The cell division cycle is surveyed by multiple checkpoints that respond to a wide range of internal and external stresses to ensure genomic integrity from one cell generation to the next. When checkpoints are triggered, CDKs are generally inhibited (the exception being the spindle assembly checkpoint in mitosis where inactivation of the CDK is inhibited); this results in cell cycle arrest and provides time for DNA repair to occur. If the damage is extensive and cannot be resolved, checkpoints trigger cell senescence or apoptosis.

There are four main checkpoints responding to different types of DNA insults, one in each cell cycle phase (Fig. 3.1). The G1 phase checkpoint prevents the replication of damaged genomic material by blocking entry into S phase, the S phase checkpoint not only responds to the presence of DNA damage but also to aberrant replication forks by stopping or slowing DNA synthesis, the G2 phase checkpoint impedes cells with damaged or entangled, catenated DNA from undergoing mitosis, and finally the spindle assembly checkpoint only allows mitotic exit if the chromosomes are properly attached to the mitotic spindle.

Two major players in checkpoint signalling in response to DNA damage are the ataxia telangiectasia mutated (ATM) and the ataxia telangiectasia and Rad3-related protein (ATR) proteins. ATM is mainly triggered by double strand DNA breaks (DSBs), while ATR primarily responds to single-stranded DNA (ssDNA) (Fig. 3.2). Once activated, these kinases phosphorylate and activate checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2) proteins, which in turn target a range of effectors that inhibit cell cycle progression, including the immediate activators of the CDK/Cyclins, the Cdc25 phosphatases. ATM/ATR and Chk2/Chk1 also phosphorylate and activate p53 protein which promotes the transcription of p21, GADD45 and 14-3-3 CDK inhibitor proteins (Elledge 1996; Ciocca and Elledge 2000; Elledge et al. 2000; Huang and Elledge 2000; Liu et al. 2000; Schulman et al. 2000; Tibbetts et al. 2000; Wang et al. 2000; Zhou and Elledge 2000), thus enhancing cell cycle arrest. Another mitotic regulatory pathway involves Plk1 protein kinase which is activated by the cooperative action of Aurora A protein kinase and its cofactor Bora (Seki et al. 2008; Takaki et al. 2008). Plk1 activity is also regulated by ATM/ATR checkpoint signalling (Smits et al. 2000; van Vugt et al. 2001). A link between this pathway and the G2 phase DNA damage checkpoint has been recently described where Bora is degraded via ATR-mediated phosphorylation following DNA damage (Qin et al. 2013), and ATM-dependent phosphorylation and activation of B56 regulatory subunit of PP2A that is responsible for dephosphorylating and inactivating Plk1 (Shouse et al. 2011).

In response to DNA lesions, cells initiate a highly coordinated cascade of events, known as the DNA damage response (DDR), which is essential for the maintenance of genomic stability and cell survival. DNA damage can interfere with essential cellular processes, such as transcription or replication, and can compromise the viability of the cells. DNA damage repair is tightly coordinated with cell cycle progression through the activation of DNA damage checkpoints. The DDR consists of detection of damage by sensor proteins, with signal transducer and effector proteins launching a cascade of events that causes cell cycle arrest, activation of DNA repair, senescence or apoptosis.

DNA damage is detected by sensor proteins, with DSBs detected by the MRE11-RAD50-NBS1 (MRN) sensor complex and ssDNA arising from DNA damage processing is signalled by Replication protein A (RPA), an ssDNA binding protein. Once DNA damage is sensed, the cell must transduce this signal down to its appropriate effectors. The MRN complex leads to the recruitment and activation of ATM and RPA recruits the ATR kinase via its partner protein, ATRIP (ATR-interacting partner) (reviewed in Elledge 1996; Zhou and Elledge 2000). ATM and ATR are related kinases that phosphorylate a number of mediators which are mostly cell cycle specific and associate with damage sensors, signal transducers and effectors at particular phases of the cell cycle, and as a consequence, provide signal transduction specificity. ATM and ATR are both members of the PI-3K-like kinase family (PIKK) of protein kinases that also includes DNA dependent protein kinase (DNA-PK) and mammalian target of rapamaycin (mTOR). The ATM signalling cascade includes 53BP1, MDC1, BRCA1, MCPH1, and PTIP, while mediators of ATR signalling include TopBP1, and Claspin (reviewed in Marechal and Zou 2013). The transducer kinases lead to the activation of the effector kinases CHK1 and CHK2, activating signalling cascades with downstream targets including transcription factors, cell cycle regulators, apoptosis and DNA repair factors (Fig. 3.2). There is crosstalk between the ATM/Chk2 and ATR/Chk1 pathways and they share many substrates.

It is estimated that each of the ~10¹³ cells within the human body incurs tens of thousands of DNA-damaging events per day (Lindahl and Barnes 2000). To avoid the deleterious consequences of damage accumulation, multiple DNA repair pathways have evolved, each associated with specific classes of lesions.

DNA is subject to a high level of endogenous damage resulting from reactive oxygen species (ROS) generated from normal cellular metabolism, in addition to a number of other factors including spontaneous hydrolysis, abasic sites, and alkylation. Endogenous sources of damage can arise from physiological DNA processing and DNA repair processes themselves, such as DNA mismatches, insertions and deletions which can be introduced as a result of misincorporation of bases by replicative DNA polymerases (McCulloch and Kunkel 2008).

There are a number of exogenous or environmental sources of DNA-damaging agents including ultraviolet radiation (UV), ionizing radiation (IR), chemical agents used as clinical and chemotherapeutic drugs, tobacco smoke and other environmental agents. Environmental stresses such as ultraviolet light (UV) from the sun, primarily causes two types of DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6–4 pyrimidine products (6–4PP) (You et al. 2001). IR is the most common DSB-inducing agent, and can originate from both natural (gamma and cosmic radiation) and artificial sources (eg. X-rays and radiotherapy). IR can also induce DNA damage indirectly through the production of ROS.

Chemical agents used as chemotherapeutic drugs, including alkylating agents (eg. temozolomide), bifunctional alkylating agents (e.g. platinum agents), and mitomycin C cause DNA damage in the form of intra-strand and inter-strand cross-links. Targeted drugs such as topoisomerase I or II inhibitors, generate ssDNA or dsDNA breaks by trapping topoisomerase-DNA covalent complexes (Sinha 1995). Nitrosamines are a class of potential carcinogens that are found in tobacco smoke, and there is a possible dose-response relationship between the amount and duration of tobacco smoke exposure and mutational burden (Govindan et al. 2012). Other naturally occurring environmental agents include N-nitrosoamines, heterocyclic amines, and polycyclic aromatic hydrocarbons which are common in the diet, producing bulky DNA adducts (reviewed in Dexheimer 2013).

Defective cell cycle checkpoint function may increase spontaneous and induced gene mutations and chromosomal aberrations by reducing the efficiency of DNA repair. Cell cycle checkpoints integrate DNA repair with cell cycle progression and are commonly deregulated in cancers leading to increased genomic instability. This provides cancer cells with an evolutionary or adaptive advantage, allowing them to modify their transcriptome and/or genome to increase their ability to thrive in new tissue environments.

Cell cycle checkpoint pathways integrate repair of specific DNA lesions with the cell cycle position. The phase of the cell cycle in which a checkpoint is induced, and the duration of the arrest is dependent on the type of triggering DNA lesion, the number of lesions and the length of time required to repair the lesions (for review see Lazzaro et al. 2009). There are some lesions that are rapidly repaired and do not require cell cycle checkpoint arrest, while in some cases the cells may keep cycling and reach a cell cycle phase where the specific damage is less toxic or more easily repaired.

G1/S phase transition is a major target of alterations in cancer. Such alterations often result in changing the Retinoblastoma protein (Rb) phosphorylation/dephosphorylation balance and consequently effecting the cell proliferation and blocking entry into S phase (Hall and Peters 1996). Rb point mutations or deletions are found in a wide variety of human cancers, particularly retinoblastomas and sarcomas. Histone deacetylase (HDAC)-Rb complex formation is also inhibited by HDAC point mutations found in human cancer (Brehm et al. 1998; Luo et al. 1998; Magnaghi-Jaulin et al. 1998).

P16-mediated senescence is another G1 phase mechanism that responds to extensive DNA damage (Robles and Adami 1998; Shapiro 2006). CDKN2A is the gene which encodes the cell cycle inhibitor p16 ^INK4A , which is commonly defective in melanoma (Bartkova et al. 1996; Castellano et al. 1997; Hayward 2003). P16, a major driver of senescence, creates G1 phase arrest by blocking Rb inactivation through CDK4/6-Cyclin D inhibition. Melanoma associated mutation of p16 disrupts its ability to promote senescence arrest (Haferkamp et al. 2008). In addition to this role, increased p16 expression has been correlated with the G2 phase checkpoint arrest in response to suberythemal UVR (Pavey et al. 1999, 2001; Abd Elmageed et al. 2009).

The tumour suppressor p53 is involved in DNA damage response and is inactivated in 50 % of all human cancers (Hollstein et al. 1991), and humans carrying a germ-line deletion of one TP53 allele are highly prone to cancer development (Malkin et al. 1990; Srivastava et al. 1990). In the face of a range of genotoxic insults, p53 is rapidly stabilized and its increased level promotes cell cycle arrests in G1 and G2 phases in cell lines in vitro and signals apoptosis in response to excessive damage. The G1 checkpoint is activated by ATM signalling to Chk2 and p53, among other targets, preventing the replication of damaged DNA by blocking entry into S-phase. ATM promotes Homologous recombination repair by recruiting BRCA1 to DSBs, but can also antagonize BRCA1 and promote NHEJ by recruiting 53BP1. Defects in the function of p53 may contribute to the genetic instability that appears to drive neoplastic evolution.

The decatenation checkpoint is another G2 phase checkpoint that has been reported to be defective in a large proportion (68 %) of melanoma cell-lines (Brooks et al. 2014). This ATM/Chk2-dependent checkpoint ensures that chromosome catenation, normally resulting from DNA replication, is resolved before the cell progresses into mitosis. The difference in checkpoint functional response was not attributable to Topoisomerase II levels. Intriguingly, despite the absence of cycle arrest, the checkpoint defective cell-lines were able to activate the checkpoint signalling following treatment with the topoisomerase II inhibitor ICRF-193, indicating participation of other players, possibly involved in checkpoint recovery, in this phenomenon (Brooks et al. 2014).

The mitotic spindle assembly checkpoint (SAC) monitors spindle defects. Many of the spindle assembly checkpoint sensors and effectors are localized to kinetochores, including the Mad and Bub gene families and Aurora kinases. This checkpoint detects defects in spindle attachment to the replicated chromosomes through the centromere localised kinetochore complex, and tension across the centromere, to ensure the fidelity of partitioning of the replicated genome into the daughter cells. Although mutations in SAC genes are uncommon, they have been reported at low frequency in a number of cancer types (Cahill et al. 1998). The microtubule direct drugs, taxane, vincalkaloids, and some of the newer targeted drugs such as Aurora inhibitors target this checkpoint. Cancer cells appear less able to maintain the mitotic arrest in the face of these drugs resulting in mitotic slippage and triggering cell death (Dowling et al. 2005; Weaver and Cleveland 2005; Gabrielli et al. 2007; Stevens et al. 2008).

Increased endogenous damage levels in cancer cells caused by defects in the cell cycle checkpoints and deregulation of DNA repair mechanisms can lead to genomic instability and contribute to the initiation and progression of cancer. This must be accommodated by the cells employing either novel mechanisms to cope with the stress, an adaptation to accommodate the stress, becoming more reliant on alternative stress response mechanisms or a combination of all three. Up-regulated DDR pathways can confer therapy resistance to DNA-damaging chemotherapy and radiotherapy, while down-regulated DDR pathways can lead to dependence on a compensatory pathway. This increased reliance on alternative mechanisms to cope with a stress makes it plausible to disarm these mechanisms to selectively target the cancer cells population while normal cells are being protected by their intact checkpoint and repair responses.