Journal articles on the topic 'CHK2'
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Maioru, Ovidiu, Lucian Pop, Viorica Radoi, Radu Ursu, Nicolae Bacalbasa, Irina Balescu, and Ioan D. Suciu. "CHEK2 gene in breast cancer." Romanian Medical Journal 69, S3 (June 20, 2022): 15–16. http://dx.doi.org/10.37897/rmj.2022.s3.4.
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Checkpoint kinases (Chks) are serine/threonine kinases that are involved in the control of the cell cycle. Two subtypes have so far been identified, Chk1 and Chk2. They are essential components to delay cell cycle progression in all cells and act at all three cell cycle checkpoints. Here we provide more information regarding the CHEK2 gene and its role in breast cancer as well as known mutations that present a higher cancer risk.
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Yan, Tao, Anand B. Desai, James W. Jacobberger, R. Michael Sramkoski, Tamalette Loh, and Timothy J. Kinsella. "CHK1 and CHK2 are differentially involved in mismatch repair–mediated 6-thioguanine-induced cell cycle checkpoint responses." Molecular Cancer Therapeutics 3, no. 9 (September 1, 2004): 1147–57. http://dx.doi.org/10.1158/1535-7163.1147.3.9.
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Abstract The DNA mismatch repair (MMR) system plays an important role in mediating a G2-M checkpoint arrest and subsequent cell death following treatment with a variety of chemotherapeutic agents. In this study, using 6-thioguanine (6-TG) as a mismatch-inducing drug, we examine the role of ataxia telangiectasia mutated (ATM)/CHK2 and ATM and Rad-3 related (ATR)/CHK1 signaling pathways in MMR-mediated cell cycle responses in MMR-proficient human colorectal cancer RKO cells. We show that, in response to 6-TG (3 μmol/L × 24 hours), activating phosphorylation of CHK1 at Ser317 [CHK1(pS317)] and CHK2 at Thr68 [CHK2(pT68)] are induced differentially during a prolonged course (up to 6 days) of MMR-mediated cell cycle arrests following 6-TG treatment, with CHK1(pS317) being induced within 1 day and CHK2(pT68) being induced later. Using chemical inhibitors and small interfering RNA of the signaling kinases, we show that a MMR-mediated 6-TG-induced G2 arrest is ATR/CHK1 dependent but ATM/CHK2 independent and that ATR/CHK1 signaling is responsible for both initiation and maintenance of the G2 arrest. However, CHK2(pT68) seems to be involved in a subsequent tetraploid G1 arrest, which blocks cells that escape from the G2-M checkpoint following 6-TG treatment. Furthermore, we show that CHK2 is hyperphosphorylated at later times following 6-TG treatment and the phosphorylation of CHK2 seems to be ATM independent but up-regulated when ATR or CHK1 is reduced. Thus, our data suggest that CHK1(pS317) is involved in a MMR-mediated 6-TG-induced G2 arrest, whereas CHK2(pT68) seems to be involved in a subsequent tetraploid G1-S checkpoint. The two signaling kinases seem to work cooperatively to ensure that 6-TG damaged cells arrest at these cell cycle checkpoints.
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Kornepati, Anand V., Yilun Deng, Eloise Dray, Clare Murray, Suresh C. Kari, Erica Osta, Zexuan Liu, et al. "Intracellular PD-L1 regulates DNA damage checkpoints and suppresses Chk1 and PARP inhibitor synthetic lethality." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 67.15. http://dx.doi.org/10.4049/jimmunol.206.supp.67.15.
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Abstract Tumor PD-L1 mediates non-canonical, intracellular signals including the DNA damage response (DDR), of unclear significance and mechanisms. We show that genetic PD-L1 depletion (PD-L1KO) destabilized the Chk2 protein, a DNA damage response (DDR) factor, resulting in ATM/Chk2 pathway defects but not ATR/Chk1 in melanoma, bladder, breast, and ovarian cancer. Consistent with ATM/Chk2 defects, PD-L1 KO led to DNA damage (γH2AX) and impaired homologous recombination (HR) (p-RPA32, BRCA1, Rad51 foci). Thus, PD-L1 KO vs control cells were significantly more sensitive to DDR inhibitors (DDRi) against ATR, Chk1, and PARP in vitro and in vivo in NSG mice. Chk2 regulation by PD-L1 was independent of PD-L1 cytoplasmic tail yet required intracellular (vs surface) PD-L1 suggesting physical PD-L1/Chk2 interaction supported by IP and imaging. PD-L1 stabilized Chk2 protein by preventing its lysosomal degradation without altering Chek2 mRNA. αPD-L1 is thought to work by protecting PD-L1 induced anti-tumor T cell suppression via PD-1, but PD-L1 DDR effects were PD-1-independent. Intracellular PD-L1 suppressed DDRi induced cGAS/STING activation by immunoblots and qRT-PCR of type 1 IFN genes. In vivo in WT mice, genetic PD-L1 depletion but not αPD-L1, sensitized highly immunotherapy resistant 4T1 breast cancer to PARPi. Strikingly, PARPi had reduced effect on PD-L1KO tumors in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi efficacy. Our work implicates a novel role of intracellular PD-L1 in DDR and tumor immunogenicity and identifies related therapeutic vulnerabilities exposed by intracellular PD-L1 targeting. Surface vs intracellular PD-L1, and specific DDR signals could be treatment response biomarkers.
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Rinaldi, Vera D., Jordana C. Bloom, and John C. Schimenti. "Oocyte Elimination Through DNA Damage Signaling from CHK1/CHK2 to p53 and p63." Genetics 215, no. 2 (April 9, 2020): 373–78. http://dx.doi.org/10.1534/genetics.120.303182.
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Eukaryotic organisms have evolved mechanisms to prevent the accumulation of cells bearing genetic aberrations. This is especially crucial for the germline, because fecundity and fitness of progeny would be adversely affected by an excessively high mutational incidence. The process of meiosis poses unique problems for mutation avoidance because of the requirement for SPO11-induced programmed double-strand breaks (DSBs) in recombination-driven pairing and segregation of homologous chromosomes. Mouse meiocytes bearing unrepaired meiotic DSBs or unsynapsed chromosomes are eliminated before completing meiotic prophase I. In previous work, we showed that checkpoint kinase 2 (CHK2; CHEK2), a canonical DNA damage response protein, is crucial for eliminating not only oocytes defective in meiotic DSB repair (e.g., Trip13Gt mutants), but also Spo11−/− oocytes that are defective in homologous chromosome synapsis and accumulate a threshold level of spontaneous DSBs. However, rescue of such oocytes by Chk2 deficiency was incomplete, raising the possibility that a parallel checkpoint pathway(s) exists. Here, we show that mouse oocytes lacking both p53 (TRP53) and the oocyte-exclusive isoform of p63, TAp63, protects nearly all Spo11−/− and Trip13Gt/Gt oocytes from elimination. We present evidence that checkpoint kinase I (CHK1; CHEK1), which is known to signal to TRP53, also becomes activated by persistent DSBs in oocytes, and to an increased degree when CHK2 is absent. The combined data indicate that nearly all oocytes reaching a threshold level of unrepaired DSBs are eliminated by a semiredundant pathway of CHK1/CHK2 signaling to TRP53/TAp63.
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Zachos, George, Michael D. Rainey, and David A. F. Gillespie. "Chk1-Dependent S-M Checkpoint Delay in Vertebrate Cells Is Linked to Maintenance of Viable Replication Structures." Molecular and Cellular Biology 25, no. 2 (January 15, 2005): 563–74. http://dx.doi.org/10.1128/mcb.25.2.563-574.2005.
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ABSTRACT We investigated mitotic delay during replication arrest (the S-M checkpoint) in DT40 B-lymphoma cells deficient in the Chk1 or Chk2 kinase. We show here that cells lacking Chk1, but not those lacking Chk2, enter mitosis with incompletely replicated DNA when DNA synthesis is blocked, but only after an initial delay. This initial delay persists when S-M checkpoint failure is induced in Chk2−/− cells with the Chk1 inhibitor UCN-01, indicating that it does not depend on Chk1 or Chk2 activity. Surprisingly, dephosphorylation of tyrosine 15 did not accompany Cdc2 activation during premature entry to mitosis in Chk1−/− cells, although mitotic phosphorylation of cyclin B2 did occur. Previous studies have shown that Chk1 is required to stabilize stalled replication forks during replication arrest, and strikingly, premature mitosis occurs only in Chk1-deficient cells which have lost the capacity to synthesize DNA as a result of progressive replication fork inactivation. These results suggest that Chk1 maintains the S-M checkpoint indirectly by preserving the viability of replication structures and that it is the continued presence of such structures, rather than the activation of Chk1 per se, which delays mitosis until DNA replication is complete.
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Ou, Yi-Hung, Pei-Han Chung, Te-Ping Sun, and Sheau-Yann Shieh. "p53 C-Terminal Phosphorylation by CHK1 and CHK2 Participates in the Regulation of DNA-Damage-induced C-Terminal Acetylation." Molecular Biology of the Cell 16, no. 4 (April 2005): 1684–95. http://dx.doi.org/10.1091/mbc.e04-08-0689.
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The tumor suppressor protein p53 mediates stress-induced growth arrest or apoptosis and plays a major role in safeguarding genome integrity. In response to DNA damage, p53 can be modified at multiple sites by phosphorylation and acetylation. We report on the characterization of p53 C-terminal phosphorylation by CHK1 and CHK2, two serine/threonine (Ser/Thr) protein kinases, previously implicated in the phosphorylation of the p53 N terminus. Using tryptic phosphopeptide mapping, we have identified six additional CHK1 and CHK2 sites residing in the final 100 amino acids of p53. Phosphorylation of at least three of these sites, Ser366, Ser378, and Thr387, was induced by DNA damage, and the induction at Ser366 and Thr387 was abrogated by small interfering RNA targeting chk1 and chk2. Furthermore, mutation of these phosphorylation sites has a different impact on p53 C-terminal acetylation and on the activation of p53-targeted promoters. Our results demonstrate a possible interplay between p53 C-terminal phosphorylation and acetylation, and they provide an additional mechanism for the control of the activity of p53 by CHK1 and CHK2.
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Martínez-Marchal, Ana, Yan Huang, Maria Teresa Guillot-Ferriols, Mònica Ferrer-Roda, Anna Guixé, Montserrat Garcia-Caldés, and Ignasi Roig. "The DNA damage response is required for oocyte cyst breakdown and follicle formation in mice." PLOS Genetics 16, no. 11 (November 18, 2020): e1009067. http://dx.doi.org/10.1371/journal.pgen.1009067.
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Mammalian oogonia proliferate without completing cytokinesis, forming cysts. Within these, oocytes differentiate and initiate meiosis, promoting double-strand break (DSBs) formation, which are repaired by homologous recombination (HR) causing the pairing and synapsis of the homologs. Errors in these processes activate checkpoint mechanisms, leading to apoptosis. At the end of prophase I, in contrast with what is observed in spermatocytes, oocytes accumulate unrepaired DSBs. Simultaneously to the cyst breakdown, there is a massive oocyte death, which has been proposed to be necessary to enable the individualization of the oocytes to form follicles. Based upon all the above-mentioned information, we hypothesize that the apparently inefficient HR occurring in the oocytes may be a requirement to first eliminate most of the oocytes and enable cyst breakdown and follicle formation. To test this idea, we compared perinatal ovaries from control and mutant mice for the effector kinase of the DNA Damage Response (DDR), CHK2. We found that CHK2 is required to eliminate ~50% of the fetal oocyte population. Nevertheless, the number of oocytes and follicles found in Chk2-mutant ovaries three days after birth was equivalent to that of the controls. These data revealed the existence of another mechanism capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2-/- mice, implying that CHK1 regulates postnatal oocyte death. Moreover, we found that CHK1 and CHK2 functions are required for the timely breakdown of the cyst and to form follicles. Thus, we uncovered a novel CHK1 function in regulating the oocyte population in mice. Based upon these data, we propose that the CHK1- and CHK2-dependent DDR controls the number of oocytes and is required to properly break down oocyte cysts and form follicles in mammals.
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Feijoo, Carmen, Clare Hall-Jackson, Rong Wu, David Jenkins, Jane Leitch, David M. Gilbert, and Carl Smythe. "Activation of mammalian Chk1 during DNA replication arrest." Journal of Cell Biology 154, no. 5 (September 3, 2001): 913–24. http://dx.doi.org/10.1083/jcb.200104099.
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Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block–induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.
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Tsoi, Ho, Wai-Chung Tsang, Ellen P. S. Man, Man-Hong Leung, Chan-Ping You, Sum-Yin Chan, Wing-Lok Chan, and Ui-Soon Khoo. "Checkpoint Kinase 2 Inhibition Can Reverse Tamoxifen Resistance in ER-Positive Breast Cancer." International Journal of Molecular Sciences 23, no. 20 (October 14, 2022): 12290. http://dx.doi.org/10.3390/ijms232012290.
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Breast cancer is a heterogeneous disease. Tamoxifen is frequently used to treat ER-positive breast cancer. Our team has identified a novel splice variant of NCOR2, BQ323636.1 (BQ), that mediates tamoxifen resistance. However, the upstream factors that modulate BQ expression are not apparent. This study reveals that tamoxifen treatment causes induction of DNA damage which can enhance BQ expression. We show that DNA damage can activate the ATM/CHK2 and ATR/CHK1 signalling cascades and confirm that ATM/CHK2 signalling is responsible for enhancing the protein stability of BQ. siRNA or a small inhibitor targeting CHK2 resulted in the reduction in BQ expression through reduced phosphorylation and enhanced poly-ubiquitination of BQ. Inhibition of CHK2 by CCT241533 could reverse tamoxifen resistance in vitro and in vivo. Using clinical samples in the tissue microarray, we confirmed that high p-CHK2 expression was significantly associated with high nuclear BQ expression, tamoxifen resistance and poorer overall and disease-specific survival. In conclusion, tamoxifen treatment can enhance BQ expression in ER-positive breast cancer by activating the ATM/CHK2 axis. Targeting CHK2 is a promising approach to overcoming tamoxifen resistance in ER-positive breast cancer.
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Kim, Hyeon Ho, Kotb Abdelmohsen, and Myriam Gorospe. "Regulation of HuR by DNA Damage Response Kinases." Journal of Nucleic Acids 2010 (2010): 1–8. http://dx.doi.org/10.4061/2010/981487.
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As many DNA-damaging conditions repress transcription, posttranscriptional processes critically influence gene expression during the genotoxic stress response. The RNA-binding protein HuR robustly influences gene expression following DNA damage. HuR function is controlled in two principal ways: (1) by mobilizing HuR from the nucleus to the cytoplasm, where it modulates the stability and translation of target mRNAs and (2) by altering its association with target mRNAs. Here, we review evidence that two main effectors of ataxia-telangiectasia-mutated/ATM- and Rad3-related (ATM/ATR), the checkpoint kinases Chk1 and Chk2, jointly influence HuR function. Chk1 affects HuR localization by phosphorylating (hence inactivating) Cdk1, a kinase that phosphorylates HuR and thereby blocks HuR's cytoplasmic export. Chk2 modulates HuR binding to target mRNAs by phosphorylating HuR's RNA-recognition motifs (RRM1 and RRM2). We discuss how HuR phosphorylation by kinases including Chk1/Cdk1 and Chk2 impacts upon gene expression patterns, cell proliferation, and survival following genotoxic injury.
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Wang, Zan-Ying, Wen-Qiong Liu, Si’e Wang, and Zeng-Tao Wei. "Fisetin induces G2/M phase cell cycle arrest by inactivating cdc25C-cdc2 via ATM-Chk1/2 activation in human endometrial cancer cells." Bangladesh Journal of Pharmacology 10, no. 2 (April 3, 2015): 279. http://dx.doi.org/10.3329/bjp.v10i2.21945.
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<p>Endometrial cancer is one of the most prevalent gynaecological malignancies where, currently available therapeutic options remain limited. Recently phytochemicals are exploited for their efficiency in cancer therapy. The present study investigates the anti-proliferative effect of fisetin, a flavonoid on human endometrial cancer cells (KLE and Hec1 A). Fisetin (20-100 µM) effectively reduced the viability of Hec1 A and KLE cells and potentially altered the cell population at G2/M stage. Expression levels of the cell cycle proteins (cyclin B1, p-Cdc2, p-Cdc25C, p-Chk1, Chk2, p-ATM, cyclin B1, H2AX, p21 and p27) were analyzed. Fisetin suppressed cyclin B1 expression and caused inactiva-tion of Cdc25C and Cdc2 by increasing their phosphorylation levels and further activated ATM, Chk1 and Chk2. Increased levels of p21 and p27 were observed as well. These results suggest that fisetin induced G2/M cell cycle arrest via inactivating Cdc25c and Cdc2 through activation of ATM, Chk1 and Chk2.</p><p> </p><p> </p>
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Stolarova, Lenka, Petra Kleiblova, Marketa Janatova, Jana Soukupova, Petra Zemankova, Libor Macurek, and Zdenek Kleibl. "CHEK2 Germline Variants in Cancer Predisposition: Stalemate Rather than Checkmate." Cells 9, no. 12 (December 12, 2020): 2675. http://dx.doi.org/10.3390/cells9122675.
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Germline alterations in many genes coding for proteins regulating DNA repair and DNA damage response (DDR) to DNA double-strand breaks (DDSB) have been recognized as pathogenic factors in hereditary cancer predisposition. The ATM-CHEK2-p53 axis has been documented as a backbone for DDR and hypothesized as a barrier against cancer initiation. However, although CHK2 kinase coded by the CHEK2 gene expedites the DDR signal, its function in activation of p53-dependent cell cycle arrest is dispensable. CHEK2 mutations rank among the most frequent germline alterations revealed by germline genetic testing for various hereditary cancer predispositions, but their interpretation is not trivial. From the perspective of interpretation of germline CHEK2 variants, we review the current knowledge related to the structure of the CHEK2 gene, the function of CHK2 kinase, and the clinical significance of CHEK2 germline mutations in patients with hereditary breast, prostate, kidney, thyroid, and colon cancers.
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Adamson, Aaron W., Dillon I. Beardsley, Wan-Ju Kim, Yajuan Gao, R. Baskaran, and Kevin D. Brown. "Methylator-induced, Mismatch Repair-dependent G2 Arrest Is Activated through Chk1 and Chk2." Molecular Biology of the Cell 16, no. 3 (March 2005): 1513–26. http://dx.doi.org/10.1091/mbc.e04-02-0089.
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SN1 DNA methylating agents such as the nitrosourea N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) elicit a G2/M checkpoint response via a mismatch repair (MMR) system-dependent mechanism; however, the exact nature of the mechanism governing MNNG-induced G2/M arrest and how MMR mechanistically participates in this process are unknown. Here, we show that MNNG exposure results in activation of the cell cycle checkpoint kinases ATM, Chk1, and Chk2, each of which has been implicated in the triggering of the G2/M checkpoint response. We document that MNNG induces a robust, dose-dependent G2 arrest in MMR and ATM-proficient cells, whereas this response is abrogated in MMR-deficient cells and attenuated in ATM-deficient cells treated with moderate doses of MNNG. Pharmacological and RNA interference approaches indicated that Chk1 and Chk2 are both required components for normal MNNG-induced G2 arrest. MNNG-induced nuclear exclusion of the cell cycle regulatory phosphatase Cdc25C occurred in an MMR-dependent manner and was compromised in cells lacking ATM. Finally, both Chk1 and Chk2 interact with the MMR protein MSH2, and this interaction is enhanced after MNNG exposure, supporting the notion that the MMR system functions as a molecular scaffold at the sites of DNA damage that facilitates activation of these kinases.
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Hines, Stephanie L., Ahmed N. Mohammad, Jessica Jackson, Sarah Macklin, and Thomas R. Caulfield. "Integrative data fusion for comprehensive assessment of a novel CHEK2 variant using combined genomics, imaging, and functional–structural assessments via protein informatics." Molecular Omics 15, no. 1 (2019): 59–66. http://dx.doi.org/10.1039/c8mo00137e.
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Boonen, Rick A. C. M., Wouter W. Wiegant, Nandi Celosse, Bas Vroling, Stephan Heijl, Zsofia Kote-Jarai, Martina Mijuskovic, et al. "Functional Analysis Identifies Damaging CHEK2 Missense Variants Associated with Increased Cancer Risk." Cancer Research 82, no. 4 (December 13, 2021): 615–31. http://dx.doi.org/10.1158/0008-5472.can-21-1845.
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Abstract Heterozygous carriers of germline loss-of-function variants in the tumor suppressor gene checkpoint kinase 2 (CHEK2) are at an increased risk for developing breast and other cancers. While truncating variants in CHEK2 are known to be pathogenic, the interpretation of missense variants of uncertain significance (VUS) is challenging. Consequently, many VUS remain unclassified both functionally and clinically. Here we describe a mouse embryonic stem (mES) cell–based system to quantitatively determine the functional impact of 50 missense VUS in human CHEK2. By assessing the activity of human CHK2 to phosphorylate one of its main targets, Kap1, in Chek2 knockout mES cells, 31 missense VUS in CHEK2 were found to impair protein function to a similar extent as truncating variants, while 9 CHEK2 missense VUS resulted in intermediate functional defects. Mechanistically, most VUS impaired CHK2 kinase function by causing protein instability or by impairing activation through (auto)phosphorylation. Quantitative results showed that the degree of CHK2 kinase dysfunction correlates with an increased risk for breast cancer. Both damaging CHEK2 variants as a group [OR 2.23; 95% confidence interval (CI), 1.62–3.07; P < 0.0001] and intermediate variants (OR 1.63; 95% CI, 1.21–2.20; P = 0.0014) were associated with an increased breast cancer risk, while functional variants did not show this association (OR 1.13; 95% CI, 0.87–1.46; P = 0.378). Finally, a damaging VUS in CHEK2, c.486A>G/p.D162G, was also identified, which cosegregated with familial prostate cancer. Altogether, these functional assays efficiently and reliably identified VUS in CHEK2 that associate with cancer. Significance: Quantitative assessment of the functional consequences of CHEK2 variants of uncertain significance identifies damaging variants associated with increased cancer risk, which may aid in the clinical management of patients and carriers.
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Dove, Alan W. "Chk1, Chk2, is the amplifier on?" Journal of Cell Biology 154, no. 5 (September 3, 2001): 903. http://dx.doi.org/10.1083/jcb1545iti3.
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Gottifredi, Vanesa, Orit Karni-Schmidt, Sheau-Yann Shieh, and Carol Prives. "p53 Down-Regulates CHK1 through p21 and the Retinoblastoma Protein." Molecular and Cellular Biology 21, no. 4 (February 15, 2001): 1066–76. http://dx.doi.org/10.1128/mcb.21.4.1066-1076.2001.
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ABSTRACT Both fission yeast and mammalian cells require the function of the checkpoint kinase CHK1 for G2 arrest after DNA damage. The tumor suppressor p53, a well-studied stress response factor, has also been shown to play a role in DNA damage G2 arrest, although in a manner that is probably independent of CHK1. p53, however, can be phosphorylated and regulated by both CHK1 as well as another checkpoint kinase, hCds1 (also called CHK2). It was therefore of interest to determine whether reciprocally, p53 affects either CHK1 or CHK2. We found that induction of p53 either by diverse stress signals or ectopically using a tetracycline-regulated promoter causes a marked reduction in CHK1 protein levels. CHK1 downregulation by p53 occurs as a result of reduced CHK1 RNA accumulation, indicating that repression occurs at the level of transcription. Repression of CHK1 by p53 requires p21, since p21 alone is sufficient for this to occur and cells lacking p21 cannot downregulate CHK1. Interestingly, pRB is also required for CHK1 downregulation, suggesting the possible involvement of E2F-dependent transcription in the regulation of CHK1. Our results identify a new repression target of p53 and suggest that p53 and CHK1 play interdependent and complementary roles in regulating both the arrest and resumption of G2 after DNA damage.
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Xu, Xingzhi, Lyuben M. Tsvetkov, and David F. Stern. "Chk2 Activation and Phosphorylation-Dependent Oligomerization." Molecular and Cellular Biology 22, no. 12 (June 15, 2002): 4419–32. http://dx.doi.org/10.1128/mcb.22.12.4419-4432.2002.
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ABSTRACT The tumor suppressor gene CHK2 encodes a versatile effector serine/threonine kinase involved in responses to DNA damage. Chk2 has an amino-terminal SQ/TQ cluster domain (SCD), followed by a forkhead-associated (FHA) domain and a carboxyl-terminal kinase catalytic domain. Mutations in the SCD or FHA domain impair Chk2 checkpoint function. We show here that autophosphorylation of Chk2 produced in a cell-free system requires trans phosphorylation by a wortmannin-sensitive kinase, probably ATM or ATR. Both SQ/TQ sites and non-SQ/TQ sites within the Chk2 SCD can be phosphorylated by active Chk2. Amino acid substitutions in the SCD and the FHA domain impair auto- and trans-kinase activities of Chk2. Chk2 forms oligomers that minimally require the FHA domain of one Chk2 molecule and the SCD within another Chk2 molecule. Chk2 oligomerization in vivo increases after DNA damage, and when damage is induced by gamma irradiation, this increase requires ATM. Chk2 oligomerization is phosphorylation dependent and can occur in the absence of other eukaryotic proteins. Chk2 can cross-phosphorylate another Chk2 molecule in an oligomeric complex. Induced oligomerization of a Chk2 chimera in vivo concomitant with limited DNA damage augments Chk2 kinase activity. These results suggest that Chk2 oligomerization regulates Chk2 activation, signal amplification, and transduction in DNA damage checkpoint pathways.
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Zhao, Wenjing, Shaobo Chen, Xianming Hou, Ge Chen, and Yupei Zhao. "CHK2 Promotes Anoikis and is Associated with the Progression of Papillary Thyroid Cancer." Cellular Physiology and Biochemistry 45, no. 4 (2018): 1590–602. http://dx.doi.org/10.1159/000487724.
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Background/Aims: Cell cycle checkpoint kinase 2 (CHK2) performs essential cellular functions and might be associated with tumorigenesis and tumor progression. Here, we explored the function and molecular mechanisms of CHK2 in the progression of papillary thyroid cancer (PTC). Methods: The expression levels of both total CHK2 and activated CHK2 (p-CHK2) in tissues from 100 PTC patients were detected and evaluated using immunohistochemistry. The roles of CHK2 on cell proliferation, invasion, migration, apoptosis and cancer stem cell (CSC) markers were investigated by CCK-8, Transwell, flow cytometry, western blot and ALDEFLOUR assay. PTC cells cultured in suspension conditions were assayed for anoikis. The anchorage-independent condition was further detected by soft agar colony formation assay. Furthermore, anoikis associated regulatory proteins were explored by western blot and verified by forced downregulation experiment, respectively. Results: We found that the levels of both CHK2 and p-CHK2 were significantly upregulated in PTC cancer tissues compared with those in tumor-adjacent tissues. The overexpression of p-CHK2 in primary tumor tissues was associated with tumor aggressiveness and metastatic potential. However, the levels of both CHK2 and p-CHK2 were decreased in metastatic lymph nodes. Our results showed that CHK2 upregulated the levels of CSC markers with no effect on cell proliferation, invasion and migration. Interestingly, we revealed a previously undescribed anoikis-promoting role for CHK2 in PTC. Specifically, the detachment of PTC cells from the extracellular matrix (ECM) triggers CHK2 degradation. Then, the forced downregulation of CHK2 rescued PTC cells from anoikis, but no effect was observed on the apoptosis of adherent PTC cells. Additionally, as a novel regulator of anoikis, CHK2 can induce cell death in a p53-independent manner via the regulation of PRAS40 activation. Conclusion: High expression levels of CHK2 and p-CHK2 were associated with the progression of PTC. Our results defined an unexpected role for CHK2 as a mediator of anoikis that functions through the regulation of PRAS40 activation, which may be associated with the survival of circulating tumor cells and metastatic behavior.
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Kawabe, Takumi. "G2 checkpoint abrogators as anticancer drugs." Molecular Cancer Therapeutics 3, no. 4 (April 1, 2004): 513–19. http://dx.doi.org/10.1158/1535-7163.513.3.4.
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Abstract Many conventional anticancer treatments kill cells irrespective of whether they are normal or cancerous, so patients suffer from adverse side effects due to the loss of healthy cells. Anticancer insights derived from cell cycle research has given birth to the idea of cell cycle G2 checkpoint abrogation as a cancer cell specific therapy, based on the discovery that many cancer cells have a defective G1 checkpoint resulting in a dependence on the G2 checkpoint during cell replication. Damaged DNA in humans is detected by sensor proteins (such as hHUS1, hRAD1, hRAD9, hRAD17, and hRAD26) that transmit a signal via ATR to CHK1, or by another sensor complex (that may include γH2AX, 53BP1, BRCA1, NBS1, hMRE11, and hRAD50), the signal of which is relayed by ATM to CHK2. Most of the damage signals originated by the sensor complexes for the G2 checkpoint are conducted to CDC25C, the activity of which is modulated by 14-3-3. There are also less extensively explored pathways involving p53, p38, PCNA, HDAC, PP2A, PLK1, WEE1, CDC25B, and CDC25A. This review will examine the available inhibitors of CHK1 (Staurosporin, UCN-01, Go6976, SB-218078, ICP-1, and CEP-3891), both CHK1 and CHK2 (TAT-S216A and debromohymenialdisine), CHK2 (CEP-6367), WEE1 (PD0166285), and PP2A (okadaic acid and fostriecin), as well as the unknown checkpoint inhibitors 13-hydroxy-15-ozoapathin and the isogranulatimides. Among these targets, CHK1 seems to be the most suitable target for therapeutic G2 abrogation to date, although an unexplored target such as 14-3-3 or the strategy of targeting multiple proteins at once may be of interest in the future.
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Lovly, Christine M., Ling Yan, Christine E. Ryan, Saeko Takada, and Helen Piwnica-Worms. "Regulation of Chk2 Ubiquitination and Signaling through Autophosphorylation of Serine 379." Molecular and Cellular Biology 28, no. 19 (July 21, 2008): 5874–85. http://dx.doi.org/10.1128/mcb.00821-08.
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ABSTRACT The Chk2 protein kinase protects genome integrity by promoting cell cycle arrest or apoptosis in response to DNA double-strand breaks, and Chk2 mutations are found in both familial and sporadic cancers. Exposure of cells to ionizing radiation (IR) or radiomimetic drugs induces Chk2 phosphorylation by ATM, followed by Chk2 oligomerization, auto-/transphosphorylation, and activation. Here we demonstrate that Chk2 is ubiquitinated upon activation and that this requires Chk2 kinase activity. Serine 379 (S379) was identified as a novel IR-inducible autophosphorylation site required for ubiquitination of Chk2 by a Cullin 1-containing E3 ligase complex. Importantly, S379 was required for Chk2 to induce apoptosis in cells with DNA double-strand breaks. Thus, auto-/transphosphorylation of S379 is required for Chk2 ubiquitination and effector function.
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Hirao, Atsushi, Alison Cheung, Gordon Duncan, Pierre-Marie Girard, Andrew J. Elia, Andrew Wakeham, Hitoshi Okada, et al. "Chk2 Is a Tumor Suppressor That Regulates Apoptosis in both an Ataxia Telangiectasia Mutated (ATM)-Dependent and an ATM-Independent Manner." Molecular and Cellular Biology 22, no. 18 (September 15, 2002): 6521–32. http://dx.doi.org/10.1128/mcb.22.18.6521-6532.2002.
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ABSTRACT In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM−/− and p53−/− mice, Chk2−/− mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2−/− mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G1/S arrest. Quantitative comparison of the G1/S checkpoint, apoptosis, and expression of p53 proteins in Chk2−/− versus ATM−/− thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2−/− thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3 + related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.
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Anamika, Dhyani, Patricia Favaro, and Sara Teresinha Olalla Saad. "ANKHD1 Silencing Delays S Phase Progression in Multiple Myeloma Cells Via Activation of ATM/ATR -CDC25a Pathway." Blood 128, no. 22 (December 2, 2016): 5624. http://dx.doi.org/10.1182/blood.v128.22.5624.5624.
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Abstract Ankyrin repeat and KH domain-containing protein 1, ANKHD1, is highly expressed in myeloma cells and plays an important role in multiple myeloma (MM) progression and growth. ANKHD1 is found to be overexpressed in S phase of cell cycle in MM cells and silencing of ANKHD1 expression leads to accumulation of cells in S phase, suggesting a role in S phase progression (1). Earlier studies by our group reported that ANKHD1 silencing downregulates all replication dependent histones and that this downregulation may be associated with replication stress and DNA damage (2). We observed increased expression of γH2AX protein (phosphorylated histone H2A variant, H2AX, at Serine 139), a marker for DNA double strand breaks (DSBs) and an early sign of DNA damage induced by replication stress, in ANKHD1 silenced MM cells. In the present study we further sought to investigate the mechanisms underlying the induction of DNA damage on ANKHD1 silencing. We first confirmed the increased expression of γH2AX by flow cytometry analysis and observed that both the mean fluorescence intensity as well as percentage of γH2AX positive cells were higher in ANKHD1 silenced MM cells as compared to control cells. Phosphorylation of histone 2AX requires activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated)andATR (ATM-Rad3-related) that serves as central components of the signaling cascade initiated by DSBs. Hence, we checked for the expression of these kinases and observed increased phosphorylation of both ATM and ATR kinases in ANKHD1 silenced MM cells. There was no difference in the expressions of DNA-PKcs in control and ANKHD1 silenced cells by western blot. We next checked for the expression of CHK1 (checkpoint kinase 1) and CHK2 (checkpoint kinase 2), essential serine threonine kinases downstream of ATM and ATR. We observed a decrease in pCHK2 (phosphorylated CHK2 at Thr 68), with no change in expression of pCHK1 (phosphorylated CHK1 at Ser 345) total CHK1 or total CHK2. We also checked for expression of CDC25a (a member of the CDC25 family of dual-specificity phosphatases), that is specifically degraded in response to DNA damage (DSBs) and delays S phase progression via activation of ATM /ATR-CHK2 signaling pathway. Expression of CDC25a was significantly decreased in ANKHD1 silencing cells, confirming the induction of DSBs, and probably accounting for S phase delay on ANKHD1 silencing. Since there was decrease in active CHK2 (pCHK2) and no change in CHK1 required for degradation of CDC25a, we assume that decrease in CDC25a in ANKHD1 silenced MM cells may be via activation of ATM/ ATR pathway independent of CHK2/CHK1. Expression of several other downstream factors of DSBs induced DNA damage response and repair such as BRCA1, PTEN, DNMT1, SP1, HDAC2 were also found to be modulated in ANKHD1 silenced MM cells. In conclusion, ANKHD1 silencing in MM cells leads to DNA damage and modulates expression of several genes implicated in DNA damage and repair. DNA damage induced after ANKHD1 silencing in MM cells activates ATM/ ATR-CDC25a pathway which may lead to the activation of S phase checkpoint in MM cells. Results however are preliminary and further studies are required to understand the role of ANKHD1 in intra S phase check point. References: 1) ANKHD1 regulates cell cycle progression and proliferation in multiple myeloma cells. Dhyani et al. FEBS letters 2012; 586: 4311-18. 2) ANKHD1 is essential for repair of DNA double strand breaks in multiple myeloma. Dhyani et al. ASH Abstract, Blood 2015; 126:1762. Disclosures No relevant conflicts of interest to declare.
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Shibata, Atsushi, Olivia Barton, Angela T. Noon, Kirsten Dahm, Dorothee Deckbar, Aaron A. Goodarzi, Markus Löbrich, and Penny A. Jeggo. "Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G2/M Checkpoint Arrest." Molecular and Cellular Biology 30, no. 13 (April 26, 2010): 3371–83. http://dx.doi.org/10.1128/mcb.01644-09.
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ABSTRACT ATM-dependent initiation of the radiation-induced G2/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G2 phase are repaired by DNA nonhomologous end joining (NHEJ), while ∼15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G2/M checkpoint is maintained in irradiated G2 cells, in light of our current understanding of G2 phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1 −/− and MDC1 −/− cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.
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Macari, Elizabeth R., Alison Taylor, David Raiser, Kavitha Siva, Katherine McGrath, Jessica M. Humphries, Johan Flygare, Benjamin L. Ebert, and Leonard I. Zon. "Calmodulin Inhibition Rescues the Effects of Ribosomal Protein Deficiency in in Vitro and In Vivo Diamond Blackfan Anemia Models." Blood 126, no. 23 (December 3, 2015): 672. http://dx.doi.org/10.1182/blood.v126.23.672.672.
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Abstract Ribosomal protein (RP) mutations are found in many diseases, including Diamond Blackfan anemia (DBA), where defective erythropoiesis, craniofacial abnormalities and increased cancer risk are major complications. RP mutations cause p53 activation through accumulation of free RPs that bind and sequester MDM2, the negative regulator of p53. We previously characterized a zebrafish mutant in rps29, a gene found mutated in DBA patients. Rps29-/- embryos have hematopoietic and endothelial defects, including decreased cmyb and flk1 expression and defects in hemoglobinization. Consistent with other animal models of RP dysfunction, p53 knockdown in rps29-/- embryos rescued these defects. To uncover novel compounds that correct the phenotypes of DBA, we performed a chemical screen in rps29-/- embryos. Several structurally distinct calmodulin (CaM) inhibitors successfully rescued hemoglobin (Hb) levels in the mutant embryo. To confirm that CaM inhibitors could rescue mammalian models of DBA, we applied them to human and murine models. Treating cord blood-derived CD34+ cells deficient in RPS19 with the CaM inhibitor, trifluoperazine (TFP), relieved the erythroid differentiation block. Injection of TFP in a DBA murine model significantly increased red blood cell number and Hb levels. Mechanistic studies in A549 cells infected with lentivirus expressing RPS19 shRNA demonstrated that TFP blocks p53 nuclear accumulation and induction of multiple p53 transcriptional target genes (p<0.05). Through p53 genetic manipulation, we determined that TFP inhibits p53 transcriptional activity through its c-terminal domain (CTD). Since this region has many residues that can be phosphorylated by CaM-dependent kinases, we hypothesized that TFP blocked phosphorylation of residues in the CTD. To test this hypothesis, phosphomimetic mutants were transfected into Saos2 cells and p53 transcriptional activity in response to TFP was evaluated using p21mRNA levels. TFP treatment of cells containing WT p53 or a transactivation domain mutant, S15D, resulted in a 4-fold reduction in p21 mRNA levels, while all four phosphomimetic mutants in the CTD had attenuated responses to TFP (<2-fold). The common CaM-dependent kinases that phosphorylate these CTD residues are Chk1 and Chk2. Investigation of the role of Chk1 and Chk2 found that a chk2 morpholino and multiple inhibitors of Chk2, but not Chk1, rescued Hb levels in the rps29-/- embryo (p<0.05). Chk2 inhibitors also mimic CaM inhibition in our in vitro assays. In conclusion, we have shown a novel mechanism by which CaM inhibitors mediate p53 activity through the CTD and can rescue the phenotypes of multiple in vitro and in vivo models of DBA. Our data strongly suggests that CaM or Chk2 inhibitors may be effective therapies for DBA patients, and a clinical trial is being planned with TFP. Disclosures Ebert: Genoptix: Consultancy, Patents & Royalties; H3 Biomedicine: Consultancy; Celgene: Consultancy. Zon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.
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Chehab, Nabil H., Asra Malikzay, Michael Appel, and Thanos D. Halazonetis. "Chk2/hCds1 functions as a DNA damage checkpoint in G1 by stabilizing p53." Genes & Development 14, no. 3 (February 1, 2000): 278–88. http://dx.doi.org/10.1101/gad.14.3.278.
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Chk2/hcds1, the human homolog of theSaccharomyces cerevisiae RAD53/SPK1 andSchizosaccharomyces pombe cds1 DNA damage checkpoint genes, encodes a protein kinase that is post-translationally modified after DNA damage. Like its yeast homologs, the Chk2/hCds1 protein phosphorylates Cdc25C in vitro, suggesting that it arrests cells in G2 in response to DNA damage. We expressed Chk2/hCds1 in human cells and analyzed their cell cycle profile. Wild-type, but not catalytically inactive, Chk2/hCds1 led to G1 arrest after DNA damage. The arrest was inhibited by cotransfection of a dominant-negative p53 mutant, indicating that Chk2/hCds1 acted upstream of p53. In vitro, Chk2/hCds1 phosphorylated p53 on Ser-20 and dissociated preformed complexes of p53 with Mdm2, a protein that targets p53 for degradation. In vivo, ectopic expression of wild-type Chk2/hCds1 led to increased p53 stabilization after DNA damage, whereas expression of a dominant-negative Chk2/hCds1 mutant abrogated both phosphorylation of p53 on Ser-20 and p53 stabilization. Thus, in response to DNA damage, Chk2/hCds1 stabilizes the p53 tumor suppressor protein leading to cell cycle arrest in G1.
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Carloni, Vinicio, Matteo Lulli, Stefania Madiai, Tommaso Mello, Andrew Hall, Tu Vinh Luong, Massimo Pinzani, Krista Rombouts, and Andrea Galli. "CHK2 overexpression and mislocalisation within mitotic structures enhances chromosomal instability and hepatocellular carcinoma progression." Gut 67, no. 2 (March 30, 2017): 348–61. http://dx.doi.org/10.1136/gutjnl-2016-313114.
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ObjectiveChromosomal instability (CIN) is the most common form of genomic instability, which promotes hepatocellular carcinoma (HCC) progression by enhancing tumour heterogeneity, drug resistance and immunity escape. CIN per se is an important factor of DNA damage, sustaining structural chromosome abnormalities but the underlying mechanisms are unknown.DesignDNA damage response protein checkpoint kinase 2 (Chk2) expression was evaluated in an animal model of diethylnitrosamine-induced HCC characterised by DNA damage and elevated mitotic errors. Chk2 was also determined in two discrete cohorts of human HCC specimens. To assess the functional role of Chk2, gain on and loss-of-function, mutagenesis, karyotyping and immunofluorescence/live imaging were performed by using HCT116, Huh7 and human hepatocytes immortalised with hTERT gene (HuS).ResultsWe demonstrate that mitotic errors during HCC tumorigenesis cause lagging chromosomes/DNA damage and activation of Chk2. Overexpression/phosphorylation and mislocalisation within the mitotic spindle of Chk2 contributes to induce lagging chromosomes. Lagging chromosomes and mitotic activity are reversed by knockdown of Chk2. Furthermore, upregulated Chk2 maintains mitotic activity interacting with Aurora B kinase for chromosome condensation and cytokinesis. The forkhead-associated domain of Chk2 is required for Chk2 mislocalisation to mitotic structures. In addition, retinoblastoma protein phosphorylation contributes to defective mitoses. A cohort and independent validation cohort show a strong cytoplasm to nuclear Chk2 translocation in a subset of patients with HCC.ConclusionsThe study reveals a new mechanistic insight in the coinvolvement of Chk2 in HCC progression. These findings propose Chk2 as a putative biomarker to detect CIN in HCC providing a valuable support for clinical/therapeutical management of patients.
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Ohba, Shigeo, Tor-Christian Johannessen, Kamalakar Chatla, Xiaodong Yang, Yuichi Hirose, Russell Pieper, and Joydeep Mukherjee. "CBMS-02 Phosphoglycerate mutase 1 (PGAM1) controls DNA damage repair via regulation of WIP1 activity." Neuro-Oncology Advances 2, Supplement_3 (November 1, 2020): ii4. http://dx.doi.org/10.1093/noajnl/vdaa143.014.
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Abstract Phosphoglycerate Mutase 1 (PGAM1) is overexpressed in different forms of cancer and has been suggested to have additional functions beyond its role in metabolism. We here report that PGAM1 is overexpressed in GBMs and indirectly regulates activation of ATM, Chk1 and Chk2 but not ATR, thereby increasing the efficiency of DNA damage repair and resistance to radiation (IR) and temozolomide (TMZ) treatment. Genetic suppression of PGAM1 in multiple GBM cell lines resulted in decrease proliferation, apoptosis and colony formation after radiation and temozolomide treatment compared to parental cells. Moreover, parental cells demonstrated DNA damage (gH2AX foci) whereas isogenic PGAM1 knockdown cells exhibited no DNA damage repair activation and a significant increase in sub-G0 apoptotic cells that expressed annexin-V, cleaved caspase-3 and cleaved PARP-1. Mechanistically, suppression of PGAM1 expression inhibited phosphorylation of ATM at s1981 and the subsequent downstream phosphorylation of Chk2 and cdc25C. Moreover, PGAM1 co-immunoprecipitated with WIP1, a phosphatase reported to bind and dephosphorylate ATM, Chk1, and Chk2. Cytoplasmic binding of WIP1 with PGAM1 prevented nuclear localization of WIP1, leaving ATM and its downstream substrates phosphorylated, which is required for DNA damage repair activity. Consistent with these observations, mice intracranially implanted with PGAM1 knockdown GBM cells and treated with TMZ and IR had longer survival than similarly treated mice implanted with matched control cells. These results therefore define PGAM1 as an activator of DNA damage repair pathway and link tumor metabolism to drug response in GBM.
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Guo, Ran, Shan-Shan Wang, Xiao-You Jiang, Ye Zhang, Yang Guo, Hong-Yan Cui, Qi-Qiang Guo, Liu Cao, and Xiao-Chen Xie. "CHK2 Promotes Metabolic Stress-Induced Autophagy through ULK1 Phosphorylation." Antioxidants 11, no. 6 (June 14, 2022): 1166. http://dx.doi.org/10.3390/antiox11061166.
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Reactive oxygen species (ROS) act as a signaling intermediate to promote cellular adaptation to maintain homeostasis by regulating autophagy during pathophysiological stress. However, the mechanism by which ROS promotes autophagy is still largely unknown. Here, we show that the ATM/CHK2/ULK1 axis initiates autophagy to maintain cellular homeostasis by sensing ROS signaling under metabolic stress. We report that ULK1 is a physiological substrate of CHK2, and that the binding of CHK2 to ULK1 depends on the ROS signal and the phosphorylation of threonine 68 of CHK2 under metabolic stress. Further, CHK2 phosphorylates ULK1 on serine 556, and this phosphorylation is dependent on the ATM/CHK2 signaling pathway. CHK2-mediated phosphorylation of ULK1 promotes autophagic flux and inhibits apoptosis induced by metabolic stress. Taken together, these results demonstrate that the ATM/CHK2/ULK1 axis initiates an autophagic adaptive response by sensing ROS, and it protects cells from metabolic stress-induced cellular damage.
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Stolz, Ailine, Norman Ertych, and Holger Bastians. "Loss of the tumour-suppressor genes CHK2 and BRCA1 results in chromosomal instability." Biochemical Society Transactions 38, no. 6 (November 24, 2010): 1704–8. http://dx.doi.org/10.1042/bst0381704.
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CHK2 (checkpoint kinase 2) and BRCA1 (breast cancer early-onset 1) are tumour-suppressor genes that have been implicated previously in the DNA damage response. Recently, we have identified CHK2 and BRCA1 as genes required for the maintenance of chromosomal stability and have shown that a Chk2-mediated phosphorylation of Brca1 is required for the proper and timely assembly of mitotic spindles. Loss of CHK2, BRCA1 or inhibition of its Chk2-mediated phosphorylation inevitably results in the transient formation of abnormal spindles that facilitate the establishment of faulty microtubule–kinetochore attachments associated with the generation of lagging chromosomes. Importantly, both CHK2 and BRCA1 are lost at very high frequency in aneuploid lung adenocarcinomas that are typically induced in knockout mice exhibiting chromosomal instability. Thus these results suggest novel roles for Chk2 and Brca1 in mitosis that might contribute to their tumour-suppressor functions.
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Ghelli Luserna Di Rorà, Andrea, Antonella Padella, Maria Chiara Fontana, Eugenio Fonzi, Anna Ferrari, Enrica Imbrogno, Martina Ghetti, et al. "The Prolonged Inhibition of Chk1/Chk2 Kinases Enhances Genetic Instability and Compromises the Efficacy of Chemotherapy Against Acute Lymphoblastic Leukemia Cells." Blood 134, Supplement_1 (November 13, 2019): 5047. http://dx.doi.org/10.1182/blood-2019-129964.
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Acute lymphoblastic leukemia (ALL) cells respond to chemotherapy, or more generally to DNA damages, by activating different DNA Damage Response (DDR) pathways. DDR-pathways regulate cell cycle progression and DNA damages repair. Molecular and functional alterations in key DDR-related genes drastically affect the effectiveness of DNA-damaging treatments in cancer cells. For this reasons selective DDR-inhibitors have been developed in order to sensitize cancer cells against conventional chemotherapy. Despite the proven efficacy of DDR-inhibitors in cancer treatment, only few studies have highlighted the biological consequences the prolonged inhibition of DDR-pathways in cancer cells. We hypothesized that the protracted inhibition of the DDR pathways may generate resistant clones characterized by an increased genetic instability. The aim of the study was to evaluate biological consequences of the prolonged inhibition of two crucial DDR-related kinase such as cell cycle checkpoint kinase 1 (Chk1) and 2 (Chk2) in B cells ALL. In particular, we investigated the consequences of Chk1/Chk2 inhibition in term of increase of genetic instability and in term of responsiveness to chemotherapy agents. Starting from B-ALL cell line NALM-6, we generated a resistant model (hereafter referred as N6R-PF8) by treating the parental cells with increasing concentration of PF-00477736 (Chk1/Chk2 inhibitor) for more than a year and increasing the IC50 value of 10-folds. From a molecular point of view, the N6R-PF8 accumulated significant molecular alterations. SNP microarray analysis highlighted different alterations in DDR-related genes and, in particular, in the ATM/CHK2 pathway. Three regions in copy number LOSS (CN=1) containing several genes involved in cell cycle checkpoint regulation (ATM and NPAT) and in the apoptosis (BIRC2, CASP1 and CASP5) were detectable only in NALM-6 parental cell lines and were copy number neutral in the resistant model. Immunoblotting analysis confirmed that in N6R-PF8 cells the ATM/CHK2 and ATR/CHK1 down-stream pathways were significant over-expressed and activated in comparison to the parental cell. Whole exome sequencing analysis showed that the two cell lines were characterized by different mutational profiles and that N6R-PF8 cells harboured significantly more genetic alterations in comparison with NALM-6 cells. Interestingly, crucial genes involved in DNA repair pathway (MLH3, NBN, POLD1 and PMS2) have been found altered only in N6R-PF8 cells. From a functional point of view, the molecular alterations characterizing the N6R-PF8 significantly compromised the cytotoxicity of PF-00477736 and of different DNA damaging agents in comparison to parental cells. Furthermore, the treatment with ATR/ATM inhibitor restored the sensitivity of N6R-PF8 to PF-00477736 and to different chemotherapy agents. In this scenario the level of expression of these two kinases seems to correlate with the sensitivity to DNA damaging agents and to PF-00477736. Finally, we confirmed that the protracted inhibition of crucial DDR-related kinase may increase the overall genetic instability in ALL cells and compromise the efficacy of DNA damaging based therapies. Disclosures Martinelli: Roche: Consultancy; ARIAD: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; BMS: Consultancy.
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Kornepati, Anand, Clare Murray, Barbara Avalos, Cody Rogers, Kavya Ramkumar, Harshita Gupta, Yilun Deng, et al. "900 Depleting non-canonical, cell-intrinsic PD-L1 signals induces synthetic lethality to small molecule DNA damage response inhibitors in an immune independent and dependent manner." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A944. http://dx.doi.org/10.1136/jitc-2021-sitc2021.900.
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BackgroundTumor surface-expressed programmed death-ligand 1 (PD-L1) suppresses immunity when it engages programmed death-1 (PD-1) on anti-tumor immune cells in canonical PD-L1/PD-1.1 Non-canonical, tumour-intrinsic PD-L1 signals can mediate treatment resistance2–6 but mechanisms remain incompletely understood. Targeting non-canonical, cell-intrinsic PD-L1 signals, especially modulation of the DNA damage response (DDR), remains largely untapped.MethodsWe made PD-L1 knockout (PD-L1 KO) murine transplantable and human cell lines representing melanoma, bladder, and breast histologies. We used biochemical, genetic, and cell-biology techniques for mechanistic insights into tumor-intrinsic PD-L1 control of specific DDR and DNA repair pathways. We generated a novel inducible melanoma GEMM lacking PD-L1 only in melanocytes to corroborate DDR alterations observed in PD-L1 KO of established tumors.ResultsGenetic tumor PD-L1 depletion destabilized Chk2 and impaired ATM/Chk2, but not ATR/Chk1 DDR. PD-L1KO increased DNA damage (γH2AX) and impaired homologous recombination DNA repair (p-RPA32, BRCA1, RAD51 nuclear foci) and function (DR-GFP reporter). PD-L1 KO cells were significantly more sensitive versus controls to DDR inhibitors (DDRi) against ATR, Chk1, and PARP but not ATM in multiple human and mouse tumor models in vitro and in vivo in NSG mice. PD-1 independent, intracellular, not surface PD-L1 stabilized Chk2 protein with minimal Chek2 mRNA effect. Mechanistically, PD-L1 could directly complex with Chk2, protecting it from PIRH2-mediated polyubiquitination. PD-L1 N-terminal domains Ig-V and Ig-C but not the PD-L1 C-terminal tail co-IP’d with Chk2 and restored Chk1 inhibitor (Chk1i) treatment resistance. Tumor PD-L1 expression correlated with Chk1i sensitivity in 44 primary human small cell lung cancer cell lines, implicating tumor-intrinsic PD-L1 as a DDRi response biomarker. In WT mice, genetic PD-L1 depletion but not surface PD-L1 blockade with αPD-L1, sensitized immunotherapy-resistant, BRCA1-WT 4T1 tumors to PARP inhibitor (PARPi). PARPi effects were reduced on PD-L1 KO tumors in RAG2KO mice indicating immune-dependent DDRi efficacy. Tumor PD-L1 depletion, likely due to impaired DDR, enhanced PARPi induced tumor-intrinsic STING activation (e.g., p-TBK1, CCL5) suggesting potential to augment immunotherapies.ConclusionsWe challenge the prevailing surface PD-L1 paradigm and establish a novel mechanism for cell-intrinsic PD-L1 control of the DDR and gene product expression. We identify therapeutic vulnerabilities from tumor PD-L1 depletion utilizing small molecule DDRi currently being tested in clinical trials. Data could explain αPD-L1/DDRi treatment resistance. Intracellular PD-L1 could be a pharmacologically targetable treatment target and/or response biomarker for selective DDRi alone plus other immunotherapies.ReferencesTopalian SL, Taube JM, Anders RA, Pardoll DM. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nat Rev Cancer 16:275–287, doi:10.1038/nrc.2016.36 (2016).Clark CA, et al. Tumor-intrinsic PD-L1 signals regulate cell growth, pathogenesis and autophagy in ovarian cancer and melanoma. Canres 0258.2016 (2016).Gupta HB et al. Tumor cell-intrinsic PD-L1 promotes tumor-initiating cell generation and functions in melanoma and ovarian cancer. 1, 16030 (2016).Zhu H, et al. BET bromodomain inhibition promotes anti-tumor immunity by suppressing PD-L1 expression. Cell Rep 16:2829–2837, doi:10.1016/j.celrep.2016.08.032 (2016)Wu B, et al. Adipose PD-L1 modulates PD-1/PD-L1 checkpoint blockade immunotherapy efficacy in breast cancer. Oncoimmunology 7:e1500107, doi:10.1080/2162402X.2018.1500107 (2018)Liang J, et al. Verteporfin inhibits PD-L1 through autophagy and the STAT1-IRF1-TRIM28 signaling axis, exerting antitumor efficacy. Cancer Immunol Res 8:952–965, doi:10.1158/2326-6066.CIR-19-0159 (2020)
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Kato, Naoko, Takeshi Kondo, Junichi Tsukada, Yoshiya Tanaka, Yasuhiro Minami, Junji Tanaka, and Masahiro Imamura. "Expression of the Chk2 Gene Is Downregulated in Hodgkin’s Lymphoma Cell Lines Via Epigenetic Mechanisms." Blood 104, no. 11 (November 16, 2004): 429. http://dx.doi.org/10.1182/blood.v104.11.429.429.
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Abstract Chk2, the mammalian homologue of the yeast Rad53 and Cds1 genes, encodes a nuclear serine/threonine kinase that plays a crucial role in the DNA damage response and helps guard the integrity of the genome by regulating cell-cycle checkpoints, DNA repair and apoptosis. Furthermore Chk2 is regarded as a tumor suppressor gene. Alterations in tumor suppressors that are involved in the DNA damage response have been reported to be frequently involved in the pathogenesis of lymphoid malignancies. We investigated the expression levels of the genes encoding Chk2 in nine cell lines from lymphoid malignancies, including three Hodgkin’s lymphoma (HL) cell lines. We found that all three HL cell lines exhibited a drastic reduction in Chk2 mRNA and protein expression compared to the other cell lines without any apparent mutation of the Chk2 gene. Accumulating evidence demonstrates the importance of posttranslational modification of histone proteins in addition to DNA methylation, as epigenetic mechanisms involved in the organization of chromosomal domains and gene regulation. It is now generally appreciated that hyperacetylated histones H3 and H4 are associated with activated genomic regions, while hypoacetylation of histones H3, H4 and methylation on H3-lysine 9 results in gene repression and silencing. Therefore we tested the possibility that epigenetic mechanisms are involved in this aberrant expression of the Chk2 gene in these cells using the histone deacetylase (HDAC) inhibitors trichostatin A (TsA) and sodium butyrate (SB). Expression of Chk2 in HL cells was restored following treatment with the TsA and SB, or 5Aza-dC. Chromatin-immunoprecipitation (Chip) assays revealed that treatment of HL cells with TsA, SB or 5Aza-dC resulted in increased levels of acetylated histones H3 and H4, and decreased levels of dimethylated H3 lysine 9 at the Chk2 promoter. These results indicate that expression of the Chk2 gene is down-regulated in HL cells via epigenetic mechanisms. Furthermore we examined whether the down regulation of Chk2 gene in HL cells may be involved in apoptosis resistance to irradiation (IR). We found that upregulation of Chk2 in HL cells following treatment by SB resulted in increased susceptibility of the cells to IR. Therefore, our findings suggested that altered epigenetic regulation of Chk2 gene in HL cells results in the down-regulation of Chk2 kinase and abrogates DNA damage response signaling in the cells
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Buscemi, Giacomo, Camilla Savio, Laura Zannini, Francesca Miccichè, Debora Masnada, Makoto Nakanishi, Hiroshi Tauchi, et al. "Chk2 Activation Dependence on Nbs1 after DNA Damage." Molecular and Cellular Biology 21, no. 15 (August 1, 2001): 5214–22. http://dx.doi.org/10.1128/mcb.21.15.5214-5222.2001.
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ABSTRACT The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G1 arrest. Here we show that the ATM-dependent activation of Chk2 by γ- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.
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Liang, Xiaobing, Yi Guo, William Douglas Figg, Antonio Tito Fojo, Michael D. Mueller, and Jing Jie Yu. "The Role of Wild-Type p53 in Cisplatin-Induced Chk2 Phosphorylation and the Inhibition of Platinum Resistance with a Chk2 Inhibitor." Chemotherapy Research and Practice 2011 (December 1, 2011): 1–8. http://dx.doi.org/10.1155/2011/715469.
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The major obstacle in platinum chemotherapy is the repair of platinum-damaged DNA that results in increased resistance, reduced apoptosis, and finally treatment failure. Our research goal is to determine and block the mechanisms of platinum resistance. Our recent studies demonstrate that several kinases in the DNA-repair pathway are activated after cells are exposed to cisplatin. These include ATM, p53, and Chk2. The increased Chk2 phosphorylation is modulated by p53 in a wild-type p53 model. Overexpression of p53 by cDNA transfection in wt-p53 (but not p53 deficient) cells doubled the amount of Chk2 phosphorylation 48 hours after cisplatin treatment. p53 knockdown by specific siRNA greatly reduced Chk2 phosphorylation. We conclude that wild-type p53, in response to cisplatin stimulation, plays a role in the upstream regulation of Chk2 phosphorylation at Thr-68. Cells without normal p53 function survive via an alternative pathway in response to the exogenous influence of cisplatin. We strongly suggest that it is very important to include the p53 mutational status in any p53 involved studies due to the functional differentiation of wt p53 and p53 mutant. Inhibition of Chk2 pathway with a Chk2 inhibitor (C3742) increased cisplatin efficacy, especially those with defective p53. Our findings suggest that inhibition of platinum resistance can be achieved with a small-molecule inhibitor of Chk2, thus improving the therapeutic indices for platinum chemotherapy.
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Buscemi, Giacomo, Luigi Carlessi, Laura Zannini, Sofia Lisanti, Enrico Fontanella, Silvana Canevari, and Domenico Delia. "DNA Damage-Induced Cell Cycle Regulation and Function of Novel Chk2 Phosphoresidues." Molecular and Cellular Biology 26, no. 21 (August 28, 2006): 7832–45. http://dx.doi.org/10.1128/mcb.00534-06.
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ABSTRACT Chk2 kinase is activated by DNA damage to regulate cell cycle arrest, DNA repair, and apoptosis. Phosphorylation of Chk2 in vivo by ataxia telangiectasia-mutated (ATM) on threonine 68 (T68) initiates a phosphorylation cascade that promotes the full activity of Chk2. We identified three serine residues (S19, S33, and S35) on Chk2 that became phosphorylated in vivo rapidly and exclusively in response to ionizing radiation (IR)-induced DNA double-strand breaks in an ATM- and Nbs1-dependent but ataxia telangiectasia- and Rad3-related-independent manner. Phosphorylation of these residues, restricted to the G1 phase of the cell cycle, was induced by a higher dose of IR (>1 Gy) than that required for phosphorylation of T68 (0.25 Gy) and declined by 45 to 90 min, concomitant with a rise in Chk2 autophosphorylation. Compared to the wild-type form, Chk2 with alanine substitutions at S19, S33, and S35 (Chk2S3A) showed impaired dimerization, defective auto- and trans-phosphorylation activities, and reduced ability to promote degradation of Hdmx, a phosphorylation target of Chk2 and regulator of p53 activity. Besides, Chk2S3A failed to inhibit cell growth and, in response to IR, to arrest G1/S progression. These findings underscore the critical roles of S19, S33, and S35 and argue that these phosphoresidues may serve to fine-tune the ATM-dependent response of Chk2 to increasing amounts of DNA damage.
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Amico, Donatella, Anna Maria Barbui, Eugenio Erba, Alessandro Rambaldi, Martino Introna, and Josée Golay. "Differential response of human acute myeloid leukemia cells to gemtuzumab ozogamicin in vitro: role of Chk1 and Chk2 phosphorylation and caspase 3." Blood 101, no. 11 (June 1, 2003): 4589–97. http://dx.doi.org/10.1182/blood-2002-07-2311.
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Abstract Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated to the anticancer agent calicheamicin, approved for the treatment of CD33+-relapsed acute myeloid leukemia. We have investigated the effects of GO on 4 human myeloid leukemia lines of different French-American-British (FAB) types (KG-1, THP-1, HL-60, and NB-4), observing 3 different types of response. Exposure to GO (10-1000 ng/mL) induced G2 arrest (up to 80% of the cells) followed by apoptosis (45% of the cells) in HL-60 and NB-4 cells. By contrast, in THP-1 cells we observed a strong G2 arrest (up to 75% of the cells) with little apoptosis. Finally, the KG-1 line was completely resistant to the same concentrations of GO. These different responses did not correlate with the levels of expression of either CD33 or multiple-drug resistance proteins, although the higher cyclosporin A (CsA)–inhibitable efflux activity of KG-1 cells may play a role in the resistance of this line to the drug. We could show that Chk1 and Chk2 phosphorylation, but not p53 or p21 expression, correlated with G2 arrest, implicating the ataxia-telangiectasia mutated/ataxia-telangiectasia related (ATM/ATR)–Chk1/Chk2 pathway in the cell cycle response to GO. However, apoptosis was associated with caspase 3 activation. Freshly isolated acute myeloid leukemia (AML) cells showed patterns of response to GO in vitro similar to those observed with the cell lines, including phosphorylation of Chk2 and caspase 3 activation. Our results suggest that the different molecular pathways induced by the drug in vitro may reflect, at least in part, the variable response to GO obtained in vivo.
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Yang, Shutong, Jae-Hoon Jeong, Alexandra L. Brown, Chang-Hun Lee, Pier Paolo Pandolfi, Jay H. Chung, and Myung K. Kim. "Promyelocytic Leukemia Activates Chk2 by Mediating Chk2 Autophosphorylation." Journal of Biological Chemistry 281, no. 36 (July 11, 2006): 26645–54. http://dx.doi.org/10.1074/jbc.m604391200.
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Bin Zhang, Xiubin Gu, Uma Uppalapati, Mark A. Ashwell, David S. Leggett, and Chiang J. Li. "High-Content Fluorescent-Based Assay for Screening Activators of DNA Damage Checkpoint Pathways." Journal of Biomolecular Screening 13, no. 6 (July 2008): 538–43. http://dx.doi.org/10.1177/1087057108318509.
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Activation of DNA damage checkpoint pathways, including Chk2, serves as an anticancer barrier in precancerous lesions. In an effort to identify small-molecule activators of Chk2, the authors developed a quantitative cell-based assay using a high-content analysis (HCA) platform. Induction of phosphorylated Chk2 was evaluated using several different parameters, including fold induction, Kolmogorov-Smirnov score, and percentage of positively stained cells. These measurements were highly correlated and provided an accurate method for compound ranking/binning, structure-activity relationship studies, and lead identification. Screening for Chk2 activators was undertaken with a target-focused library and a diversified library from ArQule chemical space. Several compounds exhibited submicromolar EC 50 values for phosphorylated Chk2 induction. These compounds were further analyzed for Chk2-dependent cytotoxicity, as assessed through a high-content cell death assay in combination with siRNA silencing of Chk2 expression. Several compounds were identified and showed specific inhibition or lethality in a target-dependent manner. Therefore, identification of DNA damage checkpoint pathway activators by HCA is an attractive approach for discovering the next generation of targeted cancer therapeutics. ( Journal of Biomolecular Screening 2008:538-543)
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Petsalaki, Eleni, and George Zachos. "Chk2 prevents mitotic exit when the majority of kinetochores are unattached." Journal of Cell Biology 205, no. 3 (May 5, 2014): 339–56. http://dx.doi.org/10.1083/jcb.201310071.
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The spindle checkpoint delays exit from mitosis in cells with spindle defects. In this paper, we show that Chk2 is required to delay anaphase onset when microtubules are completely depolymerized but not in the presence of relatively few unattached kinetochores. Mitotic exit in Chk2-deficient cells correlates with reduced levels of Mps1 protein and increased Cdk1–tyrosine 15 inhibitory phosphorylation. Chk2 localizes to kinetochores and is also required for Aurora B–serine 331 phosphorylation in nocodazole or unperturbed early prometaphase. Serine 331 phosphorylation contributed to prometaphase accumulation in nocodazole after partial Mps1 inhibition and was required for spindle checkpoint establishment at the beginning of mitosis. In addition, expression of a phosphomimetic S331E mutant Aurora B rescued chromosome alignment or segregation in Chk2-deficient cells. We propose that Chk2 stabilizes Mps1 and phosphorylates Aurora B–serine 331 to prevent mitotic exit when most kinetochores are unattached. These results highlight mechanisms of an essential function of Chk2 in mitosis.
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Khashab, Farah, Farah Al-Saleh, Nora Al-Kandari, Fatemah Fadel, and May Al-Maghrebi. "JAK Inhibition Prevents DNA Damage and Apoptosis in Testicular Ischemia-Reperfusion Injury via Modulation of the ATM/ATR/Chk Pathway." International Journal of Molecular Sciences 22, no. 24 (December 13, 2021): 13390. http://dx.doi.org/10.3390/ijms222413390.
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Testicular ischemia reperfusion injury (tIRI) causes oxidative stress-induced DNA damage leading to germ cell apoptosis (GCA). The aim of the study is to establish a direct link between JAK2 activation and the DNA damage response (DDR) signaling pathways and their role in tIRI-induced GCA using AG490, a JAK2 specific inhibitor. Male Sprague Dawley rats (n = 36) were divided into three groups: sham, unilateral tIRI and tIRI + AG490 (40 mg/kg). During tIRI, augmentation in the phosphorylation levels of the JAK2/STAT1/STAT3 was measured by immunohistochemistry. Observed spermatogenic arrest was explained by the presence of considerable levels of DSB, AP sites and 8OHdG and activation of caspase 9, caspase 3 and PARP, which were measured by colorimetric assays and TUNEL. The ATM/Chk2/H2AX and ATR/Chk1 pathways were also activated as judged by their increased phosphorylation using Western blot. These observations were all prevented by AG490 inhibition of JAK2 activity. Our findings demonstrate that JAK2 regulates tIRI-induced GCA, oxidative DNA damage and activation of the ATM/Chk2/H2AX and ATR/Chk1 DDR pathways, but the cell made the apoptosis decision despite DDR efforts.
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Ma, Xiao-Yan, Jia-Fu Zhao, Yong Ruan, Wang-Ming Zhang, Lun-Qing Zhang, Zheng-Dong Cai, and Hou-Qiang Xu. "ML216-Induced BLM Helicase Inhibition Sensitizes PCa Cells to the DNA-Crosslinking Agent Cisplatin." Molecules 27, no. 24 (December 12, 2022): 8790. http://dx.doi.org/10.3390/molecules27248790.
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Using standard DNA-damaging medicines with DNA repair inhibitors is a promising anticancer tool to achieve better therapeutic responses and reduce therapy-related side effects. Cell viability assay, neutral comet assay, western blotting (WB), and cell cycle and apoptosis analysis were used to determine the synergistic effect and mechanism of ML216, a Bloom syndrome protein (BLM) helicase inhibitor, and cisplatin (CDDP), a DNA-crosslinking agent, in PCa cells. Based on the online database research, our findings revealed that BLM was substantially expressed in PCa, which is associated with a bad prognosis for PCa patients. The combination of ML216 and CDDP improved the antiproliferative properties of three PCa cell lines. As indicated by the increased production of γH2AX and caspase-3 cleavage, ML216 significantly reduced the DNA damage-induced high expression of BLM, making PC3 more susceptible to apoptosis and DNA damage caused by CDDP. Furthermore, the combination of ML216 and CDDP increased p-Chk1 and p-Chk2 expression. The DNA damage may have triggered the ATR-Chk1 and ATM-Chk2 pathways simultaneously. Our results demonstrated that ML216 and CDDP combination therapy exhibited synergistic effects, and combination chemotherapy could be a novel anticancer tactic.
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Masrouha, Nisrine, Long Yang, Sirine Hijal, Stéphane Larochelle, and Beat Suter. "The Drosophila chk2 Gene loki Is Essential for Embryonic DNA Double-Strand-Break Checkpoints Induced in S Phase or G2." Genetics 163, no. 3 (March 1, 2003): 973–82. http://dx.doi.org/10.1093/genetics/163.3.973.
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Abstract Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.
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Yam, Candice Qiu Xia, David Boy Chia, Idina Shi, Hong Hwa Lim, and Uttam Surana. "Dun1, a Chk2-related kinase, is the central regulator of securin-separase dynamics during DNA damage signaling." Nucleic Acids Research 48, no. 11 (May 13, 2020): 6092–107. http://dx.doi.org/10.1093/nar/gkaa355.
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Abstract The DNA damage checkpoint halts cell cycle progression in G2 in response to genotoxic insults. Central to the execution of cell cycle arrest is the checkpoint-induced stabilization of securin-separase complex (yeast Pds1-Esp1). The checkpoint kinases Chk1 and Chk2 (yeast Chk1 and Rad53) are thought to critically contribute to the stability of securin-separase complex by phosphorylation of securin, rendering it resistant to proteolytic destruction by the anaphase promoting complex (APC). Dun1, a Rad53 paralog related to Chk2, is also essential for checkpoint-imposed arrest. Dun1 is required for the DNA damage-induced transcription of DNA repair genes; however, its role in the execution of cell cycle arrest remains unknown. Here, we show that Dun1′s role in checkpoint arrest is independent of its involvement in the transcription of repair genes. Instead, Dun1 is necessary to prevent Pds1 destruction during DNA damage in that the Dun1-deficient cells degrade Pds1, escape G2 arrest and undergo mitosis despite the presence of checkpoint-active Chk1 and Rad53. Interestingly, proteolytic degradation of Pds1 in the absence of Dun1 is mediated not by APC but by the HECT domain-containing E3 ligase Rsp5. Our results suggest a regulatory scheme in which Dun1 prevents chromosome segregation during DNA damage by inhibiting Rsp5-mediated proteolytic degradation of securin Pds1.
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Maiuthed, Arnatchai, Chuanpit Ninsontia, Katharina Erlenbach-Wuensch, Benardina Ndreshkjana, Julienne Muenzner, Aylin Caliskan, Husayn Ahmed P., et al. "Cytoplasmic p21 Mediates 5-Fluorouracil Resistance by Inhibiting Pro-Apoptotic Chk2." Cancers 10, no. 10 (October 9, 2018): 373. http://dx.doi.org/10.3390/cancers10100373.
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The oncogenic cytoplasmic p21 contributes to cancer aggressiveness and chemotherapeutic failure. However, the molecular mechanisms remain obscure. Here, we show for the first time that cytoplasmic p21 mediates 5-Fluorouracil (5FU) resistance by shuttling p-Chk2 out of the nucleus to protect the tumor cells from its pro-apoptotic functions. We observed that cytoplasmic p21 levels were up-regulated in 5FU-resistant colorectal cancer cells in vitro and the in vivo Chorioallantoic membrane (CAM) model. Kinase array analysis revealed that p-Chk2 is a key target of cytoplasmic p21. Importantly, cytoplasmic form of p21 mediated by p21T145D transfection diminished p-Chk2-mediated activation of E2F1 and apoptosis induction. Co-immunoprecipitation, immunofluorescence, and proximity ligation assay showed that p21 forms a complex with p-Chk2 under 5FU exposure. Using in silico computer modeling, we suggest that the p21/p-Chk2 interaction hindered the nuclear localization signal of p-Chk2, and therefore, the complex is exported out of the nucleus. These findings unravel a novel mechanism regarding an oncogenic role of p21 in regulation of resistance to 5FU-based chemotherapy. We suggest a possible value of cytoplasmic p21 as a prognosis marker and a therapeutic target in colorectal cancer patients.
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Bartek, Jiri, and Jiri Lukas. "Chk1 and Chk2 kinases in checkpoint control and cancer." Cancer Cell 3, no. 5 (May 2003): 421–29. http://dx.doi.org/10.1016/s1535-6108(03)00110-7.
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Stawinska, Magdalena, Adam Cygankiewicz, Radzislaw Trzcinski, Michal Mik, Adam Dziki, and Wanda M. Krajewska. "Alterations of Chk1 and Chk2 expression in colon cancer." International Journal of Colorectal Disease 23, no. 12 (August 5, 2008): 1243–49. http://dx.doi.org/10.1007/s00384-008-0551-8.
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Chuang, Tzu-Chao, Wei-Syun Shao, Shih-Chung Hsu, Shou-Lun Lee, Ming-Ching Kao, and Vinchi Wang. "Baicalein Induces G2/M Cell Cycle Arrest Associated with ROS Generation and CHK2 Activation in Highly Invasive Human Ovarian Cancer Cells." Molecules 28, no. 3 (January 20, 2023): 1039. http://dx.doi.org/10.3390/molecules28031039.
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Ovarian cancer is a lethal gynecological cancer because drug resistance often results in treatment failure. The CHK2, a tumor suppressor, is considered to be an important molecular target in ovarian cancer due to its role in DNA repair. Dysfunctional CHK2 impairs DNA damage-induced checkpoints, reduces apoptosis, and confers resistance to chemotherapeutic drugs and radiation therapy in ovarian cancer cells. This provides a basis for finding new effective agents targeting CHK2 upregulation or activation to treat or prevent the progression of advanced ovarian cancer. Here, the results show that baicalein (5,6,7-trihydroxyflavone) treatment inhibits the growth of highly invasive ovarian cancer cells, and that baicalein-induced growth inhibition is mediated by the cell cycle arrest in the G2/M phase. Baicalein-induced G2/M phase arrest is associated with an increased reactive oxygen species (ROS) production, DNA damage, and CHK2 upregulation and activation. Thus, baicalein modulates the expression of DNA damage response proteins and G2/M phase regulatory molecules. Blockade of CHK2 activation by CHK2 inhibitors protects cells from baicalein-mediated G2/M cell cycle arrest. All the results suggest that baicalein has another novel growth inhibitory effect on highly invasive ovarian cancer cells, which is partly related to G2/M cell cycle arrest through the ROS-mediated DNA breakage damage and CHK2 activation. Collectively, our findings provide a molecular basis for the potential of baicalein as an adjuvant therapeutic agent in the treatment of metastatic ovarian cancer.
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Liu, Lan, Jaladanki N. Rao, Tongtong Zou, Lan Xiao, Peng-Yuan Wang, Douglas J. Turner, Myriam Gorospe, and Jian-Ying Wang. "Polyamines Regulate c-Myc Translation through Chk2-dependent HuR Phosphorylation." Molecular Biology of the Cell 20, no. 23 (December 2009): 4885–98. http://dx.doi.org/10.1091/mbc.e09-07-0550.
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All mammalian cells depend on polyamines for normal growth and proliferation, but the exact roles of polyamines at the molecular level remain largely unknown. The RNA-binding protein HuR modulates the stability and translation of many target mRNAs. Here, we show that in rat intestinal epithelial cells (IECs), polyamines enhanced HuR association with the 3′-untranslated region of the c-Myc mRNA by increasing HuR phosphorylation by Chk2, in turn promoting c-Myc translation. Depletion of cellular polyamines inhibited Chk2 and reduced the affinity of HuR for c-Myc mRNA; these effects were completely reversed by addition of the polyamine putrescine or by Chk2 overexpression. In cells with high content of cellular polyamines, HuR silencing or Chk2 silencing reduced c-Myc translation and c-Myc expression levels. Our findings demonstrate that polyamines regulate c-Myc translation in IECs through HuR phosphorylation by Chk2 and provide new insight into the molecular functions of cellular polyamines.
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Abjaude, Walason, Bruna Prati, Veridiana Munford, Aline Montenegro, Vanesca Lino, Suellen Herbster, Tatiana Rabachini, Lara Termini, Carlos Frederico Martins Menck, and Enrique Boccardo. "ATM Pathway Is Essential for HPV–Positive Human Cervical Cancer-Derived Cell Lines Viability and Proliferation." Pathogens 11, no. 6 (June 1, 2022): 637. http://dx.doi.org/10.3390/pathogens11060637.
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Infection with some mucosal human papillomavirus (HPV) types is the etiological cause of cervical cancer and of a significant fraction of vaginal, vulvar, anal, penile, and head and neck carcinomas. DNA repair machinery is essential for both HPV replication and tumor cells survival suggesting that cellular DNA repair machinery may play a dual role in HPV biology and pathogenesis. Here, we silenced genes involved in DNA Repair pathways to identify genes that are essential for the survival of HPV-transformed cells. We identified that inhibition of the ATM/CHK2/BRCA1 axis selectively affects the proliferation of cervical cancer-derived cell lines, without altering normal primary human keratinocytes (PHK) growth. Silencing or chemical inhibition of ATM/CHK2 reduced the clonogenic and proliferative capacity of cervical cancer-derived cells. Using PHK transduced with HPV16 oncogenes we observed that the effect of ATM/CHK2 silencing depends on the expression of the oncogene E6 and on its ability to induce p53 degradation. Our results show that inhibition of components of the ATM/CHK2 signaling axis reduces p53-deficient cells proliferation potential, suggesting the existence of a synthetic lethal association between CHK2 and p53. Altogether, we present evidence that synthetic lethality using ATM/CHK2 inhibitors can be exploited to treat cervical cancer and other HPV-associated tumors.