Chk2 and REGγ-dependent DBC1 regulation in DNA damage induced apoptosis.
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ABSTRACT:
SUBMITTER: Magni M
PROVIDER: S-EPMC4245943 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cell cycle and apoptosis regulator 2 (CCAR2, formerly known as DBC1) is a nuclear protein largely involved in DNA damage response, apoptosis, metabolism, chromatin structure and transcription regulation. Upon DNA lesions, CCAR2 is phosphorylated by the apical kinases ATM/ATR and this phosphorylation enhances CCAR2 binding to SIRT1, leading to SIRT1 inhibition, p53 acetylation and p53-dependent apoptosis. Recently, we found that also the checkpoint kinase Chk2 and the proteasome activator REGγ are required for efficient CCAR2-mediated inhibition of SIRT1 and induction of p53-dependent apoptosis.Here, we report that CCAR2 is required for the repair of heterochromatic DNA lesions, as cells knock-out for CCAR2 retain, at late time-points after genotoxic treatment, abnormal levels of DNA damage-associated nuclear foci, whose timely resolution is reinstated by HP1β depletion. Conversely, repair of DNA damages in euchromatin are not affected by CCAR2 absence.We also report that the impairment in heterochromatic DNA repair is caused by defective Chk2 activation, detectable in CCAR2 ablated cells, which finally impacts on the phosphorylation of the Chk2 substrate KAP1 that is required for the induction of heterochromatin relaxation and DNA repair.These studies further extend and confirm the role of CCAR2 in the DNA damage response and DNA repair and illustrate a new mechanism of Chk2 activity regulation. Moreover, the involvement of CCAR2 in the repair of heterochromatic DNA breaks suggests a new role for this protein in the maintenance of chromosomal stability, which is necessary to prevent cancer formation.
| S-EPMC4627348 | biostudies-literature
Project description: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 G(1) 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 (Chk2(S3A)) 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, Chk2(S3A) failed to inhibit cell growth and, in response to IR, to arrest G(1)/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.
| S-EPMC1636737 | biostudies-literature
Project description:DNA damage is the major cause of senescence and apoptosis; however, the manner by which DNA-damaged cells become senescent remains unclear. We demonstrate that DNA damage leads to a greater level of senescence rather than apoptosis in DBC1-deficient cells. In addition, we show that BLM becomes degraded during DNA damage, which induces p21 expression and senescence. DBC1 binds to and shields BLM from degradation, thus suppressing senescence. ML216 promotes DBC1-BLM interaction, which aids in the preservation of BLM following DNA damage and suppresses senescence. ML216 enhances pulmonary function by lowering the levels of senescence and fibrosis in both aged mice and a mouse model of bleomycin-induced idiopathic pulmonary fibrosis. Our data reveal a unique mechanism preventing DNA-damaged cells from becoming senescent, which may be regulated by the use of ML216 as a potential treatment for senescence-related diseases.
| S-EPMC9818470 | biostudies-literature
Project description:BackgroundGlobally, colorectal cancer (CRC) affects more than 1 million people each year. In addition to non-modifiable and other environmental risk factors, Fusobacterium nucleatum infection has been linked to CRC recently. In this study, we explored mechanisms underlying the role of Fusobacterium nucleatum infection in the progression of CRC in a mouse model.MethodsC57BL/6 J-Adenomatous polyposis coli (APC) Min/J mice [APC (Min/+)] were treated with Fusobacterium nucleatum (109 cfu/mL, 0.2 mL/time/day, i.g., 12 weeks), saline, or FadA knockout (FadA-/-) Fusobacterium nucleatum. The number, size, and weight of CRC tumors were determined in isolated tumor masses. The human CRC cell lines HCT29 and HT116 were treated with lentiviral vectors overexpressing chk2 or silencing β-catenin. DNA damage was determined by Comet assay and γH2AX immunofluorescence assay and flow cytometry. The mRNA expression of chk2 was determined by RT-qPCR. Protein expression of FadA, E-cadherin, β-catenin, and chk2 were determined by Western blot analysis.ResultsFusobacterium nucleatum treatment promoted DNA damage in CRC in APC (Min/+) mice. Fusobacterium nucleatum also increased the number of CRC cells that were in the S phase of the cell cycle. FadA-/- reduced tumor number, size, and burden in vivo. FadA-/- also reduced DNA damage, cell proliferation, expression of E-cadherin and chk2, and cells in the S phase. Chk2 overexpression elevated DNA damage and tumor growth in APC (Min/+) mice.ConclusionsIn conclusion, this study provided evidence that Fusobacterium nucleatum induced DNA damage and cell growth in CRC through FadA-dependent activation of the E-cadherin/β-catenin pathway, leading to up-regulation of chk2.
| S-EPMC7523382 | biostudies-literature
Project description:Reactivation of the p53 tumor-suppressor protein by small molecules like Nutlin-3 and RITA (reactivation of p53 and induction of tumor cell apoptosis) is a promising strategy for cancer therapy. The molecular mechanisms involved in the responses to RITA remain enigmatic. Several groups reported the induction of a p53-dependent DNA damage response. Furthermore, the existence of a p53-dependent S-phase checkpoint has been suggested, involving the checkpoint kinase Chk1. We have recently shown synergistic induction of apoptosis by RITA in combination with Nutlin-3, and we observed concomitant Chk2 phosphorylation. Therefore, we investigated whether Chk2 contributes to the cellular responses to RITA. Strikingly, the induction of apoptosis seemed entirely Chk2 dependent. Transcriptional activity of p53 in response to RITA required the presence of Chk2. A partial rescue of apoptosis observed in Noxa knockdown cells emphasized the relevance of p53 transcriptional activity for RITA-induced apoptosis. In addition, we observed an early p53- and Chk2-dependent block of DNA replication upon RITA treatment. Replicating cells seemed more prone to entering RITA-induced apoptosis. Furthermore, the RITA-induced DNA damage response, which was not a secondary effect of apoptosis induction, was strongly attenuated in cells lacking p53 or Chk2. In conclusion, we identified Chk2 as an essential mediator of the cellular responses to RITA.
| S-EPMC3354051 | biostudies-literature
Project description:Deleted in breast cancer 1 (DBC1) was initially identified from a homozygously deleted region in human chromosome 8p21. It has been well established that DBC1 plays a dual role during cancer development. Depending on the physiological context, it can promote or inhibit tumorigenesis. Whether it plays a role in lens pathogenesis remains elusive. In the present study, we demonstrated that DBC1 is highly expressed in lens epithelial cells from different vertebrates and in retina pigment epithelial cells as well. Moreover, DBC1 is SUMOylated through SUMO1 conjugation at K591 residue in human and mouse lens epithelial cells. The SUMOylated DBC1 is localized in the nucleus and plays an essential role in promoting stress-induced apoptosis. Silence of DBC1 attenuates oxidative stress-induced apoptosis. In contrast, overexpression of DBC1 enhances oxidative stress-induced apoptosis, and this process depends on p53. Mechanistically, DBC1 interacts with p53 to regulate its phosphorylation status at multiple sites and the SUMOylation of DBC1 enhances its interaction with p53. Together, our results identify that DBC1 is an important regulator mediating stress-induced apoptosis in lens, and thus participates in control of lens cataractogenesis.
| S-EPMC10522365 | biostudies-literature
Project description:Checkpoint kinase 2 (CHK2) is a major effector of the DNA damage response pathway and although its mechanism of activation has been well studied, the attenuation of its activity following DNA damage has not been explored. Here, we identify the B'alpha subunit of protein phosphatase 2A (PP 2A) as a CHK2 binding partner and show that their interaction is modulated by DNA damage. B'alpha binds to the SQ/TQ repeat region of CHK2, which is a target of ATM phosphorylation. The induction of DNA double-strand breaks by gamma irradiation as well as treatment with doxorubicin causes dissociation of the B'alpha and CHK2 proteins. This dissociation correlates with an increase in the ATM-dependent phosphorylation of CHK2 at serines 33 and 35 in the SQ/TQ region. Indeed, mutating these sites to mimic phosphorylation increases the dissociation after irradiation. PP 2A negatively regulates CHK2 phosphorylation at multiple sites, as well as its kinase activity. These data reveal a novel mechanism for PP 2A to keep CHK2 inactive under normal conditions while also allowing for a rapid release from this regulation immediately following DNA damage. This is followed by a subsequent reconstitution of the PP 2A/CHK2 complex in later time points after damage, which may help to attenuate the signal.
| S-EPMC3040716 | biostudies-literature
Project description:DNA sequences capable of forming triplexes are prevalent in the human genome and have been found to be intrinsically mutagenic. Consequently, a balance between DNA repair and apoptosis is critical to counteract their effect on genomic integrity. Using triplex-forming oligonucleotides to synthetically create altered helical distortions, we have determined that pro-apoptotic pathways are activated by the formation of triplex structures. Moreover, the TFIIH factor, XPD, occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. Here, we show that triplexes are capable of inducing XPD-independent double strand breaks, which result in the formation of ?H2AX foci. XPD was subsequently recruited to the triplex-induced double strand breaks and co-localized with ?H2AX at the damage site. Furthermore, phosphorylation of H2AX tyrosine 142 was found to stimulate the signaling pathway of XPD-dependent apoptosis. We suggest that this mechanism may play an active role in minimizing genomic instability induced by naturally occurring noncanonical structures, perhaps protecting against cancer initiation.
| S-EPMC3799437 | biostudies-literature
Project description:Growing evidence indicates that metabolic signaling pathways are interconnected to DNA damage response (DDR). However, factors that link metabolism to DDR remain incompletely understood. SIRT1, an NAD+-dependent deacetylase that regulates metabolism and aging, has been shown to protect cells from DDR. Here, we demonstrate that SIRT1 protects cells from oxidative stress-dependent DDR by binding and deacetylating checkpoint kinase 2 (CHK2). We first showed that essential proteins in DDR were hyperacetylated in Sirt1-deficient cells and that among them, the level of acetylated CHK2 was highly increased. We found that Sirt1 formed molecular complexes with CHK2, BRCA1/BRCA2-associated helicase 1 (BACH1), tumor suppressor p53-binding protein 1 (53BP1), and H2AX, all of which are key factors in response to DNA damage. We then demonstrated that CHK2 was normally inhibited by SIRT1 via deacetylation but dissociated with SIRT1 under oxidative stress conditions. This led to acetylation and activation of CHK2, which increased cell death under oxidative stress conditions. Our data also indicated that SIRT1 deacetylated the K235 and K249 residues of CHK2, whose acetylation increased cell death in response to oxidative stress. Thus, SIRT1, a metabolic sensor, protects cells from oxidative stress-dependent DDR by the deacetylation of CHK2. Our findings suggest a crucial function of SIRT1 in inhibiting CHK2 as a potential therapeutic target for cancer treatment.
| S-EPMC6430805 | biostudies-literature
Project description:Checkpoint kinase 2 (Chk2) has been implicated in DNA damage signaling. By using BJ human fibroblasts, HCT116 colorectal cancer cells and HeLa cervical cancer cells, we further detailed phosphorylation kinetics of Chk2 under treatment with neocarcinostatin (NCS) or doxorubicin (Dox). After NCS treatment, phosphorylation of Chk2 Thr68 occurs in 3 min, followed by phosphorylation of Ser19 and Ser33/35. In ATM deficient fibroblasts, NCS does not induce phosphorylation of NBS1 Ser343 and Chk2 Ser19 and Ser33/35, however Chk2 Thr68 is still phosphorylated, indicating that ATM is essential for phosphorylation of these residues when treated with NCS. By using Chk2-deficient HCT116 cells re-expressing phospho-mutant Chk2 (T68A), we found that inhibition of Thr68 phosphorylation enhances Ser19 phosphorylation in NCS treated cells. Interestingly, in contrast to NCS, Dox does not induce Ser33/35 phosphorylation in HeLa and HCT116 cells. Phosphorylation of Thr68 is sustained until 3 to 4 hours, and phosphorylation of Ser19 occurs 70 to 80 min after Dox treatment. These results demonstrate that Chk2 s involved in the early stages of DNA damage response. Differential phosphorylation kinetics of these residues suggests that DNA damage determines intermolecular and intramolecular interaction of Chk2, which may regulate phosphorylation.
| S-EPMC4623534 | biostudies-literature