RESUMO
In response to DNA damage, the histone PARylation factor 1 (HPF1) regulates PARP1/2 activity, facilitating serine ADP-ribosylation of chromatin-associated factors. While PARP1/2 are known for their role in DNA single-strand break repair (SSBR), the significance of HPF1 in this process remains unclear. Here, we investigated the impact of HPF1 deficiency on cellular survival and SSBR following exposure to various genotoxins. We found that HPF1 loss did not generally increase cellular sensitivity to agents that typically induce DNA single-strand breaks (SSBs) repaired by PARP1. SSBR kinetics in HPF1-deficient cells were largely unaffected, though its absence partially influenced the accumulation of SSB intermediates after exposure to specific genotoxins in certain cell lines, likely due to altered ADP-ribosylation of chromatin. Despite reduced serine mono-ADP-ribosylation, HPF1-deficient cells maintained robust poly-ADP-ribosylation at SSB sites, possibly reflecting PARP1 auto-poly-ADP-ribosylation at non-serine residues. Notably, poly-ADP-ribose chains were sufficient to recruit the DNA repair factor XRCC1, which may explain the relatively normal SSBR capacity in HPF1-deficient cells. These findings suggest that HPF1 and histone serine ADP-ribosylation are largely dispensable for PARP1-dependent SSBR in response to genotoxic stress, highlighting the complexity of mechanisms that maintain genomic stability and chromatin remodeling.
RESUMO
Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Reparo do DNA , Dano ao DNA , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Endonucleases Flap/uso terapêutico , Exodesoxirribonucleases/genética , Enzimas Reparadoras do DNA/genéticaRESUMO
UV irradiation induces "bulky" DNA photodimers such as (6-4)-photoproducts and cyclobutane pyrimidine dimers that are removed by nucleotide excision repair, a complex process defective in the sunlight-sensitive and cancer-prone disease xeroderma pigmentosum. Some bacteria and lower eukaryotes can also repair photodimers by enzymatically simpler mechanisms, but such pathways have not been reported in normal human cells. Here, we have identified such a mechanism. We show that normal human cells can employ a DNA base excision repair process involving NTH1, APE1, PARP1, XRCC1, and FEN1 to rapidly remove a subset of photodimers at early times following UVC irradiation. Loss of these proteins slows the early rate of repair of photodimers in normal cells, ablates their residual repair in xeroderma pigmentosum cells, and increases UVC sensitivity â¼2-fold. These data reveal that human cells can excise photodimers using a long-patch base excision repair process that functions additively but independently of nucleotide excision repair.
Assuntos
Xeroderma Pigmentoso , Humanos , Xeroderma Pigmentoso/genética , Reparo do DNA/genética , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , Dano ao DNA/genética , DNA/genética , Raios Ultravioleta , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismoRESUMO
DNA single-strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair-defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5-chloro-2'-deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA-defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility.
Assuntos
Antineoplásicos , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Quebra Cromossômica , Reparo do DNA , Replicação do DNA , DNA , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismoRESUMO
Poly(ADP-ribose) polymerase 1 (PARP1) is implicated in the detection and processing of unligated Okazaki fragments and other DNA replication intermediates, highlighting such structures as potential sources of genome breakage induced by PARP inhibition. Here, we show that PARP1 activity is greatly elevated in chicken and human S phase cells in which FEN1 nuclease is genetically deleted and is highest behind DNA replication forks. PARP inhibitor reduces the integrity of nascent DNA strands in both wild-type chicken and human cells during DNA replication, and does so in FEN1-/- cells to an even greater extent that can be detected as postreplicative single-strand nicks or gaps. Collectively, these data show that PARP inhibitors impede the maturation of nascent DNA strands during DNA replication, and implicate unligated Okazaki fragments and other nascent strand discontinuities in the cytotoxicity of these compounds.
Assuntos
Replicação do DNA , Inibidores de Poli(ADP-Ribose) Polimerases , DNA/genética , Dano ao DNA , Reparo do DNA , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologiaRESUMO
Upon exposure to genotoxic stress, cells activate DNA damage response (DDR) that coordinates DNA repair with a temporal arrest in the cell cycle progression. DDR is triggered by activation of ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related protein kinases that phosphorylate multiple targets including tumor suppressor protein tumor suppressor p53 (p53). In addition, DNA damage can activate parallel stress response pathways [such as mitogen-activated protein kinase p38 alpha (p38)/MAPK-activated protein kinase 2 (MK2) kinases] contributing to establishing the cell cycle arrest. Wild-type p53-induced phosphatase 1 (WIP1) controls timely inactivation of DDR and is needed for recovery from the G2 checkpoint by counteracting the function of p53. Here, we developed a simple in vitro assay for testing WIP1 substrates in nuclear extracts. Whereas we did not detect any activity of WIP1 toward p38/MK2, we confirmed p53 as a substrate of WIP1. Inhibition or inactivation of WIP1 in U2OS cells increased phosphorylation of p53 at S15 and potentiated its acetylation at K382. Further, we identified Deleted in breast cancer gene 1 (DBC1) as a new substrate of WIP1 but surprisingly, depletion of DBC1 did not interfere with the ability of WIP1 to regulate p53 acetylation. Instead, we have found that WIP1 activity suppresses p53-K382 acetylation by inhibiting the interaction between p53 and the acetyltransferase p300. Newly established phosphatase assay allows an easy comparison of WIP1 ability to dephosphorylate various proteins and thus contributes to identification of its physiological substrates.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Bioensaio/métodos , Neoplasias Ósseas/patologia , Núcleo Celular/metabolismo , Osteossarcoma/patologia , Proteína Fosfatase 2C/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Núcleo Celular/genética , Dano ao DNA , Reparo do DNA , Humanos , Osteossarcoma/genética , Osteossarcoma/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 2C/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2 phase, but why active Cyclin A2-CDK2 during the S phase does not trigger mitotic kinase activation remains unclear. Here, we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in the G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclina A2/metabolismo , Citoplasma/metabolismo , Fase G2/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fase S/genética , Transdução de Sinais/genética , Proteína Quinase CDC2/deficiência , Proteína Quinase CDC2/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Ciclina A2/genética , Quinase 2 Dependente de Ciclina/deficiência , Quinase 2 Dependente de Ciclina/genética , Dano ao DNA/genética , Ativação Enzimática/genética , Células HeLa , Humanos , Mitose/genética , Fosforilação/genética , Ligação Proteica , Transfecção , Quinase 1 Polo-LikeRESUMO
Genotoxic stress triggers a combined action of DNA repair and cell cycle checkpoint pathways. Protein phosphatase 2C delta (referred to as WIP1) is involved in timely inactivation of DNA damage response by suppressing function of p53 and other targets at chromatin. Here we show that WIP1 promotes DNA repair through homologous recombination. Loss or inhibition of WIP1 delayed disappearance of the ionizing radiation-induced 53BP1 foci in S/G2 cells and promoted cell death. We identify breast cancer associated protein 1 (BRCA1) as interactor and substrate of WIP1 and demonstrate that WIP1 activity is needed for correct dynamics of BRCA1 recruitment to chromatin flanking the DNA lesion. In addition, WIP1 dephosphorylates 53BP1 at Threonine 543 that was previously implicated in mediating interaction with RIF1. Finally, we report that inhibition of WIP1 allowed accumulation of DNA damage in S/G2 cells and increased sensitivity of cancer cells to a poly-(ADP-ribose) polymerase inhibitor olaparib. We propose that inhibition of WIP1 may increase sensitivity of BRCA1-proficient cancer cells to olaparib.
Assuntos
Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteína Fosfatase 2C/antagonistas & inibidores , Proteína Fosfatase 2C/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/metabolismo , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular , Células HEK293 , Recombinação Homóloga/genética , Humanos , Proteína Fosfatase 2C/genética , Pontos de Checagem da Fase S do Ciclo Celular , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates cell response to genotoxic stress by negatively regulating the tumor suppressor p53 and other targets at chromatin. Mutations in the exon 6 of the PPM1D result in production of a highly stable, C-terminally truncated PPM1D. These gain-of-function PPM1D mutations are present in various human cancers but their role in tumorigenesis remains unresolved. Here we show that truncated PPM1D impairs activation of the cell cycle checkpoints in human non-transformed RPE cells and allows proliferation in the presence of DNA damage. Next, we developed a mouse model by introducing a truncating mutation in the PPM1D locus and tested contribution of the oncogenic PPM1DT allele to colon tumorigenesis. We found that p53 pathway was suppressed in colon stem cells harboring PPM1DT resulting in proliferation advantage under genotoxic stress condition. In addition, truncated PPM1D promoted tumor growth in the colon in Apcmin mice and diminished survival. Moreover, tumor organoids derived from colon of the ApcminPpm1dT/+ mice were less sensitive to 5-fluorouracil when compared to ApcminPpm1d+/+and the sensitivity to 5-fluorouracil was restored by inhibition of PPM1D. Finally, we screened colorectal cancer patients and identified recurrent somatic PPM1D mutations in a fraction of colon adenocarcinomas that are p53 proficient and show defects in mismatch DNA repair. In summary, we provide the first in vivo evidence that truncated PPM1D can promote tumor growth and modulate sensitivity to chemotherapy.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias do Colo/tratamento farmacológico , Proteína Fosfatase 2C/genética , Proteína Supressora de Tumor p53/genética , Animais , Carcinogênese/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Éxons/genética , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Mutação/genéticaRESUMO
BACKGROUND: Hereditary mutations in the CHEK2 gene (which encodes CHK2 kinase) contribute to a moderately increased risk of breast cancer (BC) and other cancers. Large variations in the frequency of CHEK2 mutations and the occurrence of variants of unknown clinical significance (VUS) complicate estimation of cancer risk in carriers of germline CHEK2 mutations. PATIENTS AND METHODS: We performed mutation analysis of 1,526 high-risk Czech BC patients and 3,360 Czech controls. Functional analysis was performed for identified VUS using a model system based on a human RPE1-CHEK2-KO cell line harboring biallelic inactivation of endogenous CHEK2. RESULTS: The frequency of ten truncating CHEK2 variants differed markedly between BC patients (2.26%) and controls (0.11%; p = 4.1 × 1012). We also found 23 different missense variants in 4.5% patients and in 4.0% of controls. The most common was p.I157T, which was found in patients and controls with the same frequency. Functional analysis identified nine functionally deleterious VUS, another nine functionally neutral VUS, and four intermediate VUS (including p.I157T). We found that carriers of truncating CHEK2 mutations had a high BC risk (OR 8.19; 95% CI 4.11-17.75), and that carriers of functionally deleterious missense variants had a moderate risk (OR 4.06; 95% CI, 1.37-13.39). Carriers of these mutations developed BC at 44.4 and 50.7 years, respectively. Functionally neutral and functionally intermediate missense variants did not increase the BC risk. BC in CHEK2 mutation carriers was frequently ER-positive and of higher grade. Notably, carriers of CHEK2 mutations developed second cancers more frequently than BRCA1/BRCA2/PALB2/p53 or mutation non-carriers. CONCLUSION: Hereditary CHEK2 mutations contribute to the development of hereditary BC. The associated cancer risk in mutation carriers increases with the number of affected individuals in a family. Annual follow-up with breast ultrasound, mammography, or magnetic resonance imaging is recommended for asymptomatic mutation carriers from the age of 40. Surgical prevention and specific follow-up of other tumors should be considered based on family cancer history. The work was supported by grants from the Czech Health Research Council of the Ministry of Health of the Czech Republic NR 15-28830A, 16-29959A, NV19-03-00279, projects of the PROGRES Q28/LF1, GAUK 762216, SVV2019 / 260367, PRIMUS/17/MED/9, UNCE/MED/016, Progress Q26, LQ1604 NPU II and project AVČR Qualitas. The analysis of a set of unselected controls was made possible by the existence and support of the scientific infrastructure of the National Center for Medical Genomics (LM2015091) and its project aimed at creating a reference database of genetic variants of the Czech Republic (CZ.02.1.01/0.0/0.0/16_013/0001634). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 2. 4. 2019 Accepted: 14. 5. 2019.
Assuntos
Neoplasias da Mama/genética , Quinase do Ponto de Checagem 2/genética , Predisposição Genética para Doença , Linhagem Celular , República Tcheca , Feminino , Mutação em Linhagem Germinativa , Humanos , Fatores de RiscoRESUMO
Cyclins are central engines of cell cycle progression in conjunction with cyclin-dependent kinases (CDKs). Among the different cyclins controlling cell cycle progression, cyclin F does not partner with a CDK, but instead forms via its F-box domain an SCF (Skp1-Cul1-F-box)-type E3 ubiquitin ligase module. Although various substrates of cyclin F have been identified, the vulnerabilities of cells lacking cyclin F are not known. Thus, we assessed viability of cells lacking cyclin F upon challenging them with more than 180 different kinase inhibitors. The screen revealed a striking synthetic lethality between Chk1 inhibition and cyclin F loss. Chk1 inhibition in cells lacking cyclin F leads to DNA replication catastrophe. Replication catastrophe depends on accumulation of the transcription factor E2F1 in cyclin F-depleted cells. We find that SCF-cyclin F controls E2F1 ubiquitylation and degradation during the G2/M phase of the cell cycle and upon challenging cells with Chk1 inhibitors. Thus, Cyclin F restricts E2F1 activity during the cell cycle and upon checkpoint inhibition to prevent DNA replication stress. Our findings pave the way for patient selection in the clinical use of checkpoint inhibitors.
Assuntos
Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Ciclinas/metabolismo , Fator de Transcrição E2F1/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteólise , Proteínas Ligases SKP Culina F-Box/metabolismo , Mutações Sintéticas Letais , Ciclo Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/genética , Ciclinas/genética , Replicação do DNA , Fator de Transcrição E2F1/genética , Células HeLa , Humanos , Fosforilação , Ligação Proteica , Proteínas Ligases SKP Culina F-Box/genética , UbiquitinaçãoRESUMO
Germline mutations in checkpoint kinase 2 (CHEK2), a multiple cancer-predisposing gene, increase breast cancer (BC) risk; however, risk estimates differ substantially in published studies. We analyzed germline CHEK2 variants in 1,928 high-risk Czech breast/ovarian cancer (BC/OC) patients and 3,360 population-matched controls (PMCs). For a functional classification of VUS, we developed a complementation assay in human nontransformed RPE1-CHEK2-knockout cells quantifying CHK2-specific phosphorylation of endogenous protein KAP1. We identified 10 truncations in 46 (2.39%) patients and in 11 (0.33%) PMC (p = 1.1 × 10-14 ). Two types of large intragenic rearrangements (LGR) were found in 20/46 mutation carriers. Truncations significantly increased unilateral BC risk (OR = 7.94; 95%CI 3.90-17.47; p = 1.1 × 10-14 ) and were more frequent in patients with bilateral BC (4/149; 2.68%; p = 0.003), double primary BC/OC (3/79; 3.80%; p = 0.004), male BC (3/48; 6.25%; p = 8.6 × 10-4 ), but not with OC (3/354; 0.85%; p = 0.14). Additionally, we found 26 missense VUS in 88 (4.56%) patients and 131 (3.90%) PMC (p = 0.22). Using our functional assay, 11 variants identified in 15 (0.78%) patients and 6 (0.18%) PMC were scored deleterious (p = 0.002). Frequencies of functionally intermediate and neutral variants did not differ between patients and PMC. Functionally deleterious CHEK2 missense variants significantly increased BC risk (OR = 3.90; 95%CI 1.24-13.35; p = 0.009) and marginally OC risk (OR = 4.77; 95%CI 0.77-22.47; p = 0.047); however, carriers low frequency will require evaluation in larger studies. Our study highlights importance of LGR detection for CHEK2 analysis, careful consideration of ethnicity in both cases and controls for risk estimates, and demonstrates promising potential of newly developed human nontransformed cell line assay for functional CHEK2 VUS classification.
Assuntos
Neoplasias da Mama Masculina/genética , Neoplasias da Mama/genética , Quinase do Ponto de Checagem 2/genética , Mutação em Linhagem Germinativa , Neoplasias Ovarianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Linhagem Celular , República Tcheca , Feminino , Técnicas de Inativação de Genes , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Deleção de Sequência , Adulto JovemRESUMO
The bulk of DNA damage caused by ionizing radiation (IR) is generally repaired within hours, yet a subset of DNA lesions may persist even for long periods of time. Such persisting IR-induced foci (pIRIF) co-associate with PML nuclear bodies (PML-NBs) and are among the characteristics of cellular senescence. Here we addressed some fundamental questions concerning the nature and determinants of this co-association, the role of PML-NBs at such sites, and the reason for the persistence of DNA damage in human primary cells. We show that the persistent DNA lesions are devoid of homologous recombination (HR) proteins BRCA1 and Rad51. Our super-resolution microscopy-based analysis showed that PML-NBs are juxtaposed to and partially overlap with the pIRIFs. Notably, depletion of 53BP1 resulted in decreased intersection between PML-NBs and pIRIFs implicating the RNF168-53BP1 pathway in their interaction. To test whether the formation and persistence of IRIFs is PML-dependent and to investigate the role of PML in the context of DNA repair and senescence, we genetically deleted PML in human hTERT-RPE-1 cells. Unexpectedly, upon high-dose IR treatment, cells displayed similar DNA damage signalling, repair dynamics and kinetics of cellular senescence regardless of the presence or absence of PML. In contrast, the PML knock-out cells showed increased sensitivity to low doses of IR and DNA-damaging agents mitomycin C, cisplatin and camptothecin that all cause DNA lesions requiring repair by HR. These results, along with enhanced sensitivity of the PML knock-out cells to DNA-PK and PARP inhibitors implicate PML as a factor contributing to HR-mediated DNA repair.
Assuntos
Dano ao DNA , Reparo do DNA , Corpos de Inclusão Intranuclear/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Senescência Celular/genética , Senescência Celular/efeitos da radiação , Reparo do DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Técnicas de Inativação de Genes , Humanos , Corpos de Inclusão Intranuclear/efeitos da radiação , Proteína da Leucemia Promielocítica/deficiência , Proteína da Leucemia Promielocítica/genéticaRESUMO
Orderly progressions of events in the cell division cycle are necessary to ensure the replication of DNA and cell division. Checkpoint systems allow the accurate execution of each cell-cycle phase. The precise regulation of the levels of cyclin proteins is fundamental to coordinate cell division with checkpoints, avoiding genome instability. Cyclin F has important functions in regulating the cell cycle during the G2 checkpoint; however, the mechanisms underlying the regulation of cyclin F are poorly understood. Here, we observe that cyclin F is regulated by proteolysis through ß-TrCP. ß-TrCP recognizes cyclin F through a non-canonical degron site (TSGXXS) after its phosphorylation by casein kinase II. The degradation of cyclin F mediated by ß-TrCP occurs at the G2/M transition. This event is required to promote mitotic progression and favors the activation of a transcriptional program required for mitosis.
Assuntos
Caseína Quinase II/metabolismo , Ciclinas/metabolismo , Mitose , Proteólise , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Ciclinas/química , Células HEK293 , Células HeLa , HumanosRESUMO
After DNA damage, the cell cycle is arrested to avoid propagation of mutations. Arrest in G2 phase is initiated by ATM-/ATR-dependent signaling that inhibits mitosis-promoting kinases such as Plk1. At the same time, Plk1 can counteract ATR-dependent signaling and is required for eventual resumption of the cell cycle. However, what determines when Plk1 activity can resume remains unclear. Here, we use FRET-based reporters to show that a global spread of ATM activity on chromatin and phosphorylation of ATM targets including KAP1 control Plk1 re-activation. These phosphorylations are rapidly counteracted by the chromatin-bound phosphatase Wip1, allowing cell cycle restart despite persistent ATM activity present at DNA lesions. Combining experimental data and mathematical modeling, we propose a model for how the minimal duration of cell cycle arrest is controlled. Our model shows how cell cycle restart can occur before completion of DNA repair and suggests a mechanism for checkpoint adaptation in human cells.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Proteína Fosfatase 2C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Humanos , Modelos Biológicos , Modelos Teóricos , Fosforilação , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/metabolismo , Proteína 28 com Motivo Tripartido , Quinase 1 Polo-LikeRESUMO
DNA damage response (DDR) pathway protects cells from genome instability and prevents cancer development. Tumor suppressor p53 is a key molecule that interconnects DDR, cell cycle checkpoints, and cell fate decisions in the presence of genotoxic stress. Inactivating mutations in TP53 and other genes implicated in DDR potentiate cancer development and also influence the sensitivity of cancer cells to treatment. Protein phosphatase 2C delta (referred to as WIP1) is a negative regulator of DDR and has been proposed as potential pharmaceutical target. Until recently, exploitation of WIP1 inhibition for suppression of cancer cell growth was compromised by the lack of selective small-molecule inhibitors effective at cellular and organismal levels. Here, we review recent advances in development of WIP1 inhibitors and discuss their potential use in cancer treatment.
Assuntos
Neoplasias/tratamento farmacológico , Proteína Fosfatase 2C/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Dano ao DNA , Humanos , Neoplasias/metabolismo , Oncogenes , Conformação Proteica , Proteína Fosfatase 2C/química , Proteína Fosfatase 2C/imunologia , Proteína Fosfatase 2C/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
In response to DNA damage, a cell can be forced to permanently exit the cell cycle and become senescent. Senescence provides an early barrier against tumor development by preventing proliferation of cells with damaged DNA. By studying single cells, we show that Cdk activity persists after DNA damage until terminal cell cycle exit. This low level of Cdk activity not only allows cell cycle progression, but also promotes cell cycle exit at a decision point in G2 phase. We find that residual Cdk1/2 activity is required for efficient p21 production, allowing for nuclear sequestration of Cyclin B1, subsequent APC/CCdh1 -dependent degradation of mitotic inducers and induction of senescence. We suggest that the same activity that triggers mitosis in an unperturbed cell cycle enforces senescence in the presence of DNA damage, ensuring a robust response when most needed.
Assuntos
Proteína Quinase CDC2/genética , Senescência Celular/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/genética , Etoposídeo/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Antígenos CD , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Tamanho Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclina B1/genética , Ciclina B1/metabolismo , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Regulação da Expressão Gênica , Humanos , Osteoblastos/citologia , Osteoblastos/enzimologia , Pteridinas/farmacologia , Purinas/farmacologia , Quinolinas/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/enzimologia , Transdução de Sinais , Análise de Célula Única , Tiazóis/farmacologiaRESUMO
B-Myb, a highly conserved member of the Myb transcription factor family, is expressed ubiquitously in proliferating cells and controls the cell cycle dependent transcription of G2/M-phase genes. Deregulation of B-Myb has been implicated in oncogenesis and loss of genomic stability. We have identified B-Myb as a novel interaction partner of the Mre11-Rad50-Nbs1 (MRN) complex, a key player in the repair of DNA double strand breaks. We show that B-Myb directly interacts with the Nbs1 subunit of the MRN complex and is recruited transiently to DNA-damage sites. In response to DNA-damage B-Myb is phosphorylated by protein kinase GSK3ß and released from the MRN complex. A B-Myb mutant that cannot be phosphorylated by GSK3ß disturbs the regulation of pro-mitotic B-Myb target genes and leads to inappropriate mitotic entry in response to DNA-damage. Overall, our work suggests a novel function of B-Myb in the cellular DNA-damage signalling.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Hidrolases Anidrido Ácido , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a DNA/química , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Proteína Homóloga a MRE11/química , Mitose/genética , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Nucleares/química , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transativadores/química , Transativadores/genéticaRESUMO
PP2C family serine/threonine phosphatase WIP1 acts as a negative regulator of the tumor suppressor p53 and is implicated in silencing of cellular responses to genotoxic stress. Chromosomal locus 17q23 carrying the PPM1D (coding for WIP1) is commonly amplified in breast carcinomas and WIP1 was proposed as potential pharmacological target. Here we employed a cellular model with knocked out PPM1D to validate the specificity and efficiency of GSK2830371, novel small molecule inhibitor of WIP1. We have found that GSK2830371 increased activation of the DNA damage response pathway to a comparable level as the loss of PPM1D. In addition, GSK2830371 did not affect proliferation of cells lacking PPM1D but significantly supressed proliferation of breast cancer cells with amplified PPM1D. Over time cells treated with GSK2830371 accumulated in G1 and G2 phases of the cell cycle in a p21-dependent manner and were prone to induction of senescence by a low dose of MDM2 antagonist nutlin-3. In addition, combined treatment with GSK2830371 and doxorubicin or nutlin-3 potentiated cell death through a strong induction of p53 pathway and activation of caspase 9. We conclude that efficient inhibition of WIP1 by GSK2830371 sensitizes breast cancer cells with amplified PPM1D and wild type p53 to chemotherapy.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Imidazóis/farmacologia , Piperazinas/farmacologia , Proteína Fosfatase 2C/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Aminopiridinas/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Dipeptídeos/farmacologia , Feminino , Humanos , Proteína Fosfatase 2C/genética , Proteína Fosfatase 2C/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Ataxia telangiectasia-mutated and Rad3-related (ATR) protein kinase, a master regulator of DNA-damage response, is activated by RPA-coated single-stranded DNA (ssDNA) generated at stalled replication forks or DNA double-strand breaks (DSBs). Here, we identify the mismatch-binding protein MutSß, a heterodimer of MSH2 and MSH3, as a key player in this process. MSH2 and MSH3 form a complex with ATR and its regulatory partner ATRIP, and their depletion compromises the formation of ATRIP foci and phosphorylation of ATR substrates in cells responding to replication-associated DSBs. Purified MutSß binds to hairpin loop structures that persist in RPA-ssDNA complexes and promotes ATRIP recruitment. Mutations in the mismatch-binding domain of MSH3 abolish the binding of MutSß to DNA hairpin loops and its ability to promote ATR activation by ssDNA. These results suggest that hairpin loops might form in ssDNA generated at sites of DNA damage and trigger ATR activation in a process mediated by MutSß.