Genetic instability in cancer cells by impaired cell cycle checkpoints

Abstract

Cells continuously encounter DNA damage caused either by damaging agents, including oxygen radicals and DNA replication errors caused by stalled replication forks, or by extracellular environments such as ultraviolet or ionizing irradiation. Such DNA damage poses a great threat to genome stability, potentially leading to loss or amplification of chromosome activity, which may result in cellular senescence, cancer or apoptosis. The DNA damage checkpoints coordinate an arrest in cell cycle progression with the DNA repair process, suppressing either mitotic catastrophe or proliferation of cells with damaged DNA. Numerous key players have been identified in terms of damage sensor proteins, transducer kinases and effectors, but their coordination and interconnectedness in damage control have only recently become evident. In this review, we discuss changes in chromatin structure, recruitment of mediator proteins and activation of transducer kinases in response to DNA damage. These cellular responses are important for determining the potential effects of current cancer therapies in terms of toxicity and efficacy. (Cancer Sci 2006; 97: 984–989)

53BP1

p53 binding protein 1

ATM

ataxia telangiectesia mutated

ATR

AT‐ and Rad3‐related

ATRIP

ATR‐interacting protein

BRCA1

breast cancer susceptibility gene 1

BRCT

BRCA1 C‐terminal repeat

Chk1

checkpoint kinase 1

Chk2

checkpoint kinase 2

DNA‐PK

DNA‐dependent protein kinase

H2Av

histone H2 variant

H2AX

histone 2 variant

IR

ionizing irradiation

MDC1

mediator of DNA damage checkpoint 1

MRN

Mre11/Rad50/Nbs1

NBS1

Nijmegen breakage syndrome 1

PCNA

proliferating cell nuclear antigen

PIKK

phosphoinositide 3‐kinases

PP2A

protein phosphatase 2A

RFC

replication factor C

RPA

replication protein A

SMC1

structural maintenance of chromatin 1

TopBP1

topoisomerase binding protein 1.

Changes in chromatin structure at DNA damage sites

Eukaryotic genomic DNA is packaged with histone and non‐histone proteins into highly condensed chromatin structures. Thus, repair of DNA damage as well as transcriptional regulation and cell cycle checkpoint activation requires histone modification and remodeling that would render chromatin more accessible to DNA repair enzymes (Fig. 1). Alteration of the chromatin structure can be achieved by covalent modification of histone tails or by altering histone composition.^{( 1} , ^{2 )} One of the earliest modifications of chromatin in the damage response is phosphorylation of H2AX at Ser139 by members of the PIKK protein kinase, ATM, ATR and DNA‐PK.^{( 3} , ⁴ , ^{5 )} Phosphorylated H2AX (γ‐H2AX) covers a region that may extend up to megabases away from the break sites^{( 6 )} and is required for the recruitment of mediator proteins such as MDC1, 53BP1, BRCA1 and the MRN complex.^{( 7 )} In addition to mediator proteins, γ‐H2AX is also essential for the recruitment of histone acetyltransferase, NuA4 and the ATP‐dependent chromatin‐remodeling complexes INO80 and SWR1 to a region within 2 kb of a break site in yeast.^{( 8} , ⁹ , ^{10 )} Mutations in subunits of INO80 show hypersensitivity to DNA damage, presumably due to low efficiency in converting double‐stranded DNA into single‐stranded DNA at damaged sites. SWR1 exchanges H2A–H2B histone dimers with H2AZ–H2B dimers in nucleosomes.^{( 11 )} Thus, these complexes likely unravel the packed chromatin, allowing repair enzymes to access the DNA damage sites. Interestingly, the Tip60 HAT complex is also required for DNA double‐strand break repair and exchange of γ‐H2AX with unphosphorylated H2AX in mammals. Overexpression of Tip60 lacking acetylase activity renders cells defective in DNA double‐strand break repair.^{( 12 )} In Drosophila, Tip60 acetylates the phosphorylated H2Av and replaces it with unacetylated H2Av. Together, histone modification and remodeling likely facilitate restoration to the undamaged chromatin site. Histone methylation has also been shown to be linked to the recruitment of checkpoint mediator proteins. Methylation of lysine 79 on histone H3, which is mediated by Dot1L, is important for the recruitment of 53BP1 to DNA damage sites.^{( 13 )} Thus, histone modification and chromatin remodeling likely change the higher order chromatin structure and recruit DNA repair and checkpoint proteins to the damage sites.

Figure 1.

Histone modification after DNA damage. DNA damage induces several types of histone modifications. These modifications are mediated by distinct enzymes and have unique functions.

Recognition of DNA damage by sensor proteins

The first step in the initiation of activity of DNA damage checkpoints is recognition of the DNA damage (Fig. 2). Studies in yeasts and mammals have demonstrated that Rad9, Rad1, Hus1 and Rad17 are essential factors that activate checkpoint signaling^{( 14 )} (Table 1). Rad9, Rad1 and Hus1 form a heterotrimeric complex (the 9‐1‐1 complex). Its structure resembles a PCNA‐like sliding clamp. Rad17 interacts with four small RFC subunits, Rfc2, Rfc3, Rfc4 and Rfc5, to form an RFC‐related complex that acts as a clamp‐loading complex. Once DNA is damaged, the 9‐1‐1 complex is recruited to the damage site under the regulation of the Rad17 complex.^{( 15 )} The chromatin‐bound 9‐1‐1 complex then facilitates phosphorylation mediated by ATR and ATM.

Figure 2.

Conceptual organization of the signal transduction of checkpoint responses. DNA damage is recognized by sensor proteins. The signals are transmitted to transducers (mainly kinases) and the regulated transducer molecules suppress effector kinases, such as Cdks and Cdc7, thereby arresting the cell cycle at the specific phases.

Table 1.

Classification of genes involved in cell cycle checkpoints into four classes by their functions

Function	Class	Gene
Sensor	RFC1‐like	RAD17
		RFC2‐5
	PCNA‐like	RAD9
		RAD1
		Hus1
Mediator	BRCT‐containing	BRCA1
		53BP1
		TopBP1
		MDC1
	MRN complex	Mre11
		RAD50
		NBS1
Transducer kinase	PI3 kinase like protein	ATR–ATRIP
		ATM
		DNA‐PK‐Ku
	Checkpoint kinases	Chk1
		Chk2
Effector	Transcription factor	p53
	Phosphatases	Cdc25A, B, C
	Kinases	Cyclin‐Cdks
		CDC7

Activation of transducer kinases

In mammals, signals initiated by DNA‐damage sensors very rapidly transduce to ATM and ATR kinases, which are both extremely large proteins that phosphorylate a great number of substrates. ATM is a 350 kDa oligomeric protein containing many HEAT (huntingin elongation factor 3, a subunit of protein phosphatase 2 A and TOR1) motifs.^{( 16 )} It exhibits significant homology to PIKK, but lacks lipid kinase activity and is mainly activated in response to DNA double‐strand breaks. Patients bearing an ATM mutation suffer from a devastating syndrome called ataxia telangiectasia that causes immunodeficiency, genome instability, clinical radio‐sensitivity and a predisposition to cancer. Although cells lacking ATM are viable, suggesting that ATM is not essential for normal cell cycle progression,^{( 17 )} its kinase activity is stimulated by DNA double‐strand breaks. Recently, the identification of a damage‐induced phosphorylation site (Ser1981) revealed a new mechanism for ATM regulation by which a rapid and sensitive switch for checkpoint signals is permitted.^{( 18 )} ATM under unstressed conditions exists as a homodimer in which the kinase domain is physically blocked by tight intermolecular binding to a protein domain at around Ser1981. Upon DNA double‐strand breaks, a conformational change in the ATM protein stimulates the kinase to phosphorylate Ser1981 by intermolecular autophosphorylation, resulting in dissociation of the homodimer. Recently, PP2A has been reported to regulate ATM autophosphorylation.^{( 18 )} In the absence of DNA damage, ATM associates constitutively with PP2A. DNA damage causes a rapid dissociation of the ATM–PP2A complex, leading to its autophosphorylation. In addition, ATM is acetylated by Tip60 and this modification is important for ATM kinase activity.^{( 19 )}

ATM kinase activity is also regulated by binding to MRE11, which enhances its ability to phosphorylate substrates in vitro.^{( 20 )} Given that ATM can be activated by ionizing radiation in cells lacking NBS1 or BRCA1, but fails to be recruited to the double‐strand break sites, it appears that the MRN complex enhances the accumulation of ATM at these sites.

ATR was discovered from its sequence similarity to ATM and Rad3, and was shown to play an essential role in DNA damage repair and DNA replication checkpoint activation.^{( 21 )} Mutations in ATR have been reported in a subset of patients with Sickel syndrome, which is a human autosomal recessive disorder.^{( 22 )} ATR is a 303 kDa protein with a C‐terminal kinase domain and regions of homology to other PIKK family members. As with ATM, ATR is capable of specifically phosphorylating serine or threonine residues in SQ/TQ sequences. Although DNA damage or replication fork stalling do not stimulate ATR activity, they regulate ATR subcellular localization. As a result of processing damaged lesions, ATR forms a heterodimer with ATRIP that binds to UV‐damaged DNA or to RPA‐coated single‐stranded DNA.^{( 23 )} However, the importance of RPA in the recruitment of ATR to single‐stranded DNA is still under question.

ATR is thought to be unresponsive to DNA double‐strand breaks; however, it plays a role in the response to IR‐induced DNA damage. Irradiation induces the formation of RPA‐coated single‐stranded DNA generated by nuclease resection or stalled replication forks caused by unrepaired single‐strand or double‐strand breaks. Consistent with this observation, ATM regulates the recruitment of ATR to sites of DNA damage, leading to double‐strand break‐induced Chk1 phosphorylation.^{( 24 )}

Unlike ATM, mice lacking ATR succumb to early embryonic death, indicating that ATR is essential for cell viability.^{( 25 )} The observation that RPA is involved in DNA replication^{( 26 )} and a component of the DNA replication fork led to a model in which ATR–ATRIP localizes to sites of this fork, monitoring the progression of DNA replication. Once the active ATR is translocated to DNA replication foci, it can phosphorylate and activate Chk1. This model is consistent with the observation that Chk1 is also essential for embryonic cell viability.^{( 27} , ^{28 )} In addition, ATR regulates the timing of DNA replication origin firing.^{( 29 )} Therefore, ATR appears to be a multifunctional kinase that regulates several distinct events from S phase to M phase.

Regulation of checkpoint kinases

The checkpoint kinases Chk1 and Chk2 were first identified in fission yeast as essential for cell cycle arrest before mitosis in response to DNA damage or DNA replication blockage, respectively. These kinases were also identified in vertebrate cells based on their homology with yeast scChk1 and scRad53/spCds1. Examination of mouse cells deficient in Chk1 revealed an essential role for this kinase in DNA damage and the DNA replication checkpoint response.^{( 27} , ^{28 )} Chk1 is phosphorylated at Ser317 and Ser345 in response to DNA damage. This phosphorylation is blocked in cells lacking the kinase ATR^{( 28 )} and markedly inhibited in cells with a reduced amount of Rad17^{( 30 )} or lacking Hus1.^{( 31 )} Like ATR, Chk1 plays a role at every point in the cell cycle and loss of Chk1 results in the premature onset of mitosis through the dephosphorylation of Cdc2 at Tyr15. Premature mitosis leads to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c, and the subsequent mitotic catastrophes.^{( 32 )}

In contrast to Chk1, Chk2 is dispensable for prenatal development.^{( 33} , ^{34 )} Given that Chk2 is activated by phosphorylation of its Thr68 in an ATM‐dependent manner in response to IR treatment, Chk2 is implicated in the DNA‐damage signaling pathway. This notion is further supported by a recent report that heterozygous germline mutations in the human Chk2 gene were found in a subset of patients with Li–Fraumeni syndrome without any mutation in their p53 gene.^{( 35 )} Although biochemical analyses revealed that activated Chk2 is capable of phosphorylating Cdc25A at Ser123, Cdc25C at Ser216, BRCA1 at Ser988 and p53 at several sites, including Ser20, examination of Chk2‐deficient mice and cells showed that this enzyme functions mainly in p53‐dependent apoptosis but not in G₂/M arrest upon DNA damage. Chk2‐deficient mice are resistant to IR as a result of the preservation of splenic lymphocytes, thymocytes, neurons of the developing brain whose apoptosis is known to be p53 dependent. ATM–Chk2 function at the intra‐S phase checkpoint upon DNA damage is considered to be important because phosphorylation of Cdc25A triggers its ubiquitination and degradation. However, Chk2‐deficient cells showed that it is dispensable for the intra‐S phase checkpoint response. Thus, the function of Chk2 in cell‐cycle arrest upon DNA damage remains questionable.

Checkpoint signaling

G₁/S checkpoint.  In the presence of DNA damage, the G₁/S checkpoint prevents replication of damaged DNA through several distinct signal transduction pathways (Fig. 3). One involves the rapid degradation of Cdc25A phosphatase whose activity is required for G₁/S transition. Upon DNA damage, the activated Chk1 phosphorylates Cdc25A, triggering its ubiquitination and degradation by the proteasome pathway.^{( 36} , ^{37 )} Degradation of Cdc25A results in the failure of Cdk2 activation and prevents Cdc45 from loading onto chromatin. Given that Cdc45 is essential for recruitment of DNA polymerase α, lack of Cdc45 incorporation into the chromatin structure inhibits new origin firing. This unloading of Cdc45 plays a role in the initial cell cycle arrest at the G₁/S boundary. Transcriptional responses by p53 are then required for maintaining this arrest. The amount and transcriptional activity of p53 is regulated by post‐transcriptional modification, such as phosphorylation, sumoylation, neddylation and acetylation. Phosphorylation of p53 on Ser15 by ATM or ATR and on Ser20 by Chk1 inhibits its nuclear export and degradation, resulting in the accumulation of p53 protein in the nucleus. The amount of p53 protein is regulated by ubiquitination by the ubiquitin ligase Mdm2. Interestingly, ATM also phosphorylates Mdm2 on Ser 395 and suppresses its interaction with p53, leading to p53 accumulation.^{( 38} , ^{39 )} Chk2 likely helps to stabilize p53 protein after DNA damage, although there have been conflicting reports.^{( 33} , ³⁴ , ⁴⁰ , ^{41 )}

Figure 3.

Schematic model of checkpoint signaling pathways at G₁/S in response to DNA damage. DNA damage activates ATM and ATR. The activated ATM and ATR then phosphorylate mediator proteins including MDC1, BRCA1 and 53BP1. Collaboration of the activated ATM and ATR, and the phosphorylated mediators activates Chk1. The activated Chk1 ultimately transmits signals via two distinct pathways.

p53 is thought to be essential for G₁ arrest in response to DNA damage. The key transcriptional target of p53 is the p21 Cdk inhibitor, which inhibits cyclin E‐Cdk2 activity, thereby inhibiting G₁/S transition. ^{( 42 )} p21 also binds to the cyclin D–Cdk4 complex and prevents it from phosphorylating Rb, thereby suppressing the Rb/E2F pathway. Thus, the G₁ checkpoint signal targets two independent and critical tumor suppressor pathways that are most commonly deregulated in human cancers.

Intra‐S checkpoint.  During the S‐phase, damaged DNA inhibits replicative DNA synthesis (intra‐S checkpoint). This checkpoint is regulated by two distinct pathways, namely ATM/ATR–Chk1–Cdc25A and ATM–NBS1–SMC1.^{( 43 )} Depending on the type of DNA damage, ATM or ATR phosphorylates Chk1, which in turn phosphorylates Cdc25A. Downregulation of Cdc25A subsequently causes inactivation of cyclin E–Cdk2. The phosphorylation of Nbs1 on Ser 343 by ATM is required for activation of the Nbs1–Mre11–Rad50 complex and the intra‐S checkpoint.^{( 44} , ^{45 )} Depending on the phosphorylation state of Nbs1, SMC1 is phosphorylated on Ser957 and Ser966 by ATM, which is required for the intra‐S checkpoint. Other mediator proteins, such as 53BP1, BRCA1 and MDC1, are also involved in the intra‐S checkpoint.

G₂/M checkpoint.  The G₂/M checkpoint prevents cells from entry into mitosis through the inhibition of cyclin B/Cdc2 kinase by Chk1‐ or p38‐mediated subcellular sequestration, degradation and inhibition of the Cdc25 family of phosphatases (Fig. 4). In response to DNA damage, the ATM or ATR are activated and phosphorylates Chk1. BRCT motif proteins, such as 53BP1, MDC1 and BRCA1, also play roles in the activation of Chk1. In mammals, there are four mediator‐type proteins that contain BRCT motifs that serve as protein–phosphoprotein interaction modules. 53BP1 is thought to be a homolog of fission yeast Crb2 and budding yeast Rad9. MDC1 functions as a molecular bridge between histone γ‐H2AX and Nbs1 in the MRN complex. BRCA1 is a causative gene of familial breast cancer. TopBP1 is probably a homolog of fission yeast Cut5 and has recently been reported as an important factor for Chk1 activation in the Xenopus system. Phosphorylated Cdc25A leads to its degradation and subsequent inactivation of cyclin B/Cdc2.

Figure 4.

Schematic model of checkpoint signaling at G₂/M upon DNA damage. Activated Chk1 phosphorylates Cdc25A. The phosphorylated Cdc25A is then degraded through the ubiquitin–proteasome pathway, resulting in the inhibition of Cdc2/cyclin B. The activated Chk1 also phosphorylates and stabilizes p53, leading to the induction of several downstream target genes including p21, Gadd45 and 14‐3‐3 sigma.

p53‐dependent mechanisms are also important for the maintenance of G2 arrest. The critical targets of p53 at G2/M are the Cdk inhibitor p21, GADD45, which causes the dissociation of the Cdc2 and cyclin complex, and 14‐3‐3 sigma, which sequesters the cyclin B/Cdc2 complex in the cytoplasm. In addition, p53 appears to repress the transcription of Cdc2 and cyclin B. Two isoforms of MAP kinase, p38 α and γ, are also implicated in the G2/M checkpoint.

Initially, Cdc25C and Cdc25B were thought to be the major effectors of the G₂/M checkpoint response. However, recent reports have revealed that both Cdc25C‐deficient and Cdc25B‐deficient cells have a normal G₂/M checkpoint, suggesting that Cdc25A is a critical target of the G₂/M checkpoint.

Spindle checkpoint.  Cancer cells possess an abnormal number of chromosomes (aneuploidy). The spindle checkpoint inhibits the ubiquitin ligase activity of the anaphase‐promoting complexor cyclosome (APC/C), which is essential for mitotic progression, until spindles are properly attached to all kinetochores, and thus prevents precocious chromosome segregation. Therefore, defects in spindle checkpoint function are likely to contribute to aneuploidy. The components of the spindle checkpoint include the Mad, Bub and Mps proteins. MAD (MAD1‐3) and BUB (BUB1‐3) were identified by yeast mutagenesis screens for mutants unable to survive a temporary exposure to nocodazole or benzimidazole, and Mps was initially identified as a centrosomal protein required for the assembly of bipolar spindles. Two mechanisms for sensing mitotic defects by spindle checkpoint have been proposed. The most upstream sensor monitors the occupancy status of microtubule‐binding sites on the kinetochore and the other sensor monitors the tension across the sister chromatid pair generated by the attachment of kinetochore microtubules emanating from opposite poles of the mitotic spindle. Recent genetic studies using knockout mice revealed that complete loss of spindle checkpoint function results in early embryonic lethality, but relatively minor alterations in the levels of spindle checkpoint proteins can promote tumorigenesis.^{( 46 )} Therefore, partial disruption of spindle checkpoint is more likely to be observed in cancer cells, because too frequent loss or gain of chromosomes might be rather toxic.

Perspective

Recent genetic analyses using mouse knockout technology or knock‐down methods employing small interference RNA have revealed that the majority of DNA damage checkpoint pathways during S to M phase are well conserved between mammals and yeast, although the function of Chk2 at the checkpoint is still unresolved. Many other aspects of checkpoint signaling also remain unresolved. For example, initiation of the G₂/M checkpoint response may not be as simple as presented here. Polo‐like kinases (PLK) are required for mitotic initiation function upstream of Cdc25. Therefore, PLK3^{( 47 )} and PLK1^{( 48 )} seem to be targeted by DNA damage‐induced mechanisms.

Several key works using clinical samples strongly suggest that point mutations of the checkpoint genes contribute to malignant transformation and genetic instability in cancer cells.^{( 49} , ^{50 )} However, the exact role of DNA damage checkpoints in the prevention of human carcinogenesis should be re‐evaluated. Mutations in the ATM and Chk2 genes have been detected in human cancer susceptibility syndromes. However, the phenotypes of cells deficient in these genes are in complete contrast. ATM‐deficient cells are hypersensitive to ionizing radiation, but Chk2‐deficient cells are resistant. In addition, unlike mice lacking ATM, Chk2‐deficient mice are not significantly cancer prone. BRCA1 and p53 are bona fide tumor suppressor genes and mutation or deletion of these genes results in malignant transformation. However, their proteins also regulate other cellular responses, such as apoptosis and cellular senescence, suggesting that defects in cellular responses other than those involving checkpoints might contribute to malignant transformation in BRCA1‐ or p53‐deficient cells. To clarify these issues, further investigations in the cross talk between signaling pathways are critically required.