Class Note for BME 510 at UA 3
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insight Cellcycle checkpoints and cancer Michael B Kastan 8 Jili Baltek2 Departrnent ofHernatologyeOntology StIude Children s Research Hospital 332 North Lauderdale Street Memphis Tennessee38105 USA eernail michaelkastanstjudeorg zDepartment ofCell Cycle and Cancer Institute ofCancerBiology Danish CanterSotiety Strandboulevarden 49 Copenhagen DK72100 Denmark eernailzjb an erdk Aquot life on earth must cope with constant exposure to DNAdamaging agents such as the Sun s radiation Highly conserved DNArepair and cellcycle checkpoint pathways allow cells to deal with both endogenous and exogenous sources of DNA damage How much an individual is exposed to these agents and how their cells respond to DNA damage are critical determinants of whether that individual will develop cancer These cellular responses are also important for determining toxicities and responses to current cancer therapies most of which target the DNA he DNA contained in every mammalian cell is under constant attack by agents that can either directly damage one of its three billionbases or breakthe phosphodiester backbone onwhichthe bases reside For example free oxygen radicals which can cause both base damage and DNA breakage arise as a consequence ofnormal cellular metabolism or can be created whenthe organism is exposed to external sources of ionizing radiation in the environment Life on Earth has evolved to deal with both metabolic and external sources of DNAedamaging agents through the development of elegant mechanisms that repair damage to the DNA Cellular responses to DNA damage constitute one of the most important elds in cancer biology First damage to cellular DNA causes cancer We know this from epidemiole ogical studies from animal models and from the observation that many humanecanceresusceptibility syndromes arise from mutations in genes involved in DNAedamage responsesz Second DNA damage is used to cure cancer Most therapeutic modalities that we currently use to treat malignancies target the DNA including radiation therapy and many chemoetherapeutic agents Third DNA damage is responsible for most of the side effects of therapy Bone marrow suppression gastrointestinal toxicities and hair loss are all attributable to DNAedamageeinduced cell death ofproliferatingprogenitor cells inthese tissues So from the perspective of cancer DNA damage causes the disease it is used to treat the disease and it is responsible for the toxicity oftherapies for the disease Among the mechanisms that cells have developed to cope with this constant attack on their DNA are elegantbut not perfect DNAerepair processes Because there are various types of DNA lesion that can occur a variety of different repair mechanisms exist In addition to directly repairing DNA breaks or adducts cells respond to DNA damage by halting cellecycle progression or by undergoing pro grammed cell death Although we have a limited under standing of how the processes of cellecycle arrest or apoptosis are coordinated with the process of DNA repair such coordination must take place to optimize the outcome for the cell or the organism In addition to damage to the DNA cells must cope with other stresses such as intermite tent or prolonged exposure to abnormally low levels of oxygen or nutrients Although cells use different aspects of the signalling pathways to deal with these types of change in their microenvironment there are commonalities in the steps that cells use to deal with DNA damage The term cellecycle checkpoint refers to mechanisms by which the cell actively halts progression through the cell 316 cycle until it can ensure that an earlier process such as DNA replication or mitosis is complete3 Here we focus on some of the mechanisms by which cells modulate progression through the cell cycle in the face of DNA damage and other stresses that affect DNA replication Although we focus on signalling pathways that have been characterized in mammalian cells lessons learned from studying lower eukaryotes in particular yeast have been instructive and re ect the considerable evolutionary conservation of these pathways Finally current concepts about how sporadic or inherited mutations in genes in these pathways contribute to cancer development willbe explored The signalling pathways Signal initiation by differentslresses DNA can be damaged in a variety of ways First energy released by free oxygen radicals generated either by normal metabolic processes or by exposure to an external source of ionizing radiation can break the phosphodiester bonds in the backbone of the DNA helix When two of these breaks are close to each other but on opposite DNA strands a doubleestrand break DSB is present in the DNA and the cell faces a particularly challenging situation for repair Second alkylating chemical moieties can modify purine bases and the size of the chemical adduct determines what repair process is usedz Bi inctional alkylating chemicals can cause intraestrand or inter strand crosslinks that require additional molecular interventions for themto be reversed Third inhibitors of DNA topoisomerases can lead to enhanced single or DSBs depending on which topoisome erase is inhibited and on the phase of the cell cycle Each type of DNA damage requires a speci c set of cellular responses to deal with the specific nature of the damage Different mechanisms are required to repair the damage to the DNA backbone or to the DNA bases and the challenges of repairing the DNA can vary in the different phases of the cell cycle To optimally repair DNA damage the cell must also control other cellular processes before or during the repair such as DNA replication or mitosis Cells that are damaged when they are already in the middle of the process of DNA replication face particular challenges but would still probablybenefit from halting or slowing DNA replica tion untilthe damage has been repaired So there should be advantages for a eukaryotic cell to transiently halt progression through the cell cycle after DNA damage whichpresumably include limiting heritable mutations in daughter cells and enhancing viability of the damaged cells Initiation of the activities of the PI3K phosphatidyle inositole3eOH kinaseelillte kinases PIKKs ATM ataxia NATUREiv0L432i13NOVEMBER2004iwwwmturecommture 2004 Nature Publishing Gr DNA damage Replication fork arrest ATM activation ATR reelocalization reel ocalizati on Wm transducers eV Modulation of cell fate Cell cycle arrests DNA repair chromatin remodelling apoptosis Genomic instability Cancer Figure1 General scneme of responsesto DNA damage or replicationrtork arrest and tne impact on cell fate genomic instability and cancer development Replicationrtork arrest stlmulates tne inltiation of cellular ATR activlty wnereas DNA damage can directly activate ATM and can lead to replicationrtork arrest tnereby also activating cellular ATR kinase Once active botn tne ATM and ATR kinases functioning ln combinatlon Witn otner proteins and substrates nelp determine tne outcome ottne cell lt genomic instabilityensues tnis can contribute to cellular transformation telangiectasia mutated and ATR AMT and Rad37related are the first steps characterized to date in the activation ofsignal transduce tion pathways that inhibit cellecycle progression after DNA dam age The ATM kinase seems to primarily be activated following DNA damage whereas the ATR kinase seems to be critical for cellue lar responses to the arrest of DNA replication forks i the DNA structures formed during replication This is the case whether the arrest ofreplicationefork progression is due to DNA damage or to other stresses Because many types ofDNA damage result both in the direct damage of the DNA and the arrest of DNA replication forks ATM and ATR seem to participate together in many cellular stress responses and complex joint responses must be coordinated Fig 1 Signal initiation by ATM and ATR To accomplish the physiological goal of minimizing the adverse effects of a stressful physiological situation an arrest of cellecycle progression should be engaged very rapidly after exposure to the stress ATM and ATR are both extremely large predicted molecular mass of 350 and 301 kilodaltons respectively protein kinases that phosphorylate numerous substrates to achieve their physiological goals7 It is a mechanistic challenge to tightly control the activities of these large kinases so that they do not stimulate growthesuppressive pathways in the absence of an appropriate stress but can be activated instantaneously following expo sure to the stress Patients mice and cells lacking ATM are viable suggesting that the ATM kinase is not essential for critical cellular functions such as normal cycle progression or cellular differentiations ATM kinase activity is minimal or low in unstressed cells and primarily is engaged to help cells deal with cellular stresses that affect DNA or chromatin structure The identi cation of a single major damage induced phosphorylation site serine 1981 led to the demon stration of a new mechanism of ATM regulation that permits a rapid and sensitive switch for checkpoint pathwaysB In unstressed cells ATM is present as ahomodimer in whichthe kinase domain is physically blocked by its tight binding to an internal domain of the NATURElVOL432l18NOV EMBERZOO4lwwwmturecommture protein surrounding serine 1981 The introduction of a DNA DSB leads to a conformational change in the ATM protein This stimulates the kinase to phosphorylate serine 1981 causingthe dissociation of the homodimerg The activated ATM monomer can now phose phorylate its numerous substrates whether they are nucleoplasmic like p53 or at the sites ofDNA breaks like NBSl Nijmegen breakage syndrome 1 BRCAl breast cancer 1 and SMC1 structural maintenance of chromosomes 1 The conformational change that induces the extremely rapid and extensive intermolecular autophosphorylation event in ATM does not seem to require the binding of the ATM dimer to sites of DNA damage but instead results from some change in highereorder chromatin structure that the ATM dimer can sense at some distance away from the site ofthe DNA breakg The nature of this chromatin structure change and how ATM senses this change including whether it is a direct or an indirect sensing mechanism remains to be discovered Recent observations that the multiprotein complex MRE11meiotic recombination 11RAD50NBSl MRN contributes to the active ation ofATM after ionizing radiation 7 at least at low doses of ionizing radiation 7 may shed light on the mechanisms by which this activation process occurs The phosphorylation of substrates by the ATM kinase requires more thanthe dissociation ofthe ATM homodimer and the release of the blocked ATM kinase do main The activated ATM monomer must also get to the sites in the cell where the substrates are present such as at DNA breaks It was recently demonstrated that MRE11 binds to ATM and enhances its ability to phosphorylate substrates in vitro in the presence of an appropriate mimic of DNA breaks Such a mce tion is consistent with the observation that ATM can be activated by exposure to ionizing radiation in cells lacking NBSl or BRCAl but fails to migrate to the sites ofDNA strand breaks Once recruited to the DNA break the activated ATM can then phosphorylate critical substrates like NBSl BRCA1 and SMC1 which accumulate at these sitesg IfATM is activated but fails to get recruited to DNA breaks as happens after ionizing radiation in cells lackingNBSl or BRCA 1 or if chromatin structure changes occur in the absence of DNA breaks then ATM is still able to phosphorylate nucleoplasmic substrates such as p53 Thus DNA damage leads to ATM activation and substrate phosphorylation by two distinctive steps 1 chromatin structure change induces intermolecular ATM autophosphorylation and homodimer dissociation and 2 activated ATM monomer is recruited to its substrates some of which localize to sites of DNA damage Fig 2 In this model ATM activation and recruitment of MRN and BRCA1 to sites ofDNAbreaks are two distinct events Although the activity of ATM in in vitro kinase assays is increased after immunoprecipitation from irradiated cells there is no measurable change inthe kinase activity of ATR i even in the face of stresses where ATR is required to sustain normal cellular responsess It seems that ATR kinase may be constitutively ready to phosphorye late substrates but have its cellular functions largely controlled by subcellular localization ATR exists in a complex with the ATR interacting protein ATRIP both before and after exposure to stresses such as ultraviolet irradiationw The observation that replication protein A RPA a singleestranded DNA ssDNA binding protein involved in DNA replication stimulates the in vitro binding ofATRIP to ssDNA led to a model in which ATR becomes localized to sites of replicationefork arrest by means of binding of ATRIP to RPA ref 16 lnthis model any stimulus or stress that leads to an abnormal stretchofssDNA such as an arrested replicationfork would be decorated with RPA The accumulation of RPA on the ssDNA would then lead to the recruitment ofthe ATRIP protein and its heterodimeric partner ATR Once the active ATR kinase is localized to the ssDNA region it can phosphorylate critical sub strates such as RAD17 and CHK1 Fig 2 Although the critical importance of RPA in the recruitment of ATR to ssDNA has been questioned the importance of this change in ATR localization is generally accepted 317 2004 Nature Publishing Group As with ATM the presence of an active ATR kinase in the cells is not sufficient forATRto carry out its cellular functions In additionto ATR several other proteins and protein complexes must be recruited to the ssDNA site as wdl These include the clampiloading RAD 1 77containing complex RSR which participates in the loading of the RAD97RAD17HUS1 97171 sliding clamp onto chromatin and the claspinprotein which is independently recruitedto chromatin HO All these events are required for the phosphorylation of CHKl byATR and forthe activation of the appropriate cellicycle checkpoints Fig 2 Whereas cells tolerate the absence of ATM cells and animals lacking ATR seem to be noniviablemz These observations suggest that ATR is probably required for normal progression through the cell cycle even in the absence of cellular stress Consistent with this concept recent results suggest a critical role for ATR in the normal progression of DNA replication forks Given the binding of ATR to regions of ssDNA a role in normal replicationifork progression is perhaps not surprising In addition to its apparent roles in normal replicationifork pro gression ATR is probably engaged in the cellular responses to many other types ofcellular stress because so manyofthem affect the rate of replicationifork progression ATR has been implicated in cellular responses to hypoxia and to DNAireplication inhibitorsls zs It is also critical for responses to DNAidamaging events that affect the progression of replication forks particularly agents that introduce bulky DNA adducts such as ultraviolet irradiation and alkylating agents or crosslinking agents So whereas ATM seems to become engaged in signalling pathways primarily following the introduction of DNA breaks ATR has a critical role in virtually all cellular stress responses that share inhibition of replicationifork progression as a common mechanism ATR even seems to be engaged in cellular responses to DNA breaks possibly compensating for ATM many ATM substrates eventually get phosphorylated a er exposure to ionizing radiation in cells lacking ATM protein Transducing the signal To efficiently spread the alert signal and orchestrate the global cellular response to DNA damage that is usually in icted to only a few sites within the vast genome the proximal checkpoint kinases ATM and ATR ref 26 cooperate closely with two other classes of proteins These are the soicalled checkpoint mediators also known as adaptors 27 ZB and the transducer kinases CHKl and CHK2 ref 29 Fig 3 Regulatory phosphorylations ofthe down stream checkpoint targets 7 diverse effector proteins that function at the interface between the cellicycle DNAirepair and cellideath machineries i may be carried out by the proximal kinases or transducer kinases alone Alternatively distinct residues ofthese same effectors are targeted by ATMATR and CHKl CHK2 respectivelyzs Fig 3 It is remarkable how rapidly within seconds after the focal injury to DNA the global checkpoint networks become activated and the local events at the damage site are coordinated with more distant cellular processes For example in response to only a few potentially harmful lesions such as DSBs in proliferating cells not only must the lesions beprocessed locally but the whole cell must be alerted to delay the most vulnerable processes such as DNA replication or initiation of chromosome segregation in a coordinated panicellular manner Such speed and spatio itemporal coordination re ect the fact that the initial checkpoint responses operate through postitranslational modifications reilocalizations dynamic interactions and changes of conformation and or stability of preexisting proteins all pheni omena that are jointly governed by these three classes of checkpoint regulators Exciting insights into these highly dynamic events have recently been obtained using newtechnologies for realitime imaging of uoresi centlylabelled checkpoint proteins in live cells and phosphoispecific antibodies that recognize proteins modified in response to DNA damage Furthermore the significance of proper checkpoint 318 ATM dlrner gtATM monomer Repllcatlorlrfork arrest ATRATRlP ATRATRlP bound to ssDNA RPA MDOl 538m Substrate arld Claspln MRN ATM rerlocallzatlorl RSR RADl 7 BROM 91 complex Substrate phosphorylatlorl Cellrcycle arrest Figure 2 Scheme of mechahlsms that lead to the lhducthh otATMy ahd ATRV dlrected cellular acthltles DNA strand breaks lead to the dlssoclatlon of the lhactlve ATM dlmer The approprlate locallzatlon of both the ATM monomer and the ATM substrates ls modulated by several protelhs lhcludlhg the MRN complex MDCl SSBPl ahd Brcal The ATRATRlP complex ls recrulted to sltes of ssDNA perhaps by RPA Optlmal substrate phosphorylathh ahdthe ehgagemehtotcellrcycle arrest depends on other protelhs such as clasplh the RSR complex and the Earl rl complex As lllustrated lh Flg l these pathways may often operate lh concert and there may be crossrtalk between the pathway comporlehts showh here signalling for the prevention of cancer is underscored by the fact that most checkpoint kinases and mediators are either established or emerge ing tumour suppressors 7 gene products whose decreased expression or lossiofifunction mutations contribute to tumorigenesis Fig 3 So what are the roles and the underlying molecular mechanisms of action ofthe checkpoint mediators and the signalitransducing kinases The emerging role of checkpointmediatnrs Although the precise mechanisms of action of this important class of checkpoint factors are largely unknown they seem to modulate the activity of ATMATR facilitate the interactions between ATMATR and their substrates and in a broader sense mediate spatio itemporal assembly of multiprotein complexes in the chromatin regions surf rounding the sites of DNA damage There are currently three known members of this class of checkpoint factors involved in the signalling by ATM so far only one such protein is known to modulate the response by ATR Fig 3 As most of the mediators are initially recruited to sites of DNA damage andor replication blockade independently of ATM and ATR they might also be involved in sensing such lesions Alternatively the mediator proteins could be recruited through their interaction withthe candidate DNAidamage sensors The ATMirelated mediators include MDC1 mediator of DNA damage checkpoint 1 also known as NFBDl 53BP1 p53 binding protein 1 and BRCA1 7 large multiidomain proteins that contain two tandem BRCTBrca1 carboxyiterminal domains at their C teri minus9 27 33 35 Interestingly the BRCT domains have recently been shownto serve as proteiniphosphoproteinib indingmodules u sug7 gesting a possible mechanism for how the mediator proteins could promote the transient multiple interactions of checkpoint and repair proteins near the DNAidamage sites Indeed unlike the initial largely ATMiindependent recruitment of the mediators to sites of DNA damage their accumulation into the microscopically visible foci depends on ATMimediated phosphorylation of histone H2AX1 75039 a modificationthat marks chromatin regions spanning megadaltons ofDNA anking each DSB ref 44 The MDC1 protein NATUREl VOL432l 13 NOVEMBER 2004 lwwwnaturecomnature 2004 Nature Publishing Group 3 Pi lt Mediators Delayed sustained senescence Rapid transient X Cellecycle Impact 7 Figure3 A simplified scheme of cellrcycle checkpoiht pathways ihduced rh response to DNA damage here DSBs With highlighted tumour suppressors showh rh red ahd protoOhcogehes showh rh greeh The proxrmal checkporht krhases ATNl ahd ATR phosphorylate diverse compOhehts ofthe hetvvork either directly red P or through the trahsducrhg krhases CHK2 ahd CHK1 black P For simplicity some cahdrdate damage sehsors ahd several ATMATR ahd CHK1CHK2 substrates have beeh omitted The BRCAT proterh also cOhtrrbutesto cellrcycle arrest ahd DNA repair by homologous recombrhatrOh Whereas p53 cOhtrols gehes rhvolved rh cell death ahd DNArreparr mechahrsms The cellrcycle phase ahd the duratrOh of the blockade affected bythe effector pathways are rhdrcated rhcludrhg the potehtral permaheht arrest sehescehce as mediated by p53 The global checkporht hetwork regulated by ATMATR ahd CHK2CHK1 also affects cellular respOhses other thah cell cycle progreserh rhcludrhg DNA reparr lrafTSCTlpllOTT chromatrh assembly ahd cell death mctions as a molecular bridge between the phosphorylated H2AX y7H2AX and the NBSl component ofthe MRN complex and helps provide a platform for a myriad of dynamic interactions for these and additional checkpoint and DNAirepair proteins including the activated ATM and BRCA1 within the vicinity of the damage sites Although the mediator proteins are unlikely to initially target the activated ATM to sites of DNA damage this might be the role of the candidate damage sensors such as the MRN complex quot the sustained multiprotein interactions mediated by MDC1 53BP1 NATURElVOL432l18NOV EMBERZOO4lwwwmturecommture and BRCA1 seem to facilitate ATM signalling and the prof cessingrepair of the lesions thereby contributing to the biological outcome ofthe checkpoint responses Fig 29 Z7 33 3H1 Consistent with this concept mammalian cells that lack any of these three mediators show enhanced sensitivity to DNAidamaging agents such as ionizing radiation and impaired intraASAphase and G2M cell cycle checkpoints Reminiscent ofthe roles ofMDCl 53BP1 and BRCA1 in proper localization timing and velocity of the ATMicontrolled signalling the ATRicontrolled checkpoint signalling at least towards the Chkl kinase that is activated by ATR relies on claspin Claspin is a mediatoradaptor protein that is structurally unrelated to the mediators involved in response to DSBs Claspin selectively interacts with chromatin structures created by active replication forks and is required for ATRimediated phosphorylation and so for proper activation ofCHKl Fig 2 Effector kinases CHK1 and CHK2 Prominent among the substrates of the apical checkpoint kinases ATM and ATR are the checkpointitransducer serinethreonine kinases also known as effector kinases CHK2 and CHK1 ref 29 Despite some crossitalk between ATM and CHK1 the ATM and ATR mediated pho spho rylations trigger preferentially the activation of CHK2 and CHK1 Fig 3 respectively Given that the ATM7CHK2 and ATR7CHK1 signalling modules share many sub strates among the checkpoint effector proteinsmg it is striking that ATM and CHK2 are dispensable for prenatal development whereas complete absence of either ATR or CHK1 results in early embryonic lethalityzs As mentioned above for ATR aplausible explanation for such a fundamental biological difference emerges from recent evidence that supports a role for ATR7CHK1 inthe regulation of some essential processes during unperturbed cell cycles including the control of DNA replication 8 or initiation of mitotic events on centrosomes More mechanistic insights into howthe checkpoint kinases ATM and ATR i in concert with the extremely mobile messenger kinases CHK2CHK1 refs 29 30itrigger cellicycle delays at various transi7 tions ofthe cellidivision cycle is the subject ofthe next section Affecting the cell cycle During unperturbed proliferation mammalian cells can only with draw from the cell cycle on experiencing growthifactor deprivation or growth inhibitory signals in earlyitoimid G1 phase see review in this issue by Massague page 298 This is before the cells pass through the RB retinoblastoma proteinE2F transcription factor7controlled restriction point after which they are committed to a round of DNA replication and cell divisional5 1 However the ATMATR7CHK2 CHKlicontrolled checkpoint network response to genotoxic stress can transiently delay cellicycle progression in G1 S or G2 phases or even impose prolonged durable cellicycle arrests in either G1 or G2 before entry into the subsequent S phase or mitosis respectively Given the critical significance of errorifree DNA replication and chromosome segregation for the maintenance of genomic integrity and the prevention of cancer it is not surprising that these most vulnerable stages of the cellidivision cycle are protected by a wider spectrum of checkpoint effector mechanisms the identity of which are brie y discussedbelow The G1 and 615 checkpoint responses The dominant checkpoint response to DNA damage in mammalian cells traversing through G1 is the ATMATRCHK2CHK17p53 MDM2 7p21 pathway Fig 3 which is capable of inducing sustained and sometimes even permanent G1 arrest7 29 52 Although the expression of ATM and CHK2 is relatively constant during the cell cycle the concentrations of ATR and CHK1 are low in the earlyitoimid G1 and their activities become important only closer to the G1S transition ATMATR directly phosphorylate the p53 transcription factor within its amino terminal transactivation do main particularly 319 2004 Nature Publishing Group on serine 15 Threonin 18 and serine 20 in the same domain along with probably some additional p53 sequences are also targeted by CHK1CHK2 refs 7 26 29 52 53 In addition the ubiquitin ligase MDM2 that normally binds p53 and ensures rapid p53 turnover is targeted a er DNA damage by ATMATR ref 54 as well as by CHK2CHK1 N Motoyama personal communication These modi cations of p53 and Mdm2 contribute to the stabilization and accumulation ofthe p53 protein as well as to its increased activity as a transcription factor The key transcriptional target of p53 is the p21CIP1WAF1 inhibitor of cyclinidependent kinases7 52 which silences the G 1 S pro moting cyclin ECdk2 kinase and thereby causes a G1 arrest This leads not onlyto the inabilityto initiate DNA synthesis but it also preserves the RBE2F pathway in its active growth suppressing mode thereby causing a sustained G1 blockade see also review in this issue by Massague page 298 Thus the G1 checkpoint response targets two critical tumour suppressor pathways governed by p53 and pRB These are arguably the two mechanisms that are most commonlyderegulated inhuman cancer7 5MZ In late G1 as part of the activated E2F7dependent Siphasei promoting transcriptional programme the expression of ATR and CHK1 increases Cyclins E and A and the activator of the cyclin EACDK2 kinase ithe CDC25A phosphatase 7 are also induced in late G1 The ATRCHK1 module but not ATMCHK2 through moderate constitutive phosphorylation of CDC25A on its several serine residues then maintains an appropriate abundance of CDC25A through its ubiquitinidependent proteasomeimediated turnover during unperturbed proliferationw ss In response to genoi toxic stress this physiologically operating mechanism becomes enhanced through increased activity ofCHK1 and CHK2 leading to downregulation of CDC25A and consequently to the inhibition of cyclin EACDK2 complexesw ss Importantly despite the simul taneous phosphorylation of CDC25A and p53 by checkpoint kinases Fig 3 the impact of these events oncellicycle machinery is faster in the CDC25A7degradation cascade that unlike the slowerioperating p53 pathway does not require the transcription and accumulation of newly synthesized proteins Thus the CHK1CHK27CDC25A checkpoint is implemented rapidly independently of p53 and it delays the G1 S transition only for a few hours unless the sustained p53 idependent mechanism prolongs the G1 arrest The Sphase checkpoint pathways The intraiS iphase checkpoint network activated by genotoxic insults causes largely transient reversible inhibition of the firing from those origins of DNA replication that have not yet been initiated It seems that there are at least two parallelbranches ofthis checkpoint that slow down the ongoing DNA synthesis both of which are controlled by the ATMATR signalling machinery One of these effector mechanisms operates through the CDC25A7degradation cascade described in the previous section The inhibition of CDK2 activity downstream ofthis pathway blocks the loading of CDC45 onto chromatin CDC45 is a protein required for the recruitment of DNA polymerase a into assembled preireplication complexes so the inhibition of CDK2 activityprevents the initiation of new origin firingw ss The other branch of the intraiSiphase checkpoint re ects the impact ofATMimediated phosphorylations ofNBSl on several sites in particular serine 343 refs 7 26 and serines 957 and 966 of the cohesin protein SMC1 refs 9 57 58 A better mechanistic under standing of this pathway whose proper function also depends on BRCA1 and FANCD2 Fanconi anaemia complementation group D2 proteinsg sg so should be particularly rewarding because the observed hypersensitivity to radiation in cells that are defective in NBS1 or SMC1 seems attributable to the inability ofATM to phosi phorylate the two critical residues ofthe SMC1 effectorg The concept of the two aboveimentioned parallel effector branches of the intraiSiphase checkpoint has been documented for responses to both ionizing radiation ref 61 and to ultraviolet light Whether the recently reported targeting of CDC7 7 another 320 kinase involved in regulation of DNA replication through an ATR dependent DNAidamage respo nse63 represents yet anotherparallel mechanismto delay DNA synthesis remains to be established Apart from the inhibition of replicationiorigin firing another critical function provided by Siphase checkpoints particularly the soicalled replication checkpoint activated by stalled replication is to protect the integrity of the stalled replication forks Such main tenance of fork stability achieved through yetitoibe discovered effector mechanisms helps prevent the conversion of primary lesions into DNA breaks and facilitates the subsequent recovery of DNA replication g ss The 62 checkpoint The G2 checkpoint also known as the G2M checkpoint prevents cells from initiating mitosis when they experience DNA damage during G2 or when they progress into G2 with some unrepaired damage in icted during previous S or G1 phasesms The accumulation of cells in G2 may also re ect a contribution of the soicalled DNA replication checkpoint often referred to as the SIM checkpoint that may sense some of the persistent DNA lesions from the previous S phase as being inappropriately or not fully replicated DNA The critical target of the G2 checkpoint is the mitosisipromoting activity of the cyclin BCDK1 kinase whose activation a er various stresses is inhibited by ATMATR CHK1CHK2 andor p387kinase7 mediated subcellular sequestration degradation and or inhibition ofthe CD C25 family ofphosphatases that normally activate CDK1 at the G2M boundaryss swg In addition other upstream regulators of CDC25C andor cyclin BCDK1 such as the Poloilike kinases PLK3 and PLKl seem to be targeted by DNAidamageiinduced mech7 anisms Analogous to the role of the checkpoint mediators in the S7 phase checkpoint 53BP1 and BRCA1 are also involved in the regulation ofthe G27checkpoint responses39 41 59 The maintenance phase of the G2 checkpoint probably partly relies on the transcriptional programmes regulated by BRCA1 and p53 leading to the upregulation of cellicycle inhibitors such as the CDK inhibitor p21 GADD45a growth arrest and DNAidamagei inducible 45 alpha and 147373 sigma proteinsss m The fact that even tumours defective in other checkpoints such as those with mutant p53 tend to selectivelyaccumulate in G2 after DNA damage indicates that p537independent mechanisms are sufficient to sustain the G2 arrest At the same time this phenomenon has inspired efforts to interfere with the G2 checkpoint as a potential strategy to sensitize cancer cells which are deficient in their G1 S checkpoint pathways to radiation or drugiinduced DNA damage71 Impacting cancer As the checkpoint and repair pathways facilitate cellular responses to DNA damage and because there is significant data suggesting that DNA damage from both endogenous and exogenous sources is a major contributor to the development ofhuman cancers it is reason able to speculate that alterations in these pathways increase the risk of cancer developing Data from both animal models and humans strongly support this concept Table 1 Cells with an intact DNA damage response frequently arrest or die in response to DNA damage thus reducing the likelihood of progression to malignancy Mutations in apoptosis DNAidamage responses or in mitoticicheckpoint path ways however can permit the survival or the continued growth of cells with genomic abnormalities thereby enhancing the chance of malignant transformation Although many of the DNAidamage response factors described above have been classified as tumour suppressor genes and oncogenes see also Fig 3 the dysfunction of these pathways has not been linked to cancer development in all cases Sorting out whichpathway steps are important in affecting the predisposition to malignancies versus those that are not mayprovide invaluable insights into the mechanisms responsible for human tumorigenesis Although germline mutations in mic Table 7 Human cancer susceptib ty linked to DNAdamage response Disease Gene Number of mutant Cancer predisposition Comments alleles inherited Ataxiartelangiectasia AVT ATM 2 Leukaemia lympnoma Most mutations result in null protein phenotype Niimegen breakage syndrome NBS N887 2 Leukaemia lympnoma Fragment of NBSt protein still expressed in some cell types ArTrlillte disorder ATLD Mre7 7 2 Leukaemia lympnoma l lypomorpnic mutations in M197 7 Fanconi s anaemia FA FancDZr Brca2 2 Acute myelogenous Otner FA genes not directly implicated in checkpoints also lltnown leulltaemias BICaZ 7 nypomorpnic as Fanth Familial breastr ovarian carcinoma BCa7r Brca2 t Breastr ovarian syndrome scattered otners LirFraumeni syndrome p53r CHEKZ t Sarcomasr leulltaemiasr brain tumours adrenal tumours otners This list does not include syndromes resulting rrorn DNArrepair defects which includes xeroderma pigmentosum hereditary nonrpdyposis colon cancers Bioorns syndrome and other Fanconi s anaemiacomplementation groups animals to acquiring tumours it is likely that dysfunction of these steps is also critically important in the development of sporadic tumours which constitute mo st human cancers DNAdamage signal transducers and cancer Loss of ATM strongly predisposes both humans and mice to lyme phoma developments and to alesser degree to other malignancies Because the deletion of the Rad52 protein which is required for homologous recombination significantly reduces lymphoma development in ATMede cient mice it has been suggested that excessive recombination is an important contributor to tumorigenesis in ataxia telangiectasian Similarly patients with mutations in NBS 1 or MRE11 are predisposed to develop cancer733975 In contrast to the disruption of both alleles of ATR causing embry onic lethality in micen ahuman disease Seckel syndrome was recently associated withhypomorphic mutations inATRthat leadtolowlevels of ATR expression Interestingly although these patients show growth retardation dwarfism microcephaly and mental retardation and their cells show chromosome instability a er mitomycin C exposure ahigh incidence of malignancies is not thought to be a prominent part of this inherited syndrome However ATR haploinsuf ciency enhances tumorigenesis in mice that are defective for DNAemismatch repai17 3 Certain mutations in additional components of these signalling pathways also lead to cancer predisposition Mice lacking either HZAX refs 79 80 or 53BPI ref 40 show cellecycle checkpoint defects and cancer predisposition Evenhaploinsuf ciency for HZAX results in detectable genomic instability and enhanced tumour susceptibility in the absence of p53 refs 79 80 Although HZAX maps to a cytogenetic region commonly altered in human cancers 11q23 it is not clear whether HZAX abnormalities contribute to human cancer Although Mdc1 seems to be required for cellecycle checkpoint lnctionzm mutations in the gene have not yet been linked to enhanced tumour development in mice or humans The homozygousedeficient state cannot be tested but Chk heterozygosity modestly enhances the tumorigenic phenotype of W71 t transgenic mice As the tumours in these mice did not lose the other allele of Chk a haploinsufficient tumour suppressor mechanism was suggested Potential mechanisms underlying the haploinsufficient phenotype were studied using generations of mice in which Chk was conditionally disrupted in mammary epithelial cells These cells showed inappropriate Sephase entry accumulation of DNA damage during replication and inappropriate mitotic entry These observations suggest that checkpoint defects associated with Chk haploinsufficiency can contribute to tumorigenesis Chic mice do not spontaneously develop tumours but alackof Chk2 enhances skin tumorigenesis induced by carcinogen exposure As inherited mutations in one allele of CHEKZ canbe found in some families with the extremely cancereprone LieFraumeni syndromegs and CHEKZ variants predispose individuals to breast and prostate cancer CHEKZ seems to be a complex tumour suppressor gene NATURElVOL432l18NOV EMBERZOO4lwwwmturecommture From BRCA to p53 to cancer The inheritance of a single mutated allele of either BRCA or BRCAZ markedly increases the incidence of breast and ovarian cancers in women As the tumours fromthese individuals virtually always lose the second allele both BRCA genes conform to the classic pattern of tumour suppressor genes It is now clear that both BRCA gene products participate in cellular responses to DNA damage but they seem to have distinct roles As described above BRCAl is a target of the ATM ATR and CHK2 kinases and is required for cellecycle check point responses in S phase and G2M ref 69 BRCAl also localizes to sites of DNA breakage interacts with chromatin remodelling proteins and has been implicated in transcriptional co ntrolm Which of these or other suggested functions of BRCA1 are critical for tumour suppression and which explain the relative speci city for breast and ovarian cancers associated with its mutation remain to be clarified Mouse models have suggested complex answers to this question Because bieallelic disruption of Braz in the mouse results in embryonic lethality tissue targeting and conditional disruptions have been used to assess the function of Brcal ref 88 Increased Exogenous or endogenous DNA damage Mutations In DNA damageeresponse signalling pathway DNA repair Genomic Instability Mutations In apoptosis or mitotic checkpoint pathways Clearance of damaged premalignant cells Cancer Figure 4 Schematic representation ottWO main steps that contribute to a spectrum of mutations leading to cancer development lt DNA damage is repaired efficiently the likelihood of tumour development is low lt cells have mutations in DNArdamager response signalling pathways 7 either sporadic or inherited ithls Will lead to enhanced genomic abnormalities Cells With damaged DNAtrequently arrest or do not SUlVlVe thus reducing the probability thattheyWill progress to malignancy Mutations in apoptosis pathways DNArdamage DNArrepair or mitoticrchecprint pathways can permitthe surVival or continued growth of cells With genomic abnormalities thus enhancing the likelihood of malignant transformation 321 2004 Nature Publishing Group mammary and lymphoma carcinogenesis is seen in combination with p53 disruption This suggests that p53emediated apoptosis normally eliminates cells with enhanced DNA damage associated with Braz disruption Disruption of Cth is less potent at enhancing the Brcal effects than disruption of p53 suggesting that some of the p53 tumour suppressor functions are retained in the absence ofChk2 ref 90 BRCA2 binds directly to the RADSl recombinase and has been linked to the Sephase checkpoint and to homologous recombination functions A direct link between BRCA2 and the cancereprone Fanco ni s anaemia syndrome arose when patients with the Fanconi s D1 complementation group turned out to harbour biallelic hyqpoe mo rphic mutations in the BRCA2 gene In addition BRCA1 and the Fanconi s D2 protein interact in DNAedamage signalling pathways see section The Sephase checkpoint pathways above Although mice bearing mutations in the Fanco ni s A or C genes show chromo 7 some instability and defective germecell development they do not spontaneously develop cancer In contrast mice lacking the Fanconi s D2 gene and Bra12 hypomorphic mice develop epithelial cancers such as breast ovarian and liver cancer Although mice with heterozye gous mutations in Bra12 do not develop tumours at an increased frequency mice with homozygous truncations of Bra12 develop thymic lymphomas Growth arrest and unstable chromosome structure induced by Bra12 truncation are relieved when cellecycle checkpoints that are responsive to mitotic spindle disruption are inactivated This suggests that inactivating mutations in mitotic checkpoint genes might cooperate with Brca2 deficiency in the pathogenesis ofinherited breast cancer and potentiallyother diseases of chromosomal instability such as Blooms syndrome or Fanconi anaemi This concept of mutations that cooperate with checkpoint or repair defects to enhance tumour development is likely to be a recurring theme in future studies Fig 4 The BRCA stories suggest that genetic instabilitycaus ed by altered DNAedamage response pathways may not be sufficient to lead to cancer development and that cooperating mutations must be present to facilitate continued growth or viability of preemalignant cells Similarly mice bearing hypomorphic mutations in the MreII genes show pronounced chromosomal instability but are not prone to malignancygs However tumour formation in these mice on a p537 heterozygote background is significantly enhanced suggesting that the combination of genomic instability and cellecycle checkpoint defects is a significant risk factor for tumour development One recent report demonstrated that mice bearing a mutation in p5 3 that was defective in apoptosis but retained some cellecycle checkpoint function was markedlyless prone to tumour development thanp53 7 null mice This surprising result suggests that the growth arrest and chromosome stability functions of p53 provide tumour suppressor function even in the absence of its role in apoptosisgs Finally the recent observation that ATR haploinsufficiency increases tumorie genesis on a background of mismatch repair deficiency7B may pres age a urry of new insights into how heterozygous mutations although seemingly innocuous on their own can enhance tumour formation when present in certain combinations such as those controlling checkpoint responses and repair abilities Future directions Animal models and humanecanceresusceptibility syndromes will continue to teach us about the physiological roles of the genes and pathways involved in DNAedamage responses Many questions remain such as how the crosstalk between the signalling pathways discussed here and the processes of DNA repair and apoptosis opere ate As these pathways seem to be major determinants of cellular responses to the types of cytotoxic agent that we use to treat tumours these insights may teach us new ways to more effectively treat tumours Similarly because these response pathways seem to be major protectors from cancer development the study of these pathways could lead to effective and new approaches to the reduction 322 of cancer development In addition to the prevention of cancer and more effective treatment of 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