RESUMO
The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.
Assuntos
Neoplasias , Proteínas Supressoras de Tumor , Humanos , Proteínas Supressoras de Tumor/metabolismo , Proteína BRCA1/metabolismo , Ubiquitinação , Histonas/genética , Histonas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Reparo de DNA por Recombinação , DNA , Reparo do DNARESUMO
End resection of DNA double-strand breaks (DSBs) to generate 3'-single-stranded DNA facilitates DSB repair via error-free homologous recombination (HR) while stymieing repair by the error-prone non-homologous end joining (NHEJ) pathway. Activation of DNA end resection involves phosphorylation of the 5' to 3' exonuclease EXO1 by the phosphoinositide 3-kinase-like kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related) and by the cyclin-dependent kinases 1 and 2. After activation, EXO1 must also be restrained to prevent over-resection that is known to hamper optimal HR and trigger global genomic instability. However, mechanisms by which EXO1 is restrained are still unclear. Here, we report that EXO1 is rapidly degraded by the ubiquitin-proteasome system soon after DSB induction in human cells. ATR inhibition attenuated DNA-damage-induced EXO1 degradation, indicating that ATR-mediated phosphorylation of EXO1 targets it for degradation. In accord with these results, EXO1 became resistant to degradation when its SQ motifs required for ATR-mediated phosphorylation were mutated. We show that upon the induction of DNA damage, EXO1 is ubiquitinated by a member of the Skp1-Cullin1-F-box (SCF) family of ubiquitin ligases in a phosphorylation-dependent manner. Importantly, expression of degradation-resistant EXO1 resulted in hyper-resection, which attenuated both NHEJ and HR and severely compromised DSB repair resulting in chromosomal instability. These findings indicate that the coupling of EXO1 activation with its eventual degradation is a timing mechanism that limits the extent of DNA end resection for accurate DNA repair.
Assuntos
Instabilidade Cromossômica/fisiologia , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/fisiologia , Exodesoxirribonucleases/metabolismo , Proteólise , Ubiquitinação/fisiologia , Motivos de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Enzimas Reparadoras do DNA/genética , Ativação Enzimática/fisiologia , Exodesoxirribonucleases/genética , Células HEK293 , Células HeLa , Humanos , Fosforilação/fisiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Several homology-dependent pathways can repair potentially lethal DNA double-strand breaks (DSBs). The first step common to all homologous recombination reactions is the 5'-3' degradation of DSB ends that yields the 3' single-stranded DNA required for the loading of checkpoint and recombination proteins. In yeast, the Mre11-Rad50-Xrs2 complex (Xrs2 is known as NBN or NBS1 in humans) and Sae2 (known as RBBP8 or CTIP in humans) initiate end resection, whereas long-range resection depends on the exonuclease Exo1, or the helicase-topoisomerase complex Sgs1-Top3-Rmi1 together with the endonuclease Dna2 (refs 1-6). DSBs occur in the context of chromatin, but how the resection machinery navigates through nucleosomal DNA is a process that is not well understood. Here we show that the yeast Saccharomyces cerevisiae Fun30 protein and its human counterpart SMARCAD1 (ref. 8), two poorly characterized ATP-dependent chromatin remodellers of the Snf2 ATPase family, are directly involved in the DSB response. Fun30 physically associates with DSB ends and directly promotes both Exo1- and Sgs1-dependent end resection through a mechanism involving its ATPase activity. The function of Fun30 in resection facilitates the repair of camptothecin-induced DNA lesions, although it becomes dispensable when Exo1 is ectopically overexpressed. Interestingly, SMARCAD1 is also recruited to DSBs, and the kinetics of recruitment is similar to that of EXO1. The loss of SMARCAD1 impairs end resection and recombinational DNA repair, and renders cells hypersensitive to DNA damage resulting from camptothecin or poly(ADP-ribose) polymerase inhibitor treatments. These findings unveil an evolutionarily conserved role for the Fun30 and SMARCAD1 chromatin remodellers in controlling end resection, homologous recombination and genome stability in the context of chromatin.
Assuntos
Montagem e Desmontagem da Cromatina , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Reparo do DNA , DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Camptotecina/farmacologia , Linhagem Celular , Sobrevivência Celular , DNA/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Helicases/deficiência , DNA Helicases/genética , Reparo do DNA/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Instabilidade Genômica/genética , Histonas/metabolismo , Recombinação Homóloga/genética , Humanos , Mutação , Nucleossomos/genética , Nucleossomos/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/metabolismo , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
DSS1, essential for BRCA2-RAD51 dependent homologous recombination (HR), associates with the helical domain (HD) and OB fold 1 (OB1) of the BRCA2 DSS1/DNA-binding domain (DBD) which is frequently targeted by cancer-associated pathogenic variants. Herein, we reveal robust ss/dsDNA binding abilities in HD-OB1 subdomains and find that DSS1 shuts down HD-OB1's DNA binding to enable ssDNA targeting of the BRCA2-RAD51 complex. We show that C-terminal helix mutations of DSS1, including the cancer-associated R57Q mutation, disrupt this DSS1 regulation and permit dsDNA binding of HD-OB1/BRCA2-DBD. Importantly, these DSS1 mutations impair BRCA2/RAD51 ssDNA loading and focus formation and cause decreased HR efficiency, destabilization of stalled forks and R-loop accumulation, and hypersensitize cells to DNA-damaging agents. We propose that DSS1 restrains the intrinsic dsDNA binding of BRCA2-DBD to ensure BRCA2/RAD51 targeting to ssDNA, thereby promoting optimal execution of HR, and potentially replication fork protection and R-loop suppression.
Assuntos
Proteína BRCA2 , Replicação do DNA , DNA de Cadeia Simples , DNA , Recombinação Homóloga , Mutação , Rad51 Recombinase , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/química , Humanos , DNA/metabolismo , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Homeostase , Ligação Proteica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Domínios Proteicos , Linhagem Celular Tumoral , Dano ao DNA , Complexo de Endopeptidases do ProteassomaRESUMO
DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.
Assuntos
Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Exodesoxirribonucleases/metabolismo , Recombinação Genética , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Aberrações Cromossômicas , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/fisiologia , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Rad51 Recombinase/metabolismo , Radiação Ionizante , Proteínas Supressoras de Tumor/metabolismoRESUMO
Bloom syndrome (BS) is associated with a profoundly increased cancer risk and is caused by mutations in the Bloom helicase (BLM). BLM is involved in the nucleolytic processing of the ends of DNA double-strand breaks (DSBs), to yield long 3' ssDNA tails that serve as the substrate for break repair by homologous recombination (HR). Here, we use single-molecule imaging to demonstrate that BLM mediates formation of large ssDNA loops during DNA end processing. A BLM mutant lacking the N-terminal domain (NTD) retains vigorous in vitro end processing activity but fails to generate ssDNA loops. This same mutant supports DSB end processing in cells, however, these cells do not form RAD51 DNA repair foci and the processed DSBs are channeled into synthesis-dependent strand annealing (SSA) instead of HR-mediated repair, consistent with a defect in RAD51 filament formation. Together, our results provide insights into BLM functions during homologous recombination.
Assuntos
DNA de Cadeia Simples , RecQ Helicases , DNA/genética , DNA de Cadeia Simples/genética , Recombinação Homóloga/genética , RecQ Helicases/genética , RecQ Helicases/metabolismoRESUMO
The cellular response to DNA double-strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell-cycle-dependent manner. Here, we report that the crucial checkpoint signalling proteins-p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2-are phosphorylated rapidly by ATR in an ATM/Mre11/cell-cycle-independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM-deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM-deficient cells enforce an early G2/M checkpoint that is ATR-dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM-deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP-1), a protein involved in chromatin remodelling, is mediated by DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in a spatio-temporal manner in addition to ATM. We posit that ATM substrates involved in cell-cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA-PKcs in addition to ATM.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/deficiência , Ataxia Telangiectasia/enzimologia , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Fase G2 , Humanos , Fosforilação , Transporte Proteico , Proteína de Replicação A/metabolismo , Proteínas Repressoras/metabolismo , Proteína 28 com Motivo TripartidoRESUMO
Glioblastomas (GBM) are routinely treated with ionizing radiation (IR) but inevitably recur and develop therapy resistance. During treatment, the tissue surrounding tumors is also irradiated. IR potently induces senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting factors that create a senescence-associated secretory phenotype (SASP). Here, we carried out transcriptomic and tumorigenicity analyses in irradiated mouse brains to elucidate how radiotherapy-induced senescence of non-neoplastic brain cells promotes tumor growth. Following cranial irradiation, widespread senescence in the brain occurred, with the astrocytic population being particularly susceptible. Irradiated brains showed an altered transcriptomic profile characterized by upregulation of CDKN1A (p21), a key enforcer of senescence, and several SASP factors, including HGF, the ligand of the receptor tyrosine kinase (RTK) Met. Preirradiation of mouse brains increased Met-driven growth and invasiveness of orthotopically implanted glioma cells. Importantly, irradiated p21-/- mouse brains did not exhibit senescence and consequently failed to promote tumor growth. Senescent astrocytes secreted HGF to activate Met in glioma cells and to promote their migration and invasion in vitro, which could be blocked by HGF-neutralizing antibodies or the Met inhibitor crizotinib. Crizotinib also slowed the growth of glioma cells implanted in preirradiated brains. Treatment with the senolytic drug ABT-263 (navitoclax) selectively killed senescent astrocytes in vivo, significantly attenuating growth of glioma cells implanted in preirradiated brains. These results indicate that SASP factors in the irradiated tumor microenvironment drive GBM growth via RTK activation, underscoring the potential utility of adjuvant senolytic therapy for preventing GBM recurrence after radiotherapy. SIGNIFICANCE: This study uncovers mechanisms by which radiotherapy can promote GBM recurrence by inducing senescence in non-neoplastic brain cells, suggesting that senolytic therapy can blunt recurrent GBM growth and aggressiveness.
Assuntos
Encéfalo/patologia , Senescência Celular , Raios gama/efeitos adversos , Glioblastoma/patologia , Recidiva Local de Neoplasia/patologia , Fenótipo Secretor Associado à Senescência , Microambiente Tumoral , Compostos de Anilina/farmacologia , Animais , Antineoplásicos/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/etiologia , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/etiologia , Recidiva Local de Neoplasia/metabolismo , Sulfonamidas/farmacologiaRESUMO
DNA double-strand break repair by homologous recombination begins with nucleolytic resection of the 5' DNA strand at the break ends. Long-range resection is catalyzed by EXO1 and BLM-DNA2, which likely have to navigate through ribonucleotides and damaged bases. Here, we show that a short stretch of ribonucleotides at the 5' terminus stimulates resection by EXO1. Ribonucleotides within a 5' flap are resistant to cleavage by DNA2, and extended RNA:DNA hybrids inhibit both strand separation by BLM and resection by EXO1. Moreover, 8-oxo-guanine impedes EXO1 but enhances resection by BLM-DNA2, and an apurinic/apyrimidinic site stimulates resection by BLM-DNA2 and DNA strand unwinding by BLM. Accordingly, depletion of OGG1 or APE1 leads to greater dependence of DNA resection on DNA2. Importantly, RNase H2A deficiency impairs resection overall, which we attribute to the accumulation of long RNA:DNA hybrids at DNA ends. Our results help explain why eukaryotic cells possess multiple resection nucleases.
Assuntos
Quebras de DNA de Cadeia Dupla , Ribonucleotídeos/genética , Ribonucleotídeos/metabolismo , Western Blotting , Linhagem Celular Tumoral , DNA Glicosilases/genética , Enzimas Reparadoras do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Exodesoxirribonucleases/genética , Imunofluorescência , Recombinação Homóloga/genética , Humanos , RecQ Helicases/genética , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Ions of high atomic number and energy (HZE particles) pose a significant cancer risk to astronauts on prolonged space missions. On Earth, similar ions are being used for targeted cancer therapy. The properties of these particles can be drastically altered during passage through spacecraft shielding, therapy beam modulators, or the human body. Here, we have used pertinent responses to DNA double-strand breaks (DSBs) to understand the consequences of energy loss versus nuclear fragmentation of Fe ions during passage through shielding or tissue-equivalent materials. Phosphorylation of histone H2AX and recruitment of 53BP1 were used to generate 3D reconstructions of DNA damage in human cells and to follow its repair. Human cells are unable to repair a significant portion of DNA damage induced by Fe ions. DNA-PK and ATM are required, to different extents, for the partial repair of Fe-induced DNA damage. Aluminum shielding has little effect on DNA damage or its repair, confirming that the hulls of the Space Shuttle and the International Space Station afford scant protection against these particles. Lead shielding, on the other hand, exacerbates the effects of Fe ions due to energy loss during particle traversal. In sharp contrast, polyethylene (PE), a favored hydrogenous shield, results in DNA damage that is more amenable to repair presumably due to Fe-ion fragmentation. Human cells are indeed able to efficiently repair DSBs induced by chlorine ions and protons that represent fragmentation products of Fe. Interestingly, activation of the tumor suppressor p53 in Fe-irradiated cells is uniquely biphasic and culminates in the induction of high levels of p21 (Waf1/Cip1), p16 (INK4a) and senescence-associated beta-galactosidase activity. Surprisingly, these events occur even in the absence of ATM kinase implying that ATR may be a major responder to the complex DNA damage inflicted by Fe ions. Significantly, fragmentation of the Fe beam through PE attenuates these responses and this, in turn, results in better long-term survival in a colony-forming assay. Our results help us to understand the biological consequences of ion fragmentation through materials, whether in space or in the clinic, and provide us with a biological basis for the use of hydrogenous materials like PE as effective space shields.
Assuntos
Radiação Cósmica/efeitos adversos , Dano ao DNA , Proteção Radiológica , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Senescência Celular/efeitos da radiação , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Reparo do DNA/efeitos da radiação , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/enzimologia , Fibroblastos/efeitos da radiação , Humanos , Ferro , Transferência Linear de Energia/efeitos da radiação , Polietileno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
EGFR signaling has been implicated in hypoxia-associated resistance to radiation or chemotherapy. Non-small cell lung carcinomas (NSCLC) with activating L858R or ΔE746-E750 EGFR mutations exhibit elevated EGFR activity and downstream signaling. Here, relative to wild-type (WT) EGFR, mutant (MT) EGFR expression significantly increases radiosensitivity in hypoxic cells. Gene expression profiling in human bronchial epithelial cells (HBEC) revealed that MT-EGFR expression elevated transcripts related to cell cycle and replication in aerobic and hypoxic conditions and downregulated RAD50, a critical component of nonhomologous end joining and homologous recombination DNA repair pathways. NSCLCs and HBEC with MT-EGFR revealed elevated basal and hypoxia-induced γ-H2AX-associated DNA lesions that were coincident with replication protein A in the S-phase nuclei. DNA fiber analysis showed that, relative to WT-EGFR, MT-EGFR NSCLCs harbored significantly higher levels of stalled replication forks and decreased fork velocities in aerobic and hypoxic conditions. EGFR blockade by cetuximab significantly increased radiosensitivity in hypoxic cells, recapitulating MT-EGFR expression and closely resembling synthetic lethality of PARP inhibition.Implications: This study demonstrates that within an altered DNA damage response of hypoxic NSCLC cells, mutant EGFR expression, or EGFR blockade by cetuximab exerts a synthetic lethality effect and significantly compromises radiation resistance in hypoxic tumor cells. Mol Cancer Res; 15(11); 1503-16. ©2017 AACR.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Replicação do DNA , DNA/metabolismo , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Células A549 , Hidrolases Anidrido Ácido , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cetuximab/farmacologia , Dano ao DNA , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologiaRESUMO
Unrepaired DNA double-strand breaks (DSB) are the most destructive chromosomal lesions driving genomic instability, a core hallmark of cancer. Here, we identify the antioncogenic breast cancer factor INT6/EIF3E as an essential regulator of DSB repair that promotes homologous recombination (HR)-mediated repair and, to a lesser extent, nonhomologous end-joining repair. INT6 silencing impaired the accrual of the ubiquitin ligase RNF8 at DSBs and the formation of ubiquitin conjugates at DSB sites, especially Lys63-linked polyubiquitin chains, resulting in impaired recruitment of BRCA1, BRCA2, and RAD51, which are all involved in HR repair. In contrast, INT6 deficiency did not affect the accumulation of RNF168, 53BP1, or RPA at DSBs. In INT6-silenced cells, there was also an alteration in DNA damage-induced localization of MDC1, a key target for ATM phosphorylation, which is a prerequisite for RNF8 recruitment. The attenuated DNA damage localization of RNF8 resulting from INT6 depletion could be attributed to the defective retention of ATM previously reported by us. Our findings deepen insights into how INT6 protects against breast cancer by showing how it functions in DSB repair, with potential clinical implications for cancer therapy. Cancer Res; 76(20); 6054-65. ©2016 AACR.
Assuntos
Neoplasias da Mama/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Fator de Iniciação 3 em Eucariotos/fisiologia , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA2/fisiologia , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular , Feminino , Células HeLa , Recombinação Homóloga , Humanos , Proteínas Nucleares/metabolismo , Rad51 Recombinase/metabolismo , Transativadores/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Double-strand breaks (DSBs) are the most deleterious among all types of DNA damage that can occur in the cell. These breaks arise from both endogenous (e.g., DNA replication stress) and exogenous insults (e.g., ionizing radiation). DSBs are principally repaired by one of two major pathways: nonhomologous end joining (NHEJ) or homologous recombination (HR). NHEJ is an error-prone process that can occur in all phases of the cell cycle, while HR is limited to the S and G2 phases of the cell cycle when a sister chromatid is available as a template for error-free repair. The first step in HR is "DNA end resection," a process during which the broken DNA end is converted into a long stretch of 3'-ended single-stranded DNA (ssDNA). In recent years, DNA end resection has been identified as a pivotal step that controls "repair pathway choice," i.e., the appropriate choice between NHEJ and HR for DSB repair. Therefore, methods to quantitatively or semiquantitatively assess DNA end resection have gained importance in laboratories working on DNA repair. In this chapter, we describe two simple immunofluorescence-based techniques to monitor DNA end resection in mammalian cells. The first technique involves immuno-detection of replication protein A (RPA), an ssDNA-binding protein that binds to resected DNA. The second technique involves labeling of genomic DNA with 5-bromo-2'-deoxyuridine (BrdU) that can be detected by anti-BrdU antibody only after the DNA becomes single stranded due to resection. These methods are not complicated, do not involve sophisticated instrumentation or reporter constructs, and can be applied to most mammalian cell lines and, therefore, should be of broad utility as simple ways of monitoring DNA end resection in vivo.
Assuntos
Reparo do DNA por Junção de Extremidades/genética , Imunofluorescência/métodos , Animais , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/genética , HumanosRESUMO
Resection of DNA double-strand breaks (DSBs) is a pivotal step during which the choice between NHEJ and HR DNA repair pathways is made. Although CDKs are known to control initiation of resection, their role in regulating long-range resection remains elusive. Here we show that CDKs 1/2 phosphorylate the long-range resection nuclease EXO1 at four C-terminal S/TP sites during S/G2 phases of the cell cycle. Impairment of EXO1 phosphorylation attenuates resection, chromosomal integrity, cell survival and HR, but augments NHEJ upon DNA damage. In contrast, cells expressing phospho-mimic EXO1 are proficient in resection even after CDK inhibition and favour HR over NHEJ. Mutation of cyclin-binding sites on EXO1 attenuates CDK binding and EXO1 phosphorylation, causing a resection defect that can be rescued by phospho-mimic mutations. Mechanistically, phosphorylation of EXO1 augments its recruitment to DNA breaks possibly via interactions with BRCA1. In summary, phosphorylation of EXO1 by CDKs is a novel mechanism regulating repair pathway choice.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Fosforilação/fisiologia , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Citometria de Fluxo , Imunofluorescência , Humanos , Imunoprecipitação , Fosforilação/genéticaRESUMO
PURPOSE: Inhibitors of the DNA damage response (DDR) have great potential for radiosensitization of numerous cancers, including glioblastomas, which are extremely radio- and chemoresistant brain tumors. Currently, there are no DNA double-strand break (DSB) repair inhibitors that have been successful in treating glioblastoma. Our laboratory previously demonstrated that the dual phosphoinositide 3-kinase/mTOR inhibitor NVP-BEZ235 can potently inhibit the two central DDR kinases, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated (ATM), in vitro. Here, we tested whether NVP-BEZ235 could also inhibit ATM and DNA-PKcs in tumors in vivo and assessed its potential as a radio- and chemosensitizer in preclinical mouse glioblastoma models. EXPERIMENTAL DESIGN: The radiosensitizing effect of NVP-BEZ235 was tested by following tumor growth in subcutaneous and orthotopic glioblastoma models. Tumors were generated using the radioresistant U87-vIII glioma cell line and GBM9 neurospheres in nude mice. These tumors were then treated with ionizing radiation and/or NVP-BEZ235 and analyzed for DNA-PKcs and ATM activation, DSB repair inhibition, and attenuation of growth. RESULTS: NVP-BEZ235 potently inhibited both DNA-PKcs and ATM kinases and attenuated the repair of ionizing radiation-induced DNA damage in tumors. This resulted in striking tumor radiosensitization, which extended the survival of brain tumor-bearing mice. Notably, tumors displayed a higher DSB-load when compared with normal brain tissue. NVP-BEZ235 also sensitized a subset of subcutaneous tumors to temozolomide, a drug routinely used concurrently with ionizing radiation for the treatment of glioblastoma. CONCLUSIONS: These results demonstrate that it may be possible to significantly improve glioblastoma therapy by combining ionizing radiation with potent and bioavailable DNA repair inhibitors such as NVP-BEZ235.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Imidazóis/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Quinolinas/farmacologia , Radiossensibilizantes/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Barreira Hematoencefálica/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/química , Proteína Quinase Ativada por DNA/metabolismo , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Modelos Animais de Doenças , Glioblastoma/tratamento farmacológico , Glioblastoma/mortalidade , Glioblastoma/patologia , Glioblastoma/radioterapia , Humanos , Camundongos , Camundongos Transgênicos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , TemozolomidaRESUMO
Inhibitors of PI3K/Akt signaling are being actively developed for tumor therapy owing to the frequent mutational activation of the PI3K-Akt-mTORC1 pathway in many cancers, including glioblastomas (GBMs). NVP-BEZ235 is a novel and potent dual PI3K/mTOR inhibitor that is currently in phase 1/2 clinical trials for advanced solid tumors. Here, we show that NVP-BEZ235 also potently inhibits ATM and DNA-PKcs, the two major kinases responding to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs). Consequently, NVP-BEZ235 blocks both nonhomologous end joining and homologous recombination DNA repair pathways resulting in significant attenuation of DSB repair. In addition, phosphorylation of ATMtargets and implementation of the G(2)/M cell cycle checkpoint are also attenuated by this drug. As a result, NVP-BEZ235 confers an extreme degree of radiosensitization and impairs DSB repair in a panel of GBM cell lines irrespective of their Akt activation status. NVP-BEZ235 also significantly impairs DSB repair in a mouse tumor model thereby validating the efficacy of this drug as a DNA repair inhibitor in vivo. Our results, showing that NVP-BEZ235 is a potent and novel inhibitor of ATM and DNA-PKcs, have important implications for the informed and rational design of clinical trials involving this drug and also reveal the potential utility of NVP-BEZ235 as an effective radiosensitizer for GBMs in the clinic.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Imidazóis/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinolinas/farmacologia , Radiossensibilizantes/farmacologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Separação Celular , Dano ao DNA/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Humanos , Camundongos , Neoplasias Experimentais/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The resection of DNA double-strand breaks (DSBs) to generate ssDNA tails is a pivotal event in the cellular response to these breaks. In the two-step model of resection, primarily elucidated in yeast, initial resection by Mre11-CtIP is followed by extensive resection by two distinct pathways involving Exo1 or BLM/WRN-Dna2. However, resection pathways and their exact contributions in humans in vivo are not as clearly worked out as in yeast. Here, we examined the contribution of Exo1 to DNA end resection in humans in vivo in response to ionizing radiation (IR) and its relationship with other resection pathways (Mre11-CtIP or BLM/WRN). We find that Exo1 plays a predominant role in resection in human cells along with an alternate pathway dependent on WRN. While Mre11 and CtIP stimulate resection in human cells, they are not absolutely required for this process and Exo1 can function in resection even in the absence of Mre11-CtIP. Interestingly, the recruitment of Exo1 to DNA breaks appears to be inhibited by the NHEJ protein Ku80, and the higher level of resection that occurs upon siRNA-mediated depletion of Ku80 is dependent on Exo1. In addition, Exo1 may be regulated by 53BP1 and Brca1, and the restoration of resection in BRCA1-deficient cells upon depletion of 53BP1 is dependent on Exo1. Finally, we find that Exo1-mediated resection facilitates a transition from ATM- to ATR-mediated cell cycle checkpoint signaling. Our results identify Exo1 as a key mediator of DNA end resection and DSB repair and damage signaling decisions in human cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Exodesoxirribonucleases/metabolismo , Transdução de Sinais/genética , Antígenos Nucleares/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Autoantígeno Ku , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos da radiação , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Altered expression of the INT6 gene, encoding the e subunit of the translational initiation factor eIF3, occurs in human breast cancers, but how INT6 relates to carcinogenesis remains unestablished. Here, we show that INT6 is involved in the DNA damage response. INT6 was required for cell survival following γ-irradiation and G(2)-M checkpoint control. RNA interference-mediated silencing of INT6 reduced phosphorylation of the checkpoint kinases CHK1 and CHK2 after DNA damage. In addition, INT6 silencing prevented sustained accumulation of ataxia telangiectasia mutated (ATM) at DNA damage sites in cells treated with γ-radiation or the radiomimetic drug neocarzinostatin. Mechanistically, this result could be explained by interaction of INT6 with ATM, which together with INT6 was recruited to the sites of DNA damage. Finally, INT6 silencing also reduced ubiquitylation events that promote retention of repair proteins at DNA lesions. Accordingly, accumulation of the repair factor BRCA1 was defective in the absence of INT6. Our findings reveal unexpected and striking connections of INT6 with ATM and BRCA1 and suggest that the protective action of INT6 in the onset of breast cancers relies on its involvement in the DNA damage response.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/metabolismo , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Immunoblotting , Imunoprecipitação , Microscopia ConfocalRESUMO
PURPOSE: Radiation therapy (RT) is one of the primary modalities for treatment of non-small cell lung cancer (NSCLC). However, due to the intrinsic radiation resistance of these tumors, many patients experience RT failure, which leads to considerable tumor progression including regional lymph node and distant metastasis. This preclinical study evaluated the efficacy of a new-generation cyclin-dependent kinase (Cdk) inhibitor, AZD5438, as a radiosensitizer in several NSCLC models that are specifically resistant to conventional fractionated RT. METHODS AND MATERIALS: The combined effect of ionizing radiation and AZD5438, a highly specific inhibitor of Cdk1, 2, and 9, was determined in vitro by surviving fraction, cell cycle distribution, apoptosis, DNA double-strand break (DSB) repair, and homologous recombination (HR) assays in 3 NSCLC cell lines (A549, H1299, and H460). For in vivo studies, human xenograft animal models in athymic nude mice were used. RESULTS: Treatment of NSCLC cells with AZD5438 significantly augmented cellular radiosensitivity (dose enhancement ratio rangeing from 1.4 to 1.75). The degree of radiosensitization by AZD5438 was greater in radioresistant cell lines (A549 and H1299). Radiosensitivity was enhanced specifically through inhibition of Cdk1, prolonged G(2)-M arrest, inhibition of HR, delayed DNA DSB repair, and increased apoptosis. Combined treatment with AZD5438 and irradiation also enhanced tumor growth delay, with an enhancement factor ranging from 1.2-1.7. CONCLUSIONS: This study supports the evaluation of newer generation Cdk inhibitors, such as AZD5438, as potent radiosensitizers in NSCLC models, especially in tumors that demonstrate variable intrinsic radiation responses.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/radioterapia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Imidazóis/farmacologia , Neoplasias Pulmonares/radioterapia , Pirimidinas/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Animais , Apoptose , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Feminino , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glioblastomas (GBM) are lethal brain tumors that are highly resistant to therapy. The only meaningful improvement in therapeutic response came from use of the S(N)1-type alkylating agent temozolomide in combination with ionizing radiation. However, no genetic markers that might predict a better response to DNA alkylating agents have been identified in GBMs, except for loss of O(6-)methylguanine-DNA methyltransferase via promoter methylation. In this study, using genetically defined primary murine astrocytes as well as human glioma lines, we show that loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) confers sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a functional analogue of temozolomide. We find that MNNG induces replication-associated DNA double-strand breaks (DSB), which are inefficiently repaired in PTEN-deficient astrocytes and trigger apoptosis. Mechanistically, this is because PTEN-null astrocytes are compromised in homologous recombination (HR), which is important for the repair of replication-associated DSBs. Our results suggest that reduced levels of Rad51 paralogs in PTEN-null astrocytes might underlie the HR deficiency of these cells. Importantly, the HR deficiency of PTEN-null cells renders them sensitive to the poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 due to synthetic lethality. In sum, our results tentatively suggest that patients with PTEN-null GBMs (about 36%) may especially benefit from treatment with DNA alkylating agents such as temozolomide. Significantly, our results also provide a rational basis for treating the subgroup of patients who are PTEN deficient with PARP inhibitors in addition to the current treatment regimen of radiation and temozolomide.