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Mammalian G1- and S-phase checkpoints in response to DNA damage
Jiri Bartek* and Jiri Lukas
The ability to preserve genomic integrity is a fundamental irreparable, checkpoints eliminate such potentially feature of life. Recent findings regarding the molecular basis hazardous cells by permanent cell-cycle arrest or cell death. of the cell-cycle checkpoint responses of mammalian cells togenotoxic stress have converged into a two-wave concept of Reflecting their distinct positions and functions within the the G1 checkpoint, and shed light on the so-far elusive checkpoint cascades, components of the cell-cycle check- intra-S-phase checkpoint. Rapidly operating cascades that points have been subclassified into DNA damage sensors, target the Cdc25A phosphatase appear central in both the signal transducers, and effectors [4]. To ensure faithful initiation wave of the G1 checkpoint (preceding the replication and transmission of the genome and to promote p53-mediated maintenance wave) and the transient survival, checkpoints fulfil at least four tasks: they rapidly intra-S-phase response. Multiple links between defects in induce cell-cycle delay, help activate DNA repair, maintain the G1/S checkpoints, genomic instability and oncogenesis the cell-cycle arrest until repair is complete, and then are emerging, as are new challenges and hopes raised by actively re-initiate cell-cycle progression. Mechanistic elements of the first three tasks are emerging, yet themolecular basis of the recovery from checkpoint-mediated Addresses
arrest remains unknown. The biological and (patho)physi- Department of Cell Cycle and Cancer, Institute of Cancer Biology, ological relevance of the checkpoint pathways is supported Danish Cancer Society, Strandboulevarden 49, DK-2100 by their evolutionary conservation [4], and it is evident from the consequences of checkpoint failure. Checkpoint *e-mail: bartek@biobase.dkCorrespondence: Jiri Bartek malfunction leads to accumulation of mutations and chromosomal aberrations, which in turn increase the Current Opinion in Cell Biology 2001, 13:738–747
probability of developmental malformations or genetic syndromes and diseases including cancer [3–11].
2001 Elsevier Science Ltd. All rights reserved.
Despite the response of some checkpoint cascades to DNA Abbreviations
ATM

damage in quiescent cells [12•], most checkpoint pathways operate only in cycling cells, which are at higher risk of fixing and propagating deleterious mutations [3–11]. But even among proliferating cells, the choice of checkpoint cascade(s) to be alarmed, and the outcome of such response, depends on many variables. These factors include the type, extent and duration of the DNA-damage stimulus, the typeof cell cycle (meiotic versus mitotic; early embryonic versus Introduction
‘somatic’), the cell type and differentiation stage, and the It is arguable that now and then the odd genetic mutation position of the cell within the cell cycle. Although we are can be a healthy event, particularly in germ cells. Such still largely ignorant of the impact of some of these variables mutations complement genetic recombination in providing on checkpoint control and execution, rapid advances have limited genomic plasticity necessary for the process of recently been made in understanding the molecular basis of evolution to select favourable traits for future generations.
the checkpoint pathways operating in various phases of the On the other hand, less is clearly more when it comes to mitotic cycles in mammalian somatic cells. The sensors of genetic change, and all eukaryotes have evolved a plethora DNA damage remain relatively obscure, and may include of mechanisms to minimise DNA damage. The threat of the Rad1–Rad9–Hus1 complex, Rad17, and possibly the excessive genetic change needs constant attention as DNA large ATM and ATR kinases of the PI3K family (phos- becomes damaged by inherent errors in processes such as phatidyl-inositol-3-kinase), which might recognise DNA DNA replication, as well as through genotoxic stress from lesions through so-far elusive subunits analogous to the Ku reactive cellular metabolites and exogenous stimuli (e.g.
70/80 proteins of DNA-PK (DNA-dependent protein ionising radiation, ultraviolet light, cigarette smoke). Our kinase) [4,8,13•]. The choice of transducers of the damage cells cope with the required monitoring and maintenance signal (the ATM/ATR and Chk1/Chk2 kinases) reflects the of genomic integrity by means of a complex network of type of DNA damage, though some overlap between the DNA repair pathways [1,2] and the so-called cell-cycle ATM–Chk2 axis and the ATR–Chk1 axis exists [4–7].
checkpoints. The latter are biochemical signalling These upstream elements of the checkpoint cascades are pathways that sense various types of structural defects in shared by diverse cell types and cell-cycle phases. In DNA, or in chromosome function, and induce a multi- contrast, the downstream checkpoint effectors and their faceted cellular response that activates DNA repair and final targets within the cell-cycle machinery may differ in delays cell-cycle progression [3–7]. When DNA damage is Mammalian G1- and S-phase checkpoints in response to DNA damage Bartek and Lukas 739
Ubiquitin/proteasome-mediated protein degradation determines rapid forks [66••]. In some mammalian somatic cells, whose proliferation G1 arrest in response to DNA damage. DNA damage triggers a rapid critically depends on the presence of abundant cyclin D1, another cascade of phosphorylation events involving the ATM and Chk2 (upon mechanism may contribute to initiate the rapid G1 arrest. Here, DNA IR) or ATR and Chk1 (upon UV light) kinases. These cascades culminate damage leads to unmasking of a cryptic ‘destruction box’ (RxxL) within at inhibition of the S-phase-promoting cyclin E–CDK2 kinase complex, the cyclin D1 amino-terminus, which leads to its recognition by the failure to load Cdc45 on chromatin, and rapid blockade of initiation of the anaphase-promoting complex (APC) ubiquitin ligase and priming for DNA replication origins. The key step in this pathway is the Chk2/Chk1- rapid destruction by the proteasome. The result is again inactivation of triggered phosphorylation (P) of the Cdc25A phosphatase, which primes the S-phase-promoting cyclin E–CDK2, in this case by release of the Cdc25A for ubiquitination (Ub) and rapid destruction by the proteasome.
p21 CDK2 inhibitor from the disrupted cyclin D1–Cdk4(6) complexes.
The absence of Cdc25A phosphatase activity ‘locks’ the CDK2 kinase in Critical steps in both pathways involving proteasome-dependent its inactive form phosphorylated on inhibitory threonine 14 (T14) and proteolysis are highlighted by yellow. Question marks indicate the key tyrosine 15 (Y15). This pathway operates presumably in every cell type open questions for future research, namely which ubiquitin ligase primes and appears to be conserved among vertebrates. Moreover, proteolysis Cdc25A for degradation, and what is the nature of upstream signalling of Cdc25A was also linked with the replication checkpoint, which guards which couples DNA damage with the proteolysis of cyclin D1 (this against premature entry into mitosis in the presence of stalled replication pathway has been proposed to be ATM/ATR-independent).
In this review, we discuss the progress in elucidating the G1/S control and the two-wave G1
mechanisms of the mammalian DNA-damage checkpoints checkpoint response
that guard the entry into, and progression through, the To appreciate the workings of the G1 DNA damage S phase. This focus has been motivated by the recent checkpoint(s), it is helpful to briefly consider the G1/S discoveries of the molecular basis for the rapid, p53-inde- control. G1 phase is a period when cells make critical deci- pendent initiation of the G1 checkpoint [14••,15••], and the sions about their fate, including the optional commitment intra-S-phase checkpoint [16••–20••]. Furthermore, we pro- to replicate DNA and complete the cell division cycle.
vide examples of potential cell-type-restricted checkpoint Provided mitogens are available and the cellular environ- responses, and the evidence for cancer-promoting aberrations ment is favourable for proliferation, a decision to enter in the G1- and S-phase checkpoints. Finally, we highlight S phase is made at the so-called ‘restriction point’ in mid- the conceptual significance of these new discoveries and to-late G1 [21]. In unstressed cells, this commitment to the challenges they raise for future research.
replicate DNA and divide seems irreversible until the Cell multiplication
Maintenance of the G1/S arrest after DNA damage is a delayed dephosphorylation-dependent interaction with 14-3-3 proteins), this response that requires transcription, translation and/or protein leads to accumulation of a stable and transcriptionally active p53 protein stabilisation of key checkpoint transducers. Once initiated, the G1 arrest in the cell nucleus. This in turn results in induction of a number of genes must be maintained and the entry into S phase prevented as long as the including the p21 CDK inhibitor. Exposure of epithelial cells to UV light cell detects a single unrepaired DNA lesion. As in the rapid response can lead to yet another mechanism to mobilise the cellular p21. In this (see Figure 1), the ATM/ATR and Chk2/Chk1 kinases play a pivotal role.
case this is a gradual accumulation of p16, a protein that can selectively Thus, phosphorylation of p53 stabilises the protein by preventing its disrupt cyclin D–CDK4(6) complexes and thereby release already interaction with Mdm2, which acts as a specific inhibitor of p53 existing pool of p21. When accumulated to a threshold level, p21 can transactivation domain and a p53 ubiquitin ligase. Phosphorylation of stoichiometrically bind and inhibit all cellular S-phase promoting cyclin both p53 and Mdm2 also inactivates nuclear export of p53. Furthermore, E–CDK2, and thereby secure the maintenance of the G1 arrest. Another at least some types of DNA damage can upregulate the ARF protein, a important consequence of inhibiting both CDK2 and CDK4(6) kinase specific inhibitor of Mdm2. Collectively (and together with other p53 complexes is dephosphorylation of RB and inhibition of the activating mechanisms such as sumoylation, acetylation, E2F-dependent transcription of S-phase genes.
next G1 phase. Importantly, the checkpoints alarmed by by the parallel retinoblastoma protein (RB) and Myc genotoxic stress can delay cell-cycle progression even pathways, which regulate genes critical for G1/S transition when cells have already passed this restriction point.
and coordination of S–G2–M progression ([11,21,22•,23•] Available data suggest that the restriction point switch, and references therein). Within the RB pathway, the from the growth factor-dependent early G1 to the sub- molecular switch appears to be the phosphorylation of RB sequent mitogen-independent phases, reflects the by cyclin D–CDK4(6) kinases [21,24], resulting in the induction of broad transcriptional programmes regulated derepression of the RB-regulated E2F transcription Mammalian G1- and S-phase checkpoints in response to DNA damage Bartek and Lukas 741
Molecular mechanisms of the intra-S-phasecheckpoint. In contrast to G1/S and G2/M transitions, S phase can only be delayed, andnever permanently blocked in the presence ofDNA DSBs generated by IR. ATM- and Chk2-dependent degradation of the Cdc25A phosphatase, and the consequent blockadeof de novo initiation of replication origins (seeFigure 1 for details) represent an essentialmeans to achieve rapid reduction of the rate of DNA synthesis. Other cellular factorsdirectly phosphorylated by ATM and/or Chk2,such as the Nbs1–Mre11–Rad50 complex,BRCA1 and E2F-1 (J Nevins, personal communication) also contribute to theS-phase checkpoint (see also Update). What is the exact hierarchical order among these independently on parallel pathways are among the major challenges for future research.
factors [21,22•,25]. E2F and Myc jointly activate the key implemented too slowly to account for the rapid inhibition target gene cyclin E whose product activates the CDK2 of CDK2 seen upon genotoxic stress [11]. In addition, the kinase necessary for the actual initiation of DNA repli- silencing of cyclin E–CDK2 activity in late G1 occurs even cation [11,26,27]. Consequently, the cyclin E protein in cells lacking p53 or p21 ([14••–16••], and references becomes detectable and accumulates only in late G1, a therein). These facts argue for a two-wave model of the G1 few hours after the passage through the restriction point checkpoint response in mammalian cells, in which the ini- [28•]. Both its position at the convergence of the RB and tial, rapid, transient and p53-independent response is Myc pathways, and its essential and rate-limiting function followed by the delayed yet more sustained G1 arrest in G1/S transition, makes cyclin E–CDK2 activity an ideal imposed by the p53–p21 axis. A strong experimental candidate for a DNA damage checkpoint target [11]. In support for this model comes from the recent identifica- principle, progression through G1 can be blocked either at tion of a novel pathway that underlies the rapid inhibition the restriction point (by preventing RB phosphorylation), of CDK2 upon various types of DNA damage, as discussed or closer to the G1/S transition by silencing cyclin E–CDK2 activity. Both CDK2 [14••–16••] and RB [29] areindeed targeted by the DNA damage checkpoint(s), yet Rapid, p53-independent induction of the
through temporally distinct mechanisms corresponding G1 checkpoint
to induction and maintenance of the G1 checkpoint, To be effective within minutes after DNA damage, induction of the G1 block should exploit a mechanismthat is poised to act, independent of transcription and For almost a decade, the G1 arrest induced by DNA dam- protein synthesis. Recent reports suggest that pathways age has been ascribed to another transcription factor, the which fit this definition operate by targeting Cdc25A p53 tumour suppressor protein [3,9,11]. Upon diverse [14••–16••]. The phosphatase activity of Cdc25A cancels stress stimuli, cellular p53 becomes post-translationally the inhibitory phosphorylation of CDK2 and is essential modified, stabilised, and competent to induce expression for G1/S transition [11]. Independent of the p53 status, of genes required to halt the cell-cycle progression or the abundance and activity of Cdc25A rapidly decreases trigger programmed cell death [9,30]. Among the genes when mammalian cells are exposed to ultraviolet (UV) induced by p53 is the CDK inhibitor p21Waf1/Cip1, capable of light or ionising radiation (IR), reflecting ubiquitination silencing the CDKs that are essential for entry into S phase induced by DNA damage and accelerated turnover of the [24,26]. However, the transcription-dependent and protein Cdc25A protein by proteasomes [14••,16••]. This novel synthesis-dependent role of p53 in the G1 checkpoint is checkpoint pathway (Figure 1) results in persistent Cell multiplication
Aberrations of the G1/S checkpoint components in human tumours
Truncations, missense mutations, deletions, Carcinomas of the breast, lung, colon, urinary Diverse types of mutations, deletions, reduced Missense and frameshift mutations, truncations.
Carcinoma of the breast, lymphoid tumours Cyclin D1 (O) Gene amplification, translocation, Deletions, diverse mutations, promoter silencing, Many types of cancer *Further reading can be found in references 1–11,30,56,59–63, transducers (top part, non-italicized). ‡Examples of molecular defects 67–70,71•,72•• and 73. †(S) = tumour suppressor; (O) = (proto)- (somatic or germline) found in human tumours. NR = not reported. §The oncogene; (S/?) = candidate suppressor; italics: effectors and targets list shows examples of tumour types, it is not exhaustive. NR = Not of checkpoints, as opposed to upstream checkpoint regulators and reported. #All listed syndromes are cancer prone. NR = not reported.
inhibitory phosphorylation of CDK2 on tyrosine 15, and solved by a cascade of protein–protein interactions, phos- thus inhibition of cyclin E–CDK2 activity leading to the phorylations, ubiquitination and proteolysis of the key target, the Cdc25A phosphatase (Figure 1).
The signal for ubiquitination after UV and IR exposure is Interestingly, an analogous concept based on enhanced created by phosphorylation of Cdc25A mediated by Chk1 protein degradation in response to IR has been reported [14••] and Chk2 [16••], respectively. The critical residue of to target cyclin D1, another G1 regulator [32••]. The Cdc25A targeted by Chk2 is serine 123 [16••]. This pathway rapid silencing of CDK2 by this pathway is thought to is sensitive to caffeine, an inhibitor of the ATM/ATR reflect redistribution of the p21 CDK inhibitor, from kinases [14••,16••,31•], and at least the response to IR cyclin D1–CDK4(6) complexes (for which p21 serves as depends on ATM [15••,16••] as an activator of Chk2 [4,7] an assembly factor) to cyclin E–CDK2 complexes, (Figure 1). The end-point target of this cascade is the which are inhibited by p21 [24,32••] (Figure 1). If con- inhibition of CDK2-dependent loading of Cdc45, an firmed as a cell-cycle checkpoint, this mechanism would attractant for DNA polymerases, onto DNA pre-replication be an example of an ATM-independent, cell-type- complexes ([15••]; J Falck, personal communication).
restricted response, since cyclins D2 and D3 are not Consistent with criteria for a bona fide cell-cycle check- degraded upon DNA damage, and therefore this path- point, the extent of DNA damage is enhanced and cell way would have little effect in cell types expressing survival decreased after irradiation under conditions when several D-cyclins, or lacking cyclin D1 (see Update).
the ubiquitin/proteasome-mediated degradation of Nevertheless, such a mechanism may complement the Cdc25A is experimentally prevented [14••]. Thus, the more ubiquitous Cdc25A pathway, which operates in ATM/ATR–Chk2/Chk1–Cdc25A–CDK2 pathway(s) seem many cell types, upon diverse genotoxic stimuli, and is to account for the p53-independent initiation of the G1 phylogenetically conserved at least from Xenopus [15••] checkpoint, and the need for speedy execution seems Mammalian G1- and S-phase checkpoints in response to DNA damage Bartek and Lukas 743
The p53 pathway and the maintenance of the
pathways (Figure 2) may act in a stimulus-dependent, G1 arrest
Under normal conditions, p53 is a highly unstable proteinand its DNA binding capacity is low. After DNA damage, The intra-S-phase checkpoint response
numerous post-translational modifications lead to stabil- In contrast to the key role of p53 in maintenance of the isation of the p53 protein and activation of its DNA-induced G1 arrest, no specific roles for p53 or p21 sequence-specific DNA binding [9,30]. Only then can p53 have been implicated in the control of the intra-S-phase efficiently stimulate transcription of cell-cycle inhibitors checkpoint. This is perhaps not so surprising as the such as p21 (Figure 2). Furthermore, the p21 protein has to S-phase checkpoint, manifested by a decreased rate of accumulate to levels sufficiently high to inhibit the CDK- DNA synthesis after generation of DSBs, is by definition a containing complexes, before cell-cycle progression transient phenomenon [5]. The absence of the ‘mainte- becomes efficiently blocked. Although p53 has recently nance component’ during S phase, contrary to the G1 and been described binding to 5′ untranslated region of CDK4 G2 checkpoints, might be beneficial for the cells by mRNA and inhibition of CDK4 translation seemed to be providing some delay but not permanent arrest with transcription-independent [33], even this process requires incompletely replicated genome. Long-term intra-S-phase time for stabilisation and accumulation of p53, and the blockade would limit the amount of sister chromatids and subsequent slow decay of the stable CDK4 protein. Thus, therefore reduce available template for efficient repair depending on the nature of DNA damage, the period from by homologous recombination. Moreover, work in yeast generating the DNA lesion to the effective p53-dependent suggests that complete inhibition of CDKs and prolonged cell-cycle arrest can last for several hours, consistent with intra-S-phase arrest may cause regaining of replication maintenance of the G1 block previously initiated by the competence of already fired origins, which would then make the recovery process prone to over-replication of atleast parts of the genome [45]. Finally, it is possible that The events that mobilise p53 after stress, including its the p53 activation in S phase could be detrimental per se, protein stabilisation, subcellular trafficking, and transcrip- and that there are mechanisms that operate in every tional activation, have been reviewed recently [9,30]. Yet S phase to prevent p53 from targeting at least a subset of this complex regulatory web is continuously expanding genes. It has been speculated that induction of a ‘full- [34,35,36•,37]. What needs to be emphasised in relation to scale’ p53 transcription programme within S phase, when the two-wave G1 checkpoint concept is that the key the E2F-1 transcription factor (known to cooperate with upstream regulators, the ATM/ATR and Chk2/Chk1 p53 to induce apoptosis) is highly active, could promote kinases, are shared by both waves (Figures 1,2) and target Cdc25A and p53 simultaneously within minutes afterDNA damage. Phosphorylation on serine 20 of p53 by Unexpectedly, fresh insights into the intra-S-phase Chk2/Chk1 helps stabilise p53 by uncoupling it from the checkpoint mechanisms induced by DSBs have also Mdm2 ubiquitin ligase [38••–40••], while ATM- (and implicated the above-mentioned Cdc25A-degradation likely also ATR-) mediated phosphorylations of Mdm2 pathway in slowing down ongoing S phase [16••,47]. Thus, (Ser 395) [36•] and p53 (Ser 15 and some other residues) the ATM–Chk2–Cdc25A–CDK2–Cdc45 axis emerges as a interfere with nuclear export of p53 [41•], and help activate key mechanism of not only the rapid prevention of S-phase p53 [5,7,30], respectively. But despite the fact that the entry in the G1 checkpoint [14••,15••] (Figure 1), but also initial steps along the G1 checkpoint are common for the in the transient intra-S-phase response [16••] (Figure 3), Cdc25A and p53 pathways, their impact on CDK2 activity predictably affecting both the early- and late-firing origins and G1/S blockade are separated in time, due to the of DNA replication [48•]. Inhibition of CDK2 activity dependence of the latter pathway on transcription and through Cdc25A degradation leads to a several-hour delay of S-phase progression, a timing that correlates well withthe transient intra-S-phase checkpoint response [16••].
The maintenance of cell-cycle checkpoints may be further The physiological relevance of this pathway is documented prolonged by additional mechanisms. For example, the by the fact that analogous to ATM defects, interference ARF tumour suppressor (known to sequester Mdm2 in with the Chk2–Cdc25A–CDK2 cascade at any of these response to oncogenic stimuli [42]) also becomes induced steps downstream of ATM results in radioresistant DNA with delayed kinetics after DNA double-strand breaks synthesis (RDS) [16••], a phenomenon of persistent (DSBs), and may reinforce stabilisation and activation of DNA synthesis after irradiation, originally described for p53 after DNA damage [43••]. Another example is the ataxia telangiectasia patients who harbour mutations in delayed increase of the p16INK4a CDK inhibitor in human skin keratinocytes and melanocytes following exposure tophysiological doses of UV light [44]. The UV-induced Upon IR-induced activation in S-phase cells, ATM elevation of p16 occurred 16 hours after exposure and phosphorylates several checkpoint components including peaked by 24 hours, being reversible with a decline by Chk2 [49•,50•] (which then targets Cdc25A), but also 72 hours post-irradiation. Such accessory maintenance BRCA1 [51,52], and Nbs1 [17••–20••], a component of the Cell multiplication
Mre-11–Nbs1–Rad50 complex [10,53]. The ATM-mediated into the intra-S-phase checkpoint, and the appreciation of phosphorylations of Nbs1 are required for the proper checkpoint aberrations as important determinants of execution of the intra-S-phase checkpoint, since mutating multistep tumorigenesis exemplify the recent advances in the targeted serine residues (Ser 278, 343 and 397) to this field. One of the unifying features of the G1- and alanine resulted in RDS. Reports documenting functional S-phase checkpoints is their joint targeting of the Cdc25A interplay between Chk2, BRCA1, and Nbs1 [54•,55,56], pathway and, more broadly, their rapid effects on protein Mre11 complex and E2F-1 [57•], and S-phase checkpoint turnover of the critical checkpoint effectors, namely defects in BRCA1-deficient cells [58•] are tantalising yet degradation of Cdc25A, cyclin D1, and protection from so far insufficient to judge whether or not all these regu- degradation of p53. Among the major gaps that remain lators feed into the Cdc25A pathway [16••,47], or whether in our understanding of checkpoint function is the extent parallel mechanisms co-operate to inhibit DNA replication and molecular nature of the interdependence between cell cycle effects and DNA repair, along with the sig-nalling, dynamics, and indeed the mechanistic basis of the G1/S checkpoint defects and cancer
recovery from activated checkpoints in any cell-cycle Genetic instability is one of the hallmarks of cancer, and its phase. Related to this is the somewhat contradictory issue links to aberrations in DNA repair machinery and the of whether or not cells that activate the p53 response ever cell-cycle checkpoint pathways is well documented [1–11,30,56,59–61]. Evidence to support this notion con-tinues to accumulate, and here we briefly review the known, Research on the specific features of the already known, or and particularly the recently identified, cancer-associated possibly still unknown, ‘accessory’ checkpoint pathways defects of the G1/S checkpoint components (Table 1). restricted to certain cell- and tissue-types and differentia-tion stages is in its infancy. Clarification of the identity and Except for the ATR whose lack causes early embryonic modus operandi of the ‘true’ sensors of DNA lesions lethality in mice [62,63] and whose somatic defects might should also be a fruitful area of investigation in the near result in cell death, all the major G1/S checkpoint trans- future. There is also a clear need to find the missing ducers and effectors qualify as either tumour suppressors components and connections in the web of the checkpoint or proto-oncogenes, and their loss-of-function mutations or signalling cascades, and better understand the significance overexpression have been identified in many types of of the protein–protein interactions within multiprotein human malignancies (Table 1). In addition, when mouse complexes, and their dynamic changes in response to models that mimic such defects are available, the resulting distinct types of DNA damage. These studies should phenotypes generally support the putative roles of these greatly benefit from the recently established technologies checkpoint regulators and effectors in guarding against allowing kinetic and often quantitative analyses of such genomic destabilisation and tumour development.
transient events in living mammalian cells in real time.
Hereditary mutations in at least ATM [5,7], Chk2 [64•], Yet how to study phosphorylation of particular proteins at BRCA1 [59], Mre11 [65], Nbs1 [10], p53 [9,64•], p16 [60], key residues, directly in live cells, is still a subject for and RB [60] are known to cause familial cancer and/or clinical syndromes that are cancer prone (Table 1). T heintimate involvement of cell-cycle checkpoints in molecular The link between checkpoint failure, genome destabilisation, pathogenesis of cancer, and their emerging significance for and cancer, will surely inspire exploration of more rational the outcome of chemotherapy and radiotherapy, inspired therapies based on pharmacological intervention with rate- intensive efforts to explore this new knowledge for limiting events in checkpoint pathways. Elucidation of the diagnostic purposes, and particularly to search for more importance of haploinsufficiency in checkpoint genes such rational cancer treatment strategies. Global assessment of as ATM, BRCA1 or Chk2, for cancer predisposition, is the checkpoint pathways by functional genomics and required. A closer symbiosis of basic and translational proteomics approaches may help predict therapeutic research into how the checkpoint pathways work will lead responses of individual cancers or aid in selecting a tailor- not only to many more exciting discoveries to satisfy our made treatment. In terms of new therapies, attempts to curiosity about the elementary principles of life, but hope- develop CDK inhibitors, activators of p53 and particularly fully also offer a new generation of drugs to treat cancer.
its pro-apoptotic effect, as well as attenuators of check-point responses that might presensitise tumour cells to radiation and cytotoxic drugs, are well under way [9,11,30] Experiments with transgenic mice by P Sicinski and and remain a great promise for the future.
colleagues [74••] greatly substantiate the concept thatcyclin D1 could indeed represent an important checkpoint Conclusions and future directions
target in specific tissues. An excellent review on ATM- The crude molecular anatomy of mammalian cell-cycle and ATR-mediated checkpoint signalling by R Abraham checkpoints is taking shape, and we are learning rapidly has recently been published [75]. This overview also about their physiology and pathology. The two-wave summarises the current knowledge about the sensors of concept of the G1 checkpoint, the mechanistic insights damaged DNA, including the candidacy of the Rad Mammalian G1- and S-phase checkpoints in response to DNA damage Bartek and Lukas 745
protein family members for such function, an issue not resulted in formation of incompletely assembled pre-replicative complexescontaining ORC, Cdc6, Cdc7 and MCM proteins but lacking Cdc45.
Addition of recombinant Cdc25A but not Cdc25C abrogated the checkpointand restored DNA replication.
Acknowledgements
16. Falck J, Mailand N, Syljuasen RG, Bartek J, Lukas J: The ATM-Chk2
We are grateful to JHJ Petrini, C Cordon Cardo, J Nevins, and H Nevanlinna Cdc25A checkpoint pathway guards against radioresistant DNA
for sharing their data before publication, and to the Danish Cancer Society, synthesis. Nature 2001, 410:842-847.
the Danish Medical Research Council, the John and Birthe Meyer Foundation, This paper reports the discovery of the mechanism regulating the intra- and the Nordic Cancer Union for financial support. Our apologies to S-phase checkpoint in mammalian cells exposed to ionising radiation (IR).
colleagues whose work could only be cited indirectly in this review.
IR-induced formation of DNA double stranded breaks triggered degradationof the Cdc25A phosphatase, which in turn led to inhibition of the S-phasepromoting cyclin E–Cdk2 activity and transient blockade of DNA replication.
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An intriguing concept linking the anaphase-promoting complex-dependent functionally impaired when DNA synthesis is blocked. Proc Natl
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and its role in preventing premature mitosis. EMBO Rep 2000
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induced stalled replication forks. An excess of Cdc25A reduced the cells’ Elegant in vivo study showing that in the breast epithelium, the Neu-Ras ability to arrest cell-cycle progression in the presence of hydroxyurea and oncogenic pathway is connected to the cell cycle machinery via cyclin D1.
lead to initiation of premature chromosome condensation. Thus, proteolysis This explains the absolute dependency on cyclin D1 for the Neu-Ras-medi- of Cdc25A appears to play a pivotal role in both replication and DNA dam- ated oncogenic transformation in the breast.
age checkpoints (see also annotation [14••]) and thereby contributes to themaintenance of genetic stability.
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Now in press
The work which links the ATM checkpoint kinase with E2F-regulated tran-
68. Fukuda T, Sumiyoshi T, Takahashi M, Kataoka T, Asahara T, Inui H, scription, referred to in the text as (J Nevins, personal communication), has Watatani M, Yasutomi M, Kamada N, Miyagawa K: Alterations of the
double-strand break repair gene MRE11 in cancer. Cancer Res
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Osada H, Fujii Y, Takahashi T: Histological type-selective, tumor-
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to DNA damage may operate via the Rb/E2F pathway.

Source: http://www.cbi.pku.edu.cn/chinese/documents/cell//xibaoshengwuxuecankaowenxian/cocb/13/13-6/13(6)-15.pdf

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TABLE OF CONTENTS Pray these prayers out loud, you can do this on your own, but do it whilst you are fasting, not in the beginning of your fast but when you have been fasting for 24 hours or so then you are combining fasting and prayer according to Mark 9:29 It is recommended to pray twice at different times or days, because the second time you have more understanding it is a long list but i

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Unter den Wolken kann die Freiheit nicht grenzenlos sein Bern, 30.05.2006 (GS-UVEK) - Bundespräsident Moritz Leuenberger zum 1. Schweizerischen Luftfahrtkongress vom 30. Mai 2006 am Flughafen Zürich 1. Grenzen der Luftfahrt In der langen Geschichte der Menschen blieb das Fliegen bis vor kurzem den Göttern vorbehalten, die ihre Allmacht hoch über den Wolken auslebten. Für die Mens

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