10% of breaks remaining unrepaired at 72 h post-irradiation [7]. These results suggest that a sub-category of double strand breaks remain unrepaired in A-T cells or that other lesions remain unrepaired and are eventually converted into double strand breaks with ends that are not amenable to rejoining. AT cells are defective in both the G1/S and G2/M phase checkpoints after irradiation and DNA synthesis is inhibited to a lesser extent than in controls [26]. These cells are not inhibited in their progression from G1 into S phase, and G2/M phase cells show reduced mitotic delay compared to controls but these cells are irreversibly blocked in the subsequent G2 phase where they die [26]. The p53 signal transduction pathway operating through WAF1/p21 and cyclin E-cdk2 kinase was subsequently shown to be defective in A-T cells accounting for the defect at the G1/S checkpoint [27]. This response involves, at least in part, phosphorylation of p53 on serine 15 by ATM [28±30] and phosphorylation at this site reduces the interaction of p53 with MDM2 in
vivo and in vitro [31] which suppresses the transcriptional activity of p53 by enhancing its degradation through the ubiquitin pathway. Dephosphorylation of ser 376 in the C-terminal portion of p53 has also been reported to be ATM-dependent [32]. Checkpoint activation by DNA damage at G2/M is even more complex with some evidence for the participation of the p53 signalling pathway. Lessons from yeast portray ATM in a role similar to that of the related protein rad3 (S. pombe ). In response to DNA damage chk2 (S. pombe, Cds1) is phosphorylated and activated (ATM-dependent) to in turn phosphorylate and inactivate Cdc25C (ser 216), a phosphatase required for the activation of cyclinB-cdc2 kinase and for the progression of cells into mitosis [33]. Chk2 is also involved in the S phase checkpoint in S. pombe [34]. Cdc25C can be inhibited by an additional pathway operating through rad3 and the protein kinase chk1 [35]. Furthermore, the presence of chk1 on meiotic chromosomes is dependent on ATM [36]. These pathways are outlined in Fig. 1. 5. Medical applications The most debilitating aspect of A-T is the progressive neurodegeneration primarily due to loss of functional Purkinje and granular cells. Clearly
Fig. 1. Radiation signal transduction mediated by ATM. Exposure of cells to ionizing radiation leads to breaks (ssbs at dsbs) and base modi®cation in DNA. One of these lesions, probably a dsb or DNA damage converted to a dsb, activates ATM by an unknown mechanism which may or may not depend on ATM interacting directly with DNA. Once activated ATM phosphorylates a number of substrates including p53, IkB, PHAS1, c-Abl, RPA and chk2. Phosphorylation of p53 by ATM on serine 15, one of several modi®cations to p53 to stabilize and/or activate its transcriptional activity. P53 induces a number of dierent genes including p21/WAF1, mdm2, gadd45 and bax. In this case p21/WAFI is illustrated. This protein is a cyclin-dependent kinase which when bound in excess to cycline E-cdk2 in G1 phase inhibits its kinase activity preventing the phosphorylation of substrates such as the retinoblastoma protein and thus arresting cells in G1 phase. When ATM is mutated or absent, as in A-T cells, the rapid activation of the G1/S checkpoint is defective. At later times other PI3-kinases such as ataxia-telangiectasia and rad3-related (ATR) compensate for the defect. Abnormalities in other checkpoints are also observed in A-T cells and Atmÿ/ÿ mice, they exhibit radioresistant DNA synthesis and fail to show the initial G2/M delay post-irradiation. The involvement of ATM in controlling DNA synthesis after inradiation remains unclear but RPA has been implicated since its pattern of phosphorylation is abnormal in A-T cells after irradiation and it is a substrate for ATM. In relation to the G2/M checkpoint there is evidence that some of the inhibition of cyclinB-cdc2 occurs due to increased association with p21/WAFI, implicating the p53 pathway in G2/M arrest. However, based on data from yeast mutants; and the identi®cation of candidate mammalian proteins it is evident that control of the G2/M checkpoint is complex involving multiple proteins. A simplistic form of this appears in the ®gure where ATM activates chk2 which inhibits the phosphatase activity of Cdc25c. Under normal cell cycling conditions this phosphatase dephosphorylates and activates cdc2 to assist the progress of cells through G2 phase into mitosis. Clearly when cells are damaged the G2/M checkpoint is activated to impede this progress. There is also evidence that ATM activates chkl but its downstream target(s) remain unresolved.
M. F. Lavin / The International Journal of Biochemistry & Cell Biology 31 (1999) 735±740
therapeutic intervention would involve an arrest or slowing of these changes. Targetting ATM or other constructs to Purkinje cells would appear to be an impossible task at this stage. More likely, research into the progressive loss of these cells will be more rewarding in developing therapeutic intervention. The various Atmÿ/ÿ mouse models represent a useful approach to describing the abnormalities that exist in the cerebellum and elsewhere in the brain and consequently designing treatment protocols. The predominant distribution of ATM to the cytoplasm in Purkinje cells [24] compared to proliferating cells, where it is largely present in the nucleus, suggests that ATM may play a dierent role in dierentiated cells. For example, in Purkinje cells it might be expected to play a role other than in recognizing damage in DNA, such as in cell-to-cell signalling which is important in maintaining the viability of these cells. Indeed there is evidence that ATM is involved in responding to other stimuli during growth and in B and T cell functioning. Identi®cation of the molecules involved in such signalling may allow for an interventionist approach to arresting the decline in neuronal function in these patients. The ability of anti-sense ATM cDNA to sensitize normal control cells to ionizing radiation presents a potential approach to improving the therapeutic bene®t of radiotherapy. By knocking out the function of ATM by this or other approaches it should be possible to sensitize radioresistant tumours, reduce the risk to normal tissue and provide a more eective means of killing tumour cells or arresting growth.
References [1] M.F. Lavin, Y. Shiloh, The genetic defect in ataxia-telangiectasia, Annual Review of Immunology 15 (1997) 177±202. [2] R.A. Gatti, Ataxia-telangiectasia, in: B. Vogelsteion, K.W. Kinzler (Eds.), The Genetic Basis of Human Cancer, McGraw-Hill, New York, 1998, pp. 275±300. [3] F. Hecht, B.K. Hecht, Cancer in ataxia-telangiectasia patients, Cancer Genetics Cytogenetics 46 (1990) 9±19. [4] I. Vorechovsky, L. Luo, M.J. Dyer, D. Catovsky, P.L. Amlot, J.C. Yaxley, L. Foroni, L. Hammarstrom, A.D.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
739
Webster, M.A. Yuille, Clustering of missence mutations in the ataxia-telangiectasia gene in sporadic T-cell leukaemia, Nature Genetics 17 (1997) 96±99. M.A. Yuille, L.J. Coignet, S.M. Abraham, F. Yaqub, L. Luo, E. Matutes, V. Brito-Babapulle, I. Vorechovsky, M.J. Syer, D. Catovsky, ATM is usually rearranged in T-cell prolymphocytic leukaemia, Oncogene 16 (1998) 789±796. T. Stankovic, P. Weber, G. Stewart, T. Bedenham, J. Murray, P.J. Byrd, P.A.H. Moss, A.M.R. Taylor, Inactivation of ataxia-telangiectasia mutated gene in Bcell chronic lymphocytic leukaemia, Lancet 353 (1999) 26±29. N. Foray, A. Priestley, G. Alsbeih, C. Badie, E.P. Capulas, C.F. Arlett, E.P. Malaise, Hypersensitivity of ataxia-telangiectasia ®broblasts to ionizing radiation is associated with a repair de®ciency of DNA doublestrand breaks, International Journal of Radiation Biology 72 (1997) 271±283. K. Savitsky, A. Bar-Shira, S. Gilad, G. Rotman, Y. Ziv, L. Vanagaite, D.A. Tagle, S. Smith, T. Uziel, S. Sfez, M. Ashkenazi, I. Pecker, R. Harnik, S.R. Patanjali, A. Simmons, M. Frydman, A. Sartiel, R.A. Gatti, L. Chessa, O. Sanal, M.F. Lavin, N.G.J. Jaspers, A. Malcolm, R. Taylor, C.F. Arlett, T. Miki, S.M. Weissman, M. Lovett, F.S. Collins, Y. Shiloh, A single ataxia-telangiectasia gene with a product similar to PI-3 kinase, Science 268 (1995) 1749±1753. K.K. Khanna, T. Shafman, P. Kedar, K. Spring, S. Kozlov, T. Yen, K. Hobson, M. Gatei, N. Zhang, D. Watters, M. Egerton, Y. Shiloh, S. Kharbanda, D. Kufe, M.F. Lavin, Interaction between ATM protein and c-Abl in response to DNA damage, Nature 387 (1997) 520±523. D. Watters, K.K. Khanna, H. Beamish, G. Birrell, K. Spring, P. Kedar, M. Gatei, D. Stenzel, K. Hobson, S. Kozlov, A. Farrell, J. Ramsay, R. Gatti, M.F. Lavin, Cellular localisation of the ataxia-telangiectasia (ATM) gene proteins and discrimination between mutated and normal forms, Oncogene 14 (1997) 1911±1921. M.F. Lavin, K.K. Khanna, H. Beamish, R. Williams, K. Spring, D. Watters, Y. Shiloh, Relationship of the ATM gene (mutated in ataxia-telangiectasia) to phosphalidylinositol 3-kinase, Trends in Biochemical Sciences 20 (1995) 382±383. K. Savitsky, S. Sfez, D. Tagle, Y. Ziv, A. Sartiel, F.S. Collins, Y. Shiloh, G. Rotman, The complete sequence of the coding region of the ATM gene reveals similarity to cell cycle regulators in dierent species, Human Molecular Genetics 4 (1995) 2025±2032. K. Savitsky, M. Platzer, T. Uziel, S. Gilad, A. Sartiel, A. Rosenthal, O. Elroy-Stein, Y. Shiloh, G. Rotman, Ataxia-telangiectasia: structural diversity of untranslated sequences suggests complex post-transcriptional regulation of ATM gene expression, Nucleic Acids Research 25 (1997) 1678±1684. N. Zhang, P. Chen, K.K. Khanna, S. Scott, M. Gatei,
740
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
M. F. Lavin / The International Journal of Biochemistry & Cell Biology 31 (1999) 735±740 S. Kozlov, D. Watters, K. Spring, T. Yen, M.F. Lavin, Isolation of full-length ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype, in: Proceedings of the National Academy of Sciences USA 94, 1997, pp. 8021±8026. Y. Ziv, A. Bar-Shira, I. Pecker, P. Russell, T.J. Jorgensen, I. Tsarfati, Y. Shiloh, Recombinant ATM protein complements the cellular A-T phenotype, Oncogene 15 (1997) 159±167. S.E. Morgan, C. Lovly, T.K. Pandita, Y. Shiloh, M. Kastan, Fragments of ATM which have dominant-negative or complementing activity, Molecular & Cellular Biology 17 (1997) 2020±2029. C. Barlow, S. Hirotsune, R. Paylor, M. Liyanage, M. Eckhaus, F. Collins, Y. Shiloh, J.N. Crawley, Y. Reid, D. Tagle, A. Wynshaw-Boris, ATM-de®cient mice: a paragon of ataxia-telangiectasia, Cell 86 (1996) 159±171. A. Elson, Y. Wang, C.J. Daugherty, C.C. Morton, F. Zhou, J. Campos-Torres, P. Leder, Pleiotropic defects in ataxia-telangiectasia protein-de®cient mice, in: Proceedings of the National Academy of Sciences USA 83, 1996, pp. 13084±13089. Y. Xu, T. Ashley, E.E. Brainerd, R.T. Bronson, S.M. Meyn, D. Baltimore, Targeted disruption of ATM leads to growth retardation, chromosomal fragmentation during meiosis, immune defects and thymic lymphoma, Genes and Development 10 (1996) 2411±2422. K.H. Herzog, M.J. Chong, M. Kapsetaki, J.I. Morgan, P.J. McKinnon, Requirement for Atm in ionizing radiation-induced cell death in the developing central nervous system, Science 5366 (1998) 1089±1091. R.O. Kuljis, Y. Xu, M.C. Aguila, D. Baltimore, Degeneration of neurons, synapses, and neuropil and glial activation in a murine Atm knockout model of Ataxia-telangiectasia, in: Proceedings of the National Academy of Sciences, 23, 1997, pp. 12688±12693. C.H. Westphal, S. Rowan, C. Schmaltz, A. Elson, D.E. Fisher, P. Leder, Atm and p53 cooperate in apoptosis and suppression of tumorigenesis, but not in resistance to acute radiation toxicity, Nature Genetics 4 (1997) 397±401. D-S. Lim, D.G. Kirsch, C.D. Canman, J-H. Ahn, Y. Ziv, L.S. Newman, R.B. Darnell, Y. Shiloh, M.B. Kastan, ATM binds to b-adaptin in cytoplasmic vesicles, in: Proceedings of the National Academy of Sciences USA 95, 1998, pp. 10146±10151. A. Oka, S. Takashima, Expression of the ataxia-telangiectasia gene (ATM) product in human cerebellar neurons during development, Neuroscience Letters 252 (1998) 195±198.
[25] A.M. Taylor, D.G. Harnden, C.F. Arlett, S.A. Harcourt, A.R. Lehmann, S. Stevens, B.A. Bridges, Ataxia-telangiectasia: a human mutation with abnormal radiation sensitivity, Nature 4 (1975) 427±429. [26] H. Beamish, M.F. Lavin, Radiosensitivity in ataxia-telangiectasia: anomalies in radiation-induced cell cycle delay, International Journal of Radiation Biology 65 (1994) 175±184. [27] K. Khanna, H. Beamish, J. Yan, K. Hobson, R. Williams, I. Dunn, M.F. Lavin, Nature of G1/S cell cycle checkpoint defect in ataxia-telangiectasia, Oncogene 11 (1995) 609±618. [28] S. Banin, L. Moyal, S-Y. Shieh, Y. Taya, C.W. Anderson, L. Chessa, N.I. Smorodinsky, C. Prives, Y. Reiss, Y. Shiloh, Y. Ziv, Enhanced phosphorylation of p53 by ATM in response to DNA damage, Science 281 (1998) 1674±1677. [29] C.E. Canman, D-S. Lim, K.A. Cimprich, Y. Taya, K. Tamai, K. Sakaguchi, E. Appella, M.B. Kastan, J.D. Silicano, Activation of the ATM kinase by ionizing radiation and phosphorylation of p53, Science 281 (1998) 1677±1679. [30] K.K. Khanna, K.E. Keating, S. Kozlov, S. Scott, M. Gatei, K. Hobson, Y. Taya, B. Gabrielli, D. Chan, S.P. Lees-Miller, M.F. Lavin, ATM associates with and phosphorylates p53: mapping the region of interaction, Nature Genetics 20 (1998) 398±400. [31] S.Y. Shieh, M. Ikeda, Y. Taya, C. Prives, DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2, Cell 91 (1997) 325±334. [32] M.J.F. Waterman, E.S. Stavridi, J.L.F. Waterman, T.D. Halazonetis, ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins, Nature Genetics 19 (1998) 175±178. [33] S. Matsuoka, M. Huang, S.J. Elledge, Linkage of ATM to cell cycle regulation by the Chk2 protein kinase, Science 282 (1998) 1893±1897. [34] R.G. Martinho, H.D. Lindsay, G. Flaggs, A.J. DeMaggio, M.F. Hoekstra, A.M. Carr, N.J. Bentley, Analysis of Rad3 and Chk1 protein kinases de®nes dierent checkpoint responses, The EMBO Journal 17 (1998) 7239±7249. [35] N. Walworth, R. Bernards, Rad-dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint, Science 271 (1996) 353±356. [36] G. Flaggs, R.G. Martinho, H.D. Lindsay, A.J. DeMaggio, M.F. Hoekstra, A.M. Carr, A.J. Bentley, Analysis of Rad3 and Chk1 protein kinases de®nes dierent checkpoint responses, The EMBO Journal 17 (1998) 7239±7249.
Molecules in focus
ATM: the product of the gene mutated in ataxiatelangiectasia Martin F. Lavin a, b,* a
The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, Qld 4029, Australia Department of Surgery, The University of Queensland, PO Royal Brisbane Hospital, Brisbane, Qld 4029, Australia
b
Received 5 January 1999; accepted 12 April 1999
Abstract Ataxia-telangiectasia mutated (ATM) is the product of the gene mutated in the human genetic disorder ataxiatelangeictasia (A-T). It is a 370 kDa protein that is a member of the phosphatidyl inositol 3-kinases superfamily. AT cells and those derived from Atmÿ/ÿ mice are characterized by hypersensitivity to ionizing radiation and defective cell cycle checkpoints. Defects are observed at all cell cycle checkpoints in A-T cells post-irradiation including the G1/S interface where ATM plays an important role in the activation of the tumour suppressor gene product p53. Activation leads to the induction of p21/WAF1, inhibition of cyclin-dependent kinase activity, failure to phosphorylate key substrates such as the retinoblastoma protein and consequently G1 arrest. ATM also plays an important role in the regulation and surveillance of meiotic progression. Absence of ATM gives rise to a spectrum of defects including immunode®ciency, neurodegeneration, radiosensitivity and cancer predisposition. It is clear that a better de®nition of the role of ATM in DNA damage recognition, cell cycle control and cell signalling may assist in the treatment of the progressive neurodegeneration in this syndrome. # 1999 Published by Elsevier Science Ltd. All rights reserved.
1. Introduction Ataxia-telangiectasia (A-T) is a multisystem disorder characterized by cerebellar degeneration, immunode®ciency, defective recognition of damage to DNA, radiosensitivity, cell cycle checkpoint defects and cancer predisposition * Tel.: +61 (07) 3362 0341; fax: +61 (07) 3362 0106. E-mail address: [email protected] (Martin F. Lavin)
[1,2]. A-T cells in culture are also characterized by a number of hallmarks that include slow growth rate, extreme radiosensitivity, radio resistant DNA synthesis, chromosomal instability, speci®c translocations, defective repair of a subtype of DNA strand breaks and defective cell cycle checkpoint activation [1]. These abnormalities, that reveal a defect in DNA damage recognition and as a consequence genome instability, can be correlated with the increased incidence of
1357-2725/99/$ - see front matter # 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 2 8 - X
736
M. F. Lavin / The International Journal of Biochemistry & Cell Biology 31 (1999) 735±740
cancer in A-T patients [3]. It is also of interest that mutations in the ataxia-telangiectasia mutated (ATM) gene have been detected in sporadic leukaemia in non-A-T patients [4±6]. The basis of the radio sensitivity has not yet been determined but several reports point to a slower rate of repair of DNA double strand breaks or the continued existence of residual breaks [7]. The identity of the A-T gene, ATM [8], is now ®rmly established and interacting proteins have been identi®ed that further strengthens a role for ATM in DNA damage recognition and cell cycle checkpoint control [9,10]. The identi®cation of new partner proteins will assist in elucidating the many roles that apparently exist for ATM. 2. Structure Savitsky et al. [8] cloned a partial ATM cDNA, 5.9 kb in size, that encoded a protein containing a domain related to that in the phosphatidyl inositol 3-kinases (PI3-kinases) superfamily [11]. The ATM gene was subsequently shown to extend over 150 kb of genomic DNA and to code for an mRNA of approx. 13 kb in size [12]. No evidence for alternate transcripts in the open reading frame was obtained but complex alternate splicing was observed within the 5 ' and 3'-untranslated regions of the gene [13], Fulllength cDNA was eventually cloned and shown to correct aspects of the radiosensite phenotype in A-T cells as well as the defective cell cycle checkpoints [14,15]. Partial correction by a cDNA fragment containing the PI3-kinases region was also reported [16]. That a single gene mutation was responsible for A-T was con®rmed when it was shown that Atmÿ/ÿ mice had a similar phenotype [17±20]. These mice displayed some growth retardation, mild neurological dysfunction, male and female infertility, immunode®ciency, radio sensitivity, and a prediliction to thymic lymphomas [17±19]. None of these studies with Atmÿ/ÿ mice reproduced the neurodegenerative changes observed in A-T patients, but one study employing electron microscopy revealed evidence of degeneration of neurons in the cer-
ebellar cortex of 2 month old Atmÿ/ÿ mice [21]. Mice doubly null for Atm and p53 exhibit a rapid acceleration of tumourigenesis compared to the single knockouts [22]. On the other hand a requirement for Atm in radiation-induced apoptosis in the developing CNS is mediated by p53 [20]. 3. Synthesis and degradation ATM is predominantly found in the nucleus of proliferating cells but 10±20% of the protein is present in cytoplasmic vesicles including peroxisomes and endosomes [10,23]. An alteration in this distribution in favour of the cytoplasm is observed in Purkinje cells [24] which may be related to the dierentiation state of the cells. No agent has been identi®ed that can alter this distribution in proliferilnig cells. Expression of ATM and subcellular distribution varies greatly from tissue to tissue and cell type to cell type (SoonLee et al., unpublished). No obvious nuclear localization signals are detected in ATM and neither an ectopically expressed N-terminal fragment of the protein (Keating et al., unpublished) nor a C-terminal fragment (Brown et al., unpublished) are capable of entering the nucleus. This suggests that in its normal conformation ATM interacts with another protein that facilitates its entry into the nucleus. A sequence at the extreme C-terminus of ATM, related to that observed in human catalase (a peroxisomal enzyme), has the potential for localizing the protein to peroxisomes (Watters et al., submitted). The state of dierentiation of a speci®c cell type may determine how much ATM is tracked to the nucleus. The half-life of the protein is in excess of 8 h and there is no evidence for dierent splice forms. However, complex alternate splicing is observed within the 3 ' and 5 ' UTR regions of the transcript. These splice forms point to posttranscriptional regulation of ATM transcripts [13]. This form of regulation may explain the ability of PHA to increase markedly the amount of ATM protein and its kinase activity in peripheral blood mononuclear cells (Fukao et al., in press).
M. F. Lavin / The International Journal of Biochemistry & Cell Biology 31 (1999) 735±740
DEEP FIGURE Remove frame ref and adjust check for multiple ®gure
Fig. 1. (Caption overleaf)
737
738
M. F. Lavin / The International Journal of Biochemistry & Cell Biology 31 (1999) 735±740
4. Biological function A-T patients exposed to radiotherapy exhibit an adverse response and A-T cells in culture are 3±4 times more radiosensitive than controls [25]. Rejoining of radiation-induced double strand breaks is less ecient in A-T ®broblasts than in controls, with approx. 10% of breaks remaining unrepaired at 72 h post-irradiation [7]. These results suggest that a sub-category of double strand breaks remain unrepaired in A-T cells or that other lesions remain unrepaired and are eventually converted into double strand breaks with ends that are not amenable to rejoining. AT cells are defective in both the G1/S and G2/M phase checkpoints after irradiation and DNA synthesis is inhibited to a lesser extent than in controls [26]. These cells are not inhibited in their progression from G1 into S phase, and G2/M phase cells show reduced mitotic delay compared to controls but these cells are irreversibly blocked in the subsequent G2 phase where they die [26]. The p53 signal transduction pathway operating through WAF1/p21 and cyclin E-cdk2 kinase was subsequently shown to be defective in A-T cells accounting for the defect at the G1/S checkpoint [27]. This response involves, at least in part, phosphorylation of p53 on serine 15 by ATM [28±30] and phosphorylation at this site reduces the interaction of p53 with MDM2 in
vivo and in vitro [31] which suppresses the transcriptional activity of p53 by enhancing its degradation through the ubiquitin pathway. Dephosphorylation of ser 376 in the C-terminal portion of p53 has also been reported to be ATM-dependent [32]. Checkpoint activation by DNA damage at G2/M is even more complex with some evidence for the participation of the p53 signalling pathway. Lessons from yeast portray ATM in a role similar to that of the related protein rad3 (S. pombe ). In response to DNA damage chk2 (S. pombe, Cds1) is phosphorylated and activated (ATM-dependent) to in turn phosphorylate and inactivate Cdc25C (ser 216), a phosphatase required for the activation of cyclinB-cdc2 kinase and for the progression of cells into mitosis [33]. Chk2 is also involved in the S phase checkpoint in S. pombe [34]. Cdc25C can be inhibited by an additional pathway operating through rad3 and the protein kinase chk1 [35]. Furthermore, the presence of chk1 on meiotic chromosomes is dependent on ATM [36]. These pathways are outlined in Fig. 1. 5. Medical applications The most debilitating aspect of A-T is the progressive neurodegeneration primarily due to loss of functional Purkinje and granular cells. Clearly
Fig. 1. Radiation signal transduction mediated by ATM. Exposure of cells to ionizing radiation leads to breaks (ssbs at dsbs) and base modi®cation in DNA. One of these lesions, probably a dsb or DNA damage converted to a dsb, activates ATM by an unknown mechanism which may or may not depend on ATM interacting directly with DNA. Once activated ATM phosphorylates a number of substrates including p53, IkB, PHAS1, c-Abl, RPA and chk2. Phosphorylation of p53 by ATM on serine 15, one of several modi®cations to p53 to stabilize and/or activate its transcriptional activity. P53 induces a number of dierent genes including p21/WAF1, mdm2, gadd45 and bax. In this case p21/WAFI is illustrated. This protein is a cyclin-dependent kinase which when bound in excess to cycline E-cdk2 in G1 phase inhibits its kinase activity preventing the phosphorylation of substrates such as the retinoblastoma protein and thus arresting cells in G1 phase. When ATM is mutated or absent, as in A-T cells, the rapid activation of the G1/S checkpoint is defective. At later times other PI3-kinases such as ataxia-telangiectasia and rad3-related (ATR) compensate for the defect. Abnormalities in other checkpoints are also observed in A-T cells and Atmÿ/ÿ mice, they exhibit radioresistant DNA synthesis and fail to show the initial G2/M delay post-irradiation. The involvement of ATM in controlling DNA synthesis after inradiation remains unclear but RPA has been implicated since its pattern of phosphorylation is abnormal in A-T cells after irradiation and it is a substrate for ATM. In relation to the G2/M checkpoint there is evidence that some of the inhibition of cyclinB-cdc2 occurs due to increased association with p21/WAFI, implicating the p53 pathway in G2/M arrest. However, based on data from yeast mutants; and the identi®cation of candidate mammalian proteins it is evident that control of the G2/M checkpoint is complex involving multiple proteins. A simplistic form of this appears in the ®gure where ATM activates chk2 which inhibits the phosphatase activity of Cdc25c. Under normal cell cycling conditions this phosphatase dephosphorylates and activates cdc2 to assist the progress of cells through G2 phase into mitosis. Clearly when cells are damaged the G2/M checkpoint is activated to impede this progress. There is also evidence that ATM activates chkl but its downstream target(s) remain unresolved.
M. F. Lavin / The International Journal of Biochemistry & Cell Biology 31 (1999) 735±740
therapeutic intervention would involve an arrest or slowing of these changes. Targetting ATM or other constructs to Purkinje cells would appear to be an impossible task at this stage. More likely, research into the progressive loss of these cells will be more rewarding in developing therapeutic intervention. The various Atmÿ/ÿ mouse models represent a useful approach to describing the abnormalities that exist in the cerebellum and elsewhere in the brain and consequently designing treatment protocols. The predominant distribution of ATM to the cytoplasm in Purkinje cells [24] compared to proliferating cells, where it is largely present in the nucleus, suggests that ATM may play a dierent role in dierentiated cells. For example, in Purkinje cells it might be expected to play a role other than in recognizing damage in DNA, such as in cell-to-cell signalling which is important in maintaining the viability of these cells. Indeed there is evidence that ATM is involved in responding to other stimuli during growth and in B and T cell functioning. Identi®cation of the molecules involved in such signalling may allow for an interventionist approach to arresting the decline in neuronal function in these patients. The ability of anti-sense ATM cDNA to sensitize normal control cells to ionizing radiation presents a potential approach to improving the therapeutic bene®t of radiotherapy. By knocking out the function of ATM by this or other approaches it should be possible to sensitize radioresistant tumours, reduce the risk to normal tissue and provide a more eective means of killing tumour cells or arresting growth.
References [1] M.F. Lavin, Y. Shiloh, The genetic defect in ataxia-telangiectasia, Annual Review of Immunology 15 (1997) 177±202. [2] R.A. Gatti, Ataxia-telangiectasia, in: B. Vogelsteion, K.W. Kinzler (Eds.), The Genetic Basis of Human Cancer, McGraw-Hill, New York, 1998, pp. 275±300. [3] F. Hecht, B.K. Hecht, Cancer in ataxia-telangiectasia patients, Cancer Genetics Cytogenetics 46 (1990) 9±19. [4] I. Vorechovsky, L. Luo, M.J. Dyer, D. Catovsky, P.L. Amlot, J.C. Yaxley, L. Foroni, L. Hammarstrom, A.D.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
739
Webster, M.A. Yuille, Clustering of missence mutations in the ataxia-telangiectasia gene in sporadic T-cell leukaemia, Nature Genetics 17 (1997) 96±99. M.A. Yuille, L.J. Coignet, S.M. Abraham, F. Yaqub, L. Luo, E. Matutes, V. Brito-Babapulle, I. Vorechovsky, M.J. Syer, D. Catovsky, ATM is usually rearranged in T-cell prolymphocytic leukaemia, Oncogene 16 (1998) 789±796. T. Stankovic, P. Weber, G. Stewart, T. Bedenham, J. Murray, P.J. Byrd, P.A.H. Moss, A.M.R. Taylor, Inactivation of ataxia-telangiectasia mutated gene in Bcell chronic lymphocytic leukaemia, Lancet 353 (1999) 26±29. N. Foray, A. Priestley, G. Alsbeih, C. Badie, E.P. Capulas, C.F. Arlett, E.P. Malaise, Hypersensitivity of ataxia-telangiectasia ®broblasts to ionizing radiation is associated with a repair de®ciency of DNA doublestrand breaks, International Journal of Radiation Biology 72 (1997) 271±283. K. Savitsky, A. Bar-Shira, S. Gilad, G. Rotman, Y. Ziv, L. Vanagaite, D.A. Tagle, S. Smith, T. Uziel, S. Sfez, M. Ashkenazi, I. Pecker, R. Harnik, S.R. Patanjali, A. Simmons, M. Frydman, A. Sartiel, R.A. Gatti, L. Chessa, O. Sanal, M.F. Lavin, N.G.J. Jaspers, A. Malcolm, R. Taylor, C.F. Arlett, T. Miki, S.M. Weissman, M. Lovett, F.S. Collins, Y. Shiloh, A single ataxia-telangiectasia gene with a product similar to PI-3 kinase, Science 268 (1995) 1749±1753. K.K. Khanna, T. Shafman, P. Kedar, K. Spring, S. Kozlov, T. Yen, K. Hobson, M. Gatei, N. Zhang, D. Watters, M. Egerton, Y. Shiloh, S. Kharbanda, D. Kufe, M.F. Lavin, Interaction between ATM protein and c-Abl in response to DNA damage, Nature 387 (1997) 520±523. D. Watters, K.K. Khanna, H. Beamish, G. Birrell, K. Spring, P. Kedar, M. Gatei, D. Stenzel, K. Hobson, S. Kozlov, A. Farrell, J. Ramsay, R. Gatti, M.F. Lavin, Cellular localisation of the ataxia-telangiectasia (ATM) gene proteins and discrimination between mutated and normal forms, Oncogene 14 (1997) 1911±1921. M.F. Lavin, K.K. Khanna, H. Beamish, R. Williams, K. Spring, D. Watters, Y. Shiloh, Relationship of the ATM gene (mutated in ataxia-telangiectasia) to phosphalidylinositol 3-kinase, Trends in Biochemical Sciences 20 (1995) 382±383. K. Savitsky, S. Sfez, D. Tagle, Y. Ziv, A. Sartiel, F.S. Collins, Y. Shiloh, G. Rotman, The complete sequence of the coding region of the ATM gene reveals similarity to cell cycle regulators in dierent species, Human Molecular Genetics 4 (1995) 2025±2032. K. Savitsky, M. Platzer, T. Uziel, S. Gilad, A. Sartiel, A. Rosenthal, O. Elroy-Stein, Y. Shiloh, G. Rotman, Ataxia-telangiectasia: structural diversity of untranslated sequences suggests complex post-transcriptional regulation of ATM gene expression, Nucleic Acids Research 25 (1997) 1678±1684. N. Zhang, P. Chen, K.K. Khanna, S. Scott, M. Gatei,
740
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
M. F. Lavin / The International Journal of Biochemistry & Cell Biology 31 (1999) 735±740 S. Kozlov, D. Watters, K. Spring, T. Yen, M.F. Lavin, Isolation of full-length ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype, in: Proceedings of the National Academy of Sciences USA 94, 1997, pp. 8021±8026. Y. Ziv, A. Bar-Shira, I. Pecker, P. Russell, T.J. Jorgensen, I. Tsarfati, Y. Shiloh, Recombinant ATM protein complements the cellular A-T phenotype, Oncogene 15 (1997) 159±167. S.E. Morgan, C. Lovly, T.K. Pandita, Y. Shiloh, M. Kastan, Fragments of ATM which have dominant-negative or complementing activity, Molecular & Cellular Biology 17 (1997) 2020±2029. C. Barlow, S. Hirotsune, R. Paylor, M. Liyanage, M. Eckhaus, F. Collins, Y. Shiloh, J.N. Crawley, Y. Reid, D. Tagle, A. Wynshaw-Boris, ATM-de®cient mice: a paragon of ataxia-telangiectasia, Cell 86 (1996) 159±171. A. Elson, Y. Wang, C.J. Daugherty, C.C. Morton, F. Zhou, J. Campos-Torres, P. Leder, Pleiotropic defects in ataxia-telangiectasia protein-de®cient mice, in: Proceedings of the National Academy of Sciences USA 83, 1996, pp. 13084±13089. Y. Xu, T. Ashley, E.E. Brainerd, R.T. Bronson, S.M. Meyn, D. Baltimore, Targeted disruption of ATM leads to growth retardation, chromosomal fragmentation during meiosis, immune defects and thymic lymphoma, Genes and Development 10 (1996) 2411±2422. K.H. Herzog, M.J. Chong, M. Kapsetaki, J.I. Morgan, P.J. McKinnon, Requirement for Atm in ionizing radiation-induced cell death in the developing central nervous system, Science 5366 (1998) 1089±1091. R.O. Kuljis, Y. Xu, M.C. Aguila, D. Baltimore, Degeneration of neurons, synapses, and neuropil and glial activation in a murine Atm knockout model of Ataxia-telangiectasia, in: Proceedings of the National Academy of Sciences, 23, 1997, pp. 12688±12693. C.H. Westphal, S. Rowan, C. Schmaltz, A. Elson, D.E. Fisher, P. Leder, Atm and p53 cooperate in apoptosis and suppression of tumorigenesis, but not in resistance to acute radiation toxicity, Nature Genetics 4 (1997) 397±401. D-S. Lim, D.G. Kirsch, C.D. Canman, J-H. Ahn, Y. Ziv, L.S. Newman, R.B. Darnell, Y. Shiloh, M.B. Kastan, ATM binds to b-adaptin in cytoplasmic vesicles, in: Proceedings of the National Academy of Sciences USA 95, 1998, pp. 10146±10151. A. Oka, S. Takashima, Expression of the ataxia-telangiectasia gene (ATM) product in human cerebellar neurons during development, Neuroscience Letters 252 (1998) 195±198.
[25] A.M. Taylor, D.G. Harnden, C.F. Arlett, S.A. Harcourt, A.R. Lehmann, S. Stevens, B.A. Bridges, Ataxia-telangiectasia: a human mutation with abnormal radiation sensitivity, Nature 4 (1975) 427±429. [26] H. Beamish, M.F. Lavin, Radiosensitivity in ataxia-telangiectasia: anomalies in radiation-induced cell cycle delay, International Journal of Radiation Biology 65 (1994) 175±184. [27] K. Khanna, H. Beamish, J. Yan, K. Hobson, R. Williams, I. Dunn, M.F. Lavin, Nature of G1/S cell cycle checkpoint defect in ataxia-telangiectasia, Oncogene 11 (1995) 609±618. [28] S. Banin, L. Moyal, S-Y. Shieh, Y. Taya, C.W. Anderson, L. Chessa, N.I. Smorodinsky, C. Prives, Y. Reiss, Y. Shiloh, Y. Ziv, Enhanced phosphorylation of p53 by ATM in response to DNA damage, Science 281 (1998) 1674±1677. [29] C.E. Canman, D-S. Lim, K.A. Cimprich, Y. Taya, K. Tamai, K. Sakaguchi, E. Appella, M.B. Kastan, J.D. Silicano, Activation of the ATM kinase by ionizing radiation and phosphorylation of p53, Science 281 (1998) 1677±1679. [30] K.K. Khanna, K.E. Keating, S. Kozlov, S. Scott, M. Gatei, K. Hobson, Y. Taya, B. Gabrielli, D. Chan, S.P. Lees-Miller, M.F. Lavin, ATM associates with and phosphorylates p53: mapping the region of interaction, Nature Genetics 20 (1998) 398±400. [31] S.Y. Shieh, M. Ikeda, Y. Taya, C. Prives, DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2, Cell 91 (1997) 325±334. [32] M.J.F. Waterman, E.S. Stavridi, J.L.F. Waterman, T.D. Halazonetis, ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins, Nature Genetics 19 (1998) 175±178. [33] S. Matsuoka, M. Huang, S.J. Elledge, Linkage of ATM to cell cycle regulation by the Chk2 protein kinase, Science 282 (1998) 1893±1897. [34] R.G. Martinho, H.D. Lindsay, G. Flaggs, A.J. DeMaggio, M.F. Hoekstra, A.M. Carr, N.J. Bentley, Analysis of Rad3 and Chk1 protein kinases de®nes dierent checkpoint responses, The EMBO Journal 17 (1998) 7239±7249. [35] N. Walworth, R. Bernards, Rad-dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint, Science 271 (1996) 353±356. [36] G. Flaggs, R.G. Martinho, H.D. Lindsay, A.J. DeMaggio, M.F. Hoekstra, A.M. Carr, A.J. Bentley, Analysis of Rad3 and Chk1 protein kinases de®nes dierent checkpoint responses, The EMBO Journal 17 (1998) 7239±7249.