Checkpoint kinase 1, commonly referred to as Chk1, is a serine/threonine-specific protein kinase that, in humans, is encoded by the CHEK1 gene. [5] [6] Chk1 coordinates the DNA damage response (DDR) and cell cycle checkpoint response. [7] Activation of Chk1 results in the initiation of cell cycle checkpoints, cell cycle arrest, DNA repair and cell death to prevent damaged cells from progressing through the cell cycle.
In 1993, Beach and associates initially identified Chk1 as a serine/threonine kinase which regulates the G2/M phase transition in fission yeast. [8] Constitutive expression of Chk1 in fission yeast was shown to induce cell cycle arrest. The same gene called Rad27 was identified in budding yeast by Carr and associates. In 1997, homologs were identified in more complex organisms including the fruit fly, human and mouse. [9] Through these findings, it is apparent Chk1 is highly conserved from yeast to humans. [5]
Human Chk1 is located on chromosome 11 on the cytogenic band 11q22-23. Chk1 has a N-terminal kinase domain, a linker region, a regulatory SQ/TQ domain and a C-terminal domain. [9] Chk1 contains four Ser/Gln residues. [8] Chk 1 activation occurs primarily through the phosphorylation of the conserved sites, Ser-317, Ser-345 and less often at Ser-366. [8] [10]
Checkpoint kinases (Chks) are protein kinases that are involved in cell cycle control. Two checkpoint kinase subtypes have been identified, Chk1 and Chk2. Chk1 is a central component of genome surveillance pathways and is a key regulator of the cell cycle and cell survival. Chk1 is required for the initiation of DNA damage checkpoints and has recently been shown to play a role in the normal (unperturbed) cell cycle. [9] Chk1 impacts various stages of the cell cycle including the S phase, G2/M transition and M phase. [8]
In addition to mediating cell cycle checkpoints, Chk1 also contributes to DNA repair processes, gene transcription, egg production, embryo development, cellular responses to HIV infection and somatic cell viability. [8] [11]
Chk1 is essential for the maintenance of genomic integrity. Chk1 monitors DNA replication in unperturbed cell cycles and responds to genotoxic stress if present. [9] Chk1 recognizes DNA strand instability during replication and can stall DNA replication in order to allow time for DNA repair mechanisms to restore the genome. [8] Recently, Chk1 has shown to mediate DNA repair mechanisms and does so by activating various repair factors. Furthermore, Chk1 has been associated with three particular aspects of the S-phase, which includes the regulation of late origin firing, controlling the elongation process and maintenance of DNA replication fork stability. [8]
In response to DNA damage, Chk1 is an important signal transducer for G2/M checkpoint activation. Activation of Chk1 holds the cell in the G2 phase until ready to enter the mitotic phase. This delay allows time for DNA to repair or cell death to occur if DNA damage is irreversible. [12] Chk1 must inactivate in order for the cell to transition from the G2 phase into mitosis, Chk1 expression levels are mediated by regulatory proteins.
Chk1 has a regulatory role in the spindle checkpoint however the relationship is less clear as compared to checkpoints in other cell cycle stages. During this phase the Chk1 activating element of ssDNA can not be generated suggesting an alternate form of activation. Studies on Chk1 deficient chicken lymphoma cells have shown increased levels of genomic instability and failure to arrest during the spindle checkpoint phase in mitosis. [8] Furthermore, haploinsufficient mammary epithelial cells illustrated misaligned chromosomes and abnormal segregation. These studies suggest Chk1 depletion can lead to defects in the spindle checkpoint resulting in mitotic abnormalities.
DNA damage induces the activation of Chk1 which facilitates the initiation of the DNA damage response (DDR) and cell cycle checkpoints. The DNA damage response is a network of signaling pathways that leads to activation of checkpoints, DNA repair and apoptosis to inhibit damaged cells from progressing through the cell cycle.
Chk1 is regulated by ATR through phosphorylation, forming the ATR-Chk1 pathway. This pathway recognizes single strand DNA (ssDNA) which can be a result of UV-induced damage, replication stress and inter-strand cross linking. [8] [9] Often ssDNA can be a result of abnormal replication during S phase through the uncoupling of replication enzymes helicase and DNA polymerase. [8] These ssDNA structures attract ATR and eventually activates the checkpoint pathway.
However, activation of Chk1 is not solely dependent on ATR, intermediate proteins involved in DNA replication are often necessary. Regulatory proteins such as replication protein A, Claspin, Tim/Tipin, Rad 17, TopBP1 may be involved to facilitate Chk1 activation. Additional protein interactions are involved to induce maximal phosphorylation of Chk1. Chk1 activation can also be ATR-independent through interactions with other protein kinases such as PKB/AKT, MAPKAPK and p90/RSK. [8]
Also, Chk1 has been shown to be activated by the Scc1 subunit of the protein cohesin, in zygotes. [13]
Chk1 interacts with many downstream effectors to induce cell cycle arrest. In response to DNA damage, Chk1 primarily phosphorylates Cdc25 which results in its proteasomal degradation. [9] The degradation has an inhibitory effect on the formation of cyclin-dependent kinase complexes, which are key drivers of the cell cycle. [14] Through targeting Cdc25, cell cycle arrest can occur at multiple time points including the G1/S transition, S phase and G2/M transition. [8] Furthermore, Chk1 can target Cdc25 indirectly through phosphorylating Nek11.
WEE1 kinase and PLK1 are also targeted by Chk1 to induce cell cycle arrest. Phosphorylation of WEE1 kinase inhibits cdk1 which results in cell cycle arrest at the G2 phase. [8]
Chk1 has a role in the spindle checkpoint during mitosis thus interacts with spindle assembly proteins Aurora A kinase and Aurora B kinase. [9]
Recently, Chk1 has shown to mediate DNA repair mechanisms and does so by activating repair factors such as proliferating cell nuclear antigen (PCNA), FANCE, Rad51 and TLK. [8] Chk1 facilitates replication fork stabilization during DNA replication and repair however more research is necessary to define the underlying interactions. [9]
Chk1 has a central role in coordinating the DNA damage response and therefore is an area of great interest in oncology and the development of cancer therapeutics. [15] Initially Chk1 was thought to function as a tumor suppressor due to the regulatory role it serves amongst cells with DNA damage. However, there has been no evidence of homozygous loss of function mutants for Chk1 in human tumors. [8] Instead, Chk1 has been shown to be overexpressed in numerous tumors including breast, colon, liver, gastric and nasopharyngeal carcinoma. [8] There is a positive correlation with Chk1 expression and tumor grade and disease recurrence suggesting Chk1 may promote tumor growth. [8] [9] [15] Chk1 is essential for cell survival and through high levels of expressions in tumors the function may be inducing tumor cell proliferation. Further, a study has demonstrated that targeting Chk1 reactivates the tumour suppressive activity of protein phosphtase 2A (PP2A) complex in cancer cells. [16] Studies have shown complete loss of Chk1 suppresses chemically induce carcinogenesis however Chk1 haploinsufficiency results in tumor progression. [9] Due to the possibility of Chk1 involvement in tumor promotion, the kinase and related signaling molecules may be potentially effective therapeutic targets. Cancer therapies utilize DNA damaging therapies such as chemotherapies and ionizing radiation to inhibit tumor cell proliferation and induce cell cycle arrest. [17] Tumor cells with increased levels of Chk1 acquire survival advantages due to the ability to tolerate a higher level of DNA damage. Therefore, Chk1 may contribute to chemotherapy resistance. [18] In order to optimize chemotherapies, Chk1 must be inhibited to reduce the survival advantage. [7] Chk1 gene can be effectively silenced by siRNA knockdown for further analysis based on an independent validation. [19] By inhibiting Chk1, cancer cells lose the ability to repair damaged DNA which allows chemotherapeutic agents to work more effectively. Combining DNA damaging therapies such as chemotherapy or radiation treatment with Chk1 inhibition enhances targeted cell death and provides synthetic lethality. [20] Many cancers rely on Chk1 mediated cell cycle arrest heavily especially if cancers are deficient in p53. [21] Approximately 50% of cancers possess p53 mutations illustrating the dependence that many cancers may have on the Chk1 pathway. [22] [23] [24] Inhibition of Chk1 allows selective targeting of p53 mutant cells as Chk1 levels are more likely to highly expressed in tumor cells with p53 deficiencies. [15] [25] Even though this method of inhibition is highly targeted, recent research has shown Chk1 also has a role in the normal cell cycle. [26] Therefore, off-target effects and toxicity associated with combination therapies using Chk1 inhibitors must be considered during development of novel therapies. [27]
In a combined computational approach, a set of in-house plant-based semi-synthetic aminoarylbenzosuberene molecules selected for analysis, from these Bch10 regarded as a potential CHK1 inhibitor compared to the top five co-crystallized inhibitors based on their binding affinity and toxicity profile. [28]
During meiosis in human and mouse, CHEK1 protein kinase is important for integrating DNA damage repair with cell cycle arrest. [29] CHEK1 is expressed in the testes and associates with meiotic synaptonemal complexes during the zygonema and pachynema stages. [29] CHEK1 likely acts as an integrator for ATM and ATR signals and may be involved in monitoring meiotic recombination. [29] In mouse oocytes CHEK1 appears to be indispensable for prophase I arrest and to function at the G2/M checkpoint. [30]
The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA and some of its organelles, and subsequently the partitioning of its cytoplasm and other components into two daughter cells in a process called cell division.
DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA damage, resulting in tens of thousands of individual molecular lesions per cell per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. When normal repair processes fail, and when cellular apoptosis does not occur, irreparable DNA damage may occur, including double-strand breaks and DNA crosslinkages. This can eventually lead to malignant tumors, or cancer as per the two hit hypothesis.
S phase (Synthesis Phase) is the phase of the cell cycle in which DNA is replicated, occurring between G1 phase and G2 phase. Since accurate duplication of the genome is critical to successful cell division, the processes that occur during S-phase are tightly regulated and widely conserved.
G2 phase, Gap 2 phase, or Growth 2 phase, is the third subphase of interphase in the cell cycle directly preceding mitosis. It follows the successful completion of S phase, during which the cell’s DNA is replicated. G2 phase ends with the onset of prophase, the first phase of mitosis in which the cell’s chromatin condenses into chromosomes.
ATM serine/threonine kinase, symbol ATM, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks. It phosphorylates several key proteins that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis. Several of these targets, including p53, CHK2, BRCA1, NBS1 and H2AX are tumor suppressors.
Cell cycle checkpoints are control mechanisms in the eukaryotic cell cycle which ensure its proper progression. Each checkpoint serves as a potential termination point along the cell cycle, during which the conditions of the cell are assessed, with progression through the various phases of the cell cycle occurring only when favorable conditions are met. There are many checkpoints in the cell cycle, but the three major ones are: the G1 checkpoint, also known as the Start or restriction checkpoint or Major Checkpoint; the G2/M checkpoint; and the metaphase-to-anaphase transition, also known as the spindle checkpoint. Progression through these checkpoints is largely determined by the activation of cyclin-dependent kinases by regulatory protein subunits called cyclins, different forms of which are produced at each stage of the cell cycle to control the specific events that occur therein.
p21Cip1, also known as cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1, is a cyclin-dependent kinase inhibitor (CKI) that is capable of inhibiting all cyclin/CDK complexes, though is primarily associated with inhibition of CDK2. p21 represents a major target of p53 activity and thus is associated with linking DNA damage to cell cycle arrest. This protein is encoded by the CDKN1A gene located on chromosome 6 (6p21.2) in humans.
The G1/S transition is a stage in the cell cycle at the boundary between the G1 phase, in which the cell grows, and the S phase, during which DNA is replicated. It is governed by cell cycle checkpoints to ensure cell cycle integrity and the subsequent S phase can pause in response to improperly or partially replicated DNA. During this transition the cell makes decisions to become quiescent, differentiate, make DNA repairs, or proliferate based on environmental cues and molecular signaling inputs. The G1/S transition occurs late in G1 and the absence or improper application of this highly regulated check point can lead to cellular transformation and disease states such as cancer
Serine/threonine-protein kinase ATR also known as ataxia telangiectasia and Rad3-related protein (ATR) or FRAP-related protein 1 (FRP1) is an enzyme that, in humans, is encoded by the ATR gene. It is a large kinase of about 301.66 kDa. ATR belongs to the phosphatidylinositol 3-kinase-related kinase protein family. ATR is activated in response to single strand breaks, and works with ATM to ensure genome integrity.
CHEK2 is a tumor suppressor gene that encodes the protein CHK2, a serine-threonine kinase. CHK2 is involved in DNA repair, cell cycle arrest or apoptosis in response to DNA damage. Mutations to the CHEK2 gene have been linked to a wide range of cancers.
DNA-dependent protein kinase, catalytic subunit, also known as DNA-PKcs, is an enzyme that in humans is encoded by the gene designated as PRKDC or XRCC7. DNA-PKcs belongs to the phosphatidylinositol 3-kinase-related kinase protein family. The DNA-Pkcs protein is a serine/threonine protein kinase comprising a single polypeptide chain of 4,128 amino acids.
Mediator of DNA damage checkpoint protein 1 is a 2080 amino acid long protein that in humans is encoded by the MDC1 gene located on the short arm (p) of chromosome 6. MDC1 protein is a regulator of the Intra-S phase and the G2/M cell cycle checkpoints and recruits repair proteins to the site of DNA damage. It is involved in determining cell survival fate in association with tumor suppressor protein p53. This protein also goes by the name Nuclear Factor with BRCT Domain 1 (NFBD1).
Claspin is a protein that in humans is encoded by the CLSPN gene.
Wee1 is a nuclear kinase belonging to the Ser/Thr family of protein kinases in the fission yeast Schizosaccharomyces pombe. Wee1 has a molecular mass of 96 kDa and is a key regulator of cell cycle progression. It influences cell size by inhibiting the entry into mitosis, through inhibiting Cdk1. Wee1 has homologues in many other organisms, including mammals.
The meiotic recombination checkpoint monitors meiotic recombination during meiosis, and blocks the entry into metaphase I if recombination is not efficiently processed.
The G2-M DNA damage checkpoint is an important cell cycle checkpoint in eukaryotic organisms that ensures that cells don't initiate mitosis until damaged or incompletely replicated DNA is sufficiently repaired. Cells which have a defective G2-M checkpoint, if they enter M phase before repairing their DNA, it leads to apoptosis or death after cell division. The defining biochemical feature of this checkpoint is the activation of M-phase cyclin-CDK complexes, which phosphorylate proteins that promote spindle assembly and bring the cell to metaphase.
DNA re-replication is an undesirable and possibly fatal occurrence in eukaryotic cells in which the genome is replicated more than once per cell cycle. Rereplication is believed to lead to genomic instability and has been implicated in the pathologies of a variety of human cancers. To prevent rereplication, eukaryotic cells have evolved multiple, overlapping mechanisms to inhibit chromosomal DNA from being partially or fully rereplicated in a given cell cycle. These control mechanisms rely on cyclin-dependent kinase (CDK) activity. DNA replication control mechanisms cooperate to prevent the relicensing of replication origins and to activate cell cycle and DNA damage checkpoints. DNA rereplication must be strictly regulated to ensure that genomic information is faithfully transmitted through successive generations.
DNA damage is distinctly different from mutation, although both are types of error in DNA. DNA damage is an abnormal chemical structure in DNA, while a mutation is a change in the sequence of base pairs. DNA damages cause changes in the structure of the genetic material and prevents the replication mechanism from functioning and performing properly.
DNA replication stress refers to the state of a cell whose genome is exposed to various stresses. The events that contribute to replication stress occur during DNA replication, and can result in a stalled replication fork.
Telomeres, the caps on the ends of eukaryotic chromosomes, play critical roles in cellular aging and cancer. An important facet to how telomeres function in these roles is their involvement in cell cycle regulation.
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