RESUMO
The eukaryotic ssDNA-binding protein, Replication protein A (RPA), was first discovered almost three decades ago. Since then, much progress has been made to elucidate the critical roles for RPA in DNA metabolic pathways that help promote genomic stability. The canonical RPA heterotrimer (RPA1-3) is an essential coordinator of DNA metabolism that interacts with ssDNA and numerous protein partners to coordinate its roles in DNA replication, repair, recombination and telomere maintenance. An alternative form of RPA, termed aRPA, is formed by a complex of RPA4 with RPA1 and RPA3. aRPA is expressed differentially in cells compared to canonical RPA and has been shown to inhibit canonical RPA function while allowing for regular maintenance of cell viability. Interestingly, while aRPA is defective in DNA replication and cell cycle progression, it was shown to play a supporting role in nucleotide excision repair and recombination. The binding domains of canonical RPA interact with a growing number of partners involved in numerous genome maintenance processes. The protein interactions of the RPA-ssDNA complex are not only governed by competition between the binding proteins but also by post-translation modifications such as phosphorylation. Phosphorylation of RPA2 is an important post-translational modification of the RPA complex, and is essential for directing context-specific functions of the RPA complex in the DNA damage response. Due to the importance of RPA in cellular metabolism, it was identified as an appealing target for chemotherapeutic drug development that could be used in future cancer treatment regimens.
Assuntos
DNA/genética , DNA/metabolismo , Genoma/genética , Instabilidade Genômica , Proteína de Replicação A/química , Proteína de Replicação A/metabolismo , Replicação do DNA/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , FosforilaçãoRESUMO
Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms.
Assuntos
Replicação do DNA , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/metabolismo , Western Blotting , Proliferação de Células , Células Cultivadas , Células HeLa , Humanos , Mutação/genética , Fosforilação , Estrutura Terciária de Proteína , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteína de Replicação A/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Técnicas do Sistema de Duplo-HíbridoRESUMO
Replication protein A (RPA), essential for DNA replication, repair and DNA damage signalling, possesses six ssDNA-binding domains (DBDs), including DBD-F on the N-terminus of the largest subunit, RPA70. This domain functions as a binding site for p53 and other DNA damage and repair proteins that contain amphipathic alpha helical domains. Here, we demonstrate direct binding of both ssDNA and the transactivation domain 2 of p53 (p53TAD2) to DBD-F, as well as DBD-F-directed dsDNA strand separation by RPA, all of which are inhibited by fumaropimaric acid (FPA). FPA binds directly to RPA, resulting in a conformational shift as determined through quenching of intrinsic tryptophan fluorescence in full length RPA. Structural analogues of FPA provide insight on chemical properties that are required for inhibition. Finally, we confirm the inability of RPA possessing R41E and R43E mutations to bind to p53, destabilize dsDNA and quench tryptophan fluorescence by FPA, suggesting that protein binding, DNA modulation and inhibitor binding all occur within the same site on DBD-F. The disruption of p53-RPA interactions by FPA may disturb the regulatory functions of p53 and RPA, thereby inhibiting cellular pathways that control the cell cycle and maintain the integrity of the human genome.
Assuntos
Diterpenos/farmacologia , Proteína de Replicação A/química , Proteína Supressora de Tumor p53/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Ligação Competitiva , DNA de Cadeia Simples/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína , Proteína de Replicação A/efeitos dos fármacos , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Proteína Supressora de Tumor p53/químicaRESUMO
Cells respond to DNA double-strand breaks by initiating DSB repair and ensuring a cell cycle checkpoint. The primary responder to DSB repair is non-homologous end joining, which is an error-prone repair pathway. However, when DSBs are generated after DNA replication in the G2 phase of the cell cycle, a second DSB repair pathway, homologous recombination, can come into action. Both ATM and ATR are important for DSB-induced DSB repair and checkpoint responses. One method of ATM and ATR working together is through the DNA end resection of DSBs. As a readout and marker of DNA end resection, RPA is phosphorylated at Ser4/Ser8 of the N-terminus of RPA32 in response to DSBs. Here, the significance of RPA32 Ser4/Ser8 phosphorylation in response to DNA damage, specifically in the S phase to G2 phase of the cell cycle, is examined. RPA32 Ser4/Ser8 phosphorylation in G2 synchronized cells is necessary for increases in TopBP1 and Rad9 accumulation on chromatin and full activation of the ATR-dependent G2 checkpoint. In addition, our data suggest that RPA Ser4/Ser8 phosphorylation modulates ATM-dependent KAP-1 phosphorylation and Rad51 chromatin loading in G2 cells. Through the phosphorylation of RPA Ser4/Ser8, ATM acts as a partner with ATR in the G2 phase checkpoint response, regulating key downstream events including Rad9, TopBP1 phosphorylation and KAP-1 phosphorylation/activation via the targeting of RPA32 Ser4/Ser8.
Assuntos
Proteínas de Ligação a DNA , DNA , Fosforilação , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA/metabolismo , CromatinaRESUMO
During palatogenesis, the palatal mesenchyme undergoes increased cell proliferation resulting in palatal growth, elevation and fusion of the two palatal shelves. Interestingly, the palatal mesenchyme expresses all three transforming growth factor (TGF) ß isoforms (1, 2, and 3) throughout these steps of palatogenesis. However, the role of TGFß in promoting proliferation of palatal mesenchymal cells has never been explored. The purpose of this study was to identify the effect of TGFß on human embryonic palatal mesenchymal (HEPM) cell proliferation. Our results showed that all isoforms of TGFß, especially TGFß3, increased HEPM cell proliferation by up-regulating the expression of cyclins and cyclin-dependent kinases as well as c-Myc oncogene. TGFß activated both Smad-dependent and Smad-independent pathways to induce c-Myc gene expression. Furthermore, TBE1 is the only functional Smad binding element (SBE) in the c-Myc promoter and Smad4, activated by TGFß, binds to the TBE1 to induce c-Myc gene activity. We conclude that HEPM proliferation is manifested by the induction of c-Myc in response to TGFß signaling, which is essential for complete palatal confluency. Our data highlights the potential role of TGFß as a therapeutic molecule to correct cleft palate by promoting growth.
Assuntos
Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Palato/embriologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ativação Transcricional , Fator de Crescimento Transformador beta3/farmacologia , Animais , Apoptose , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Feminino , Humanos , Masculino , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Palato/citologia , Palato/efeitos dos fármacos , Palato/metabolismo , Gravidez , Regiões Promotoras Genéticas , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Proteína Smad4/genética , Proteína Smad4/metabolismo , Transfecção , Fator de Crescimento Transformador beta3/metabolismoRESUMO
Hyaluronan (HA) production has been functionally implicated in prostate tumorigenesis and metastasis. We previously used prostate tumor cells overexpressing the HA synthesizing enzyme HAS3 or the clinically relevant hyaluronidase Hyal1 to show that excess HA production suppresses tumor growth, while HA turnover accelerates spontaneous metastasis from the prostate. Here, we examined pathways responsible for effects of HAS3 and Hyal1 on tumor cell phenotype. Detailed characterization of cell cycle progression revealed that expression of Hyal1 accelerated cell cycle re-entry following synchronization, whereas HAS3 alone delayed entry. Hyal1 expressing cells exhibited a significant reduction in their ability to sustain ERK phosphorylation upon stimulation by growth factors, and in their expression of the cyclin-dependent kinase inhibitor p21. In contrast, HAS3 expressing cells showed prolonged ERK phosphorylation and increased expression of both p21 and p27, in asynchronous and synchronized cultures. Changes in cell cycle regulatory proteins were accompanied by HA-induced suppression of N-cadherin, while E-cadherin expression and ß-catenin expression and distribution remained unchanged. Our results are consistent with a model in which excess HA synthesis suppresses cell proliferation by promoting homotypic E-cadherin mediated cell-cell adhesion, consequently signaling to elevate cell cycle inhibitor expression and suppress G1- to S-phase transition.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Proliferação de Células , Ácido Hialurônico/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais/fisiologia , Antígenos CD/genética , Caderinas/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Fatores de Crescimento/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Platinum-based chemotherapy is the standard first-line treatment for oral squamous cell carcinoma (OSCC) that is inoperable, recurrent, or metastatic. Platinum sensitivity is a major determinant of patient survival in advanced OSCC. Here, we investigated the involvement of MASTL, a cell cycle kinase that mediates ENSA/ARPP19 phosphorylation and PP2A/B55 inhibition, in OSCC therapy. Interestingly, upregulation of MASTL and ENSA/ARPP19, and downregulation of PP2A/B55, were common in OSCC. MASTL expression was in association with poor patient survival. In established OSCC cell lines, upregulation of MASTL and ENSA, and downregulation of B55 genes, correlated with cisplatin resistance. We further confirmed that stable expression of MASTL in OSCC cells promoted cell survival and proliferation under cisplatin treatment, in an ENSA-dependent manner. Conversely, deletion of MASTL or ENSA, or overexpression of B55α, sensitized cisplatin response, consistent with increased DNA damage accumulation, signaling, and caspase activation. Moreover, GKI-1, the first-in-class small molecule inhibitor of MASTL kinase, phenocopied MASTL depletion in enhancing the outcome of cisplatin treatment in OSCC cells, at a dose substantially lower than that needed to disrupt mitotic entry. Finally, GKI-1 exhibited promising efficacy in a mouse tumor xenograft model, in conjunction with cisplatin therapy.
RESUMO
The pharmacological suppression of the DNA damage response and DNA repair can increase the therapeutic indices of conventional chemotherapeutics. Replication Protein A (RPA), the major single-stranded DNA binding protein in eukaryotes, is required for DNA replication, DNA repair, DNA recombination, and DNA damage response signaling. Through the use of high-throughput screening of 1500 compounds, we have identified a small molecule inhibitor, 15-carboxy-13-isopropylatis-13-ene-17,18-dioic acid (NSC15520), that inhibited both the binding of Rad9-GST and p53-GST fusion proteins to the RPA N-terminal DNA binding domain (DBD), interactions that are essential for robust DNA damage signaling. NSC15520 competitively inhibited the binding of p53-GST peptide with an IC(50) of 10 µM. NSC15520 also inhibited helix destabilization of a duplex DNA (dsDNA) oligonucleotide, an activity dependent on the N-terminal domain of RPA70. NSC15520 did not inhibit RPA from binding single-stranded oligonucleotides, suggesting that the action of this inhibitor is specific for the N-terminal DBD of RPA, and does not bind to DBDs essential for single-strand DNA binding. Computer modeling implicates direct competition between NSC15520 and Rad9 for the same binding surface on RPA. Inhibitors of protein-protein interactions within the N-terminus of RPA are predicted to act synergistically with DNA damaging agents and inhibitors of DNA repair. Novel compounds such as NSC15520 have the potential to serve as chemosensitizing agents.
Assuntos
Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteína de Replicação A/antagonistas & inibidores , Sítios de Ligação , Simulação por Computador , Reparo do DNA , DNA de Cadeia Simples , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Humanos , OligonucleotídeosRESUMO
Accumulation of extracellular hyaluronan (HA) and its processing enzyme, the hyaluronidase Hyal1, predicts invasive, metastatic progression of human prostate cancer. To dissect the roles of hyaluronan synthases (HAS) and Hyal1 in tumorigenesis and metastasis, we selected nonmetastatic 22Rv1 prostate tumor cells that overexpress HAS2, HAS3, or Hyal1 individually, and compared these cells with co-transfectants expressing Hyal1 + HAS2 or Hyal1 + HAS3. Cells expressing only HAS were less tumorigenic than vector control transfectants on orthotopic injection into mice. In contrast, cells co-expressing Hyal1 + HAS2 or Hyal1 + HAS3 showed greater than sixfold and twofold increases in tumorigenesis, respectively. Fluorescence and histological quantification revealed spontaneous lymph node metastasis in all Hyal1 transfectant-implanted mice, and node burden increased an additional twofold when Hyal1 and HAS were co-expressed. Cells only expressing HAS were not metastatic. Thus, excess HA synthesis and processing in concert accelerate the acquisition of a metastatic phenotype by prostate tumor cells. Intratumoral vascularity did not correlate with either tumor size or metastatic potential. Analysis of cell cycle progression revealed shortened doubling times of Hyal1-expressing cells. Both adhesion and motility on extracellular matrix were diminished in HA-overproducing cells; however, motility was increased twofold by Hyal1 expression and fourfold to sixfold by Hyal1/HAS co-expression, in close agreement with observed metastatic potential. This is the first comprehensive examination of these enzymes in a relevant prostate cancer microenvironment.
Assuntos
Adenocarcinoma/patologia , Ácido Hialurônico/metabolismo , Metástase Neoplásica/patologia , Neoplasias da Próstata/patologia , Adenocarcinoma/metabolismo , Animais , Ciclo Celular , Movimento Celular , Matriz Extracelular/patologia , Humanos , Ácido Hialurônico/biossíntese , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Neoplasias da Próstata/metabolismo , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
BACKGROUND: Helicobacter hepaticus, the prototype for enterohepatic Helicobacter species, colonizes the lower intestinal and hepatobiliary tracts of mice and causes typhlocolitis, hepatitis, and hepatocellular carcinoma in susceptible mouse strains. Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus. CDT of several Gram-negative bacteria is associated with double-stranded DNA breaks resulting in cell cycle arrest and death of a wide range of eukaryotic cells in vitro. We previously observed H. hepaticus CDT (HhCDT) mediated apoptosis in INT407 cells. However, the exact mechanism for the induction of the apoptotic pathway by HhCDT is unknown. The objective of this study was to identify the apoptotic signaling pathway induced by HhCDT in INT407 cells. MATERIALS AND METHODS: INT407 cells were incubated with or without recombinant HhCDT for 0-72 hours. H2AX phosphorylation and apoptotic parameters were analyzed. RESULTS: H2AX was phosphorylated 24 hours postexposure to HhCDT. Expression of pro-apoptotic Bax protein was upregulated after 24 hours, while Bcl(2) expression decreased. Cytochrome c was released from mitochondria after 12-24 hours of exposure. Concurrently, caspase 3/7 and 9 were activated. However, pretreatment of INT407 cells with caspase inhibitor (Z-VAD-FMK) inhibited the activation of caspase 3/7 and 9. Significant activity of caspase 8 was not observed in toxin treated cells. Activation of caspase 3/7 and caspase 9 confirms the involvement of the mitochondrial apoptotic pathway in HhCDT-treated cells. CONCLUSION: These findings show, for the first time, the ability of HhCDT to induce apoptosis via the mitochondrial pathway.
Assuntos
Apoptose , Toxinas Bacterianas/toxicidade , Células Epiteliais/microbiologia , Helicobacter hepaticus/patogenicidade , Mitocôndrias/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular , Citocromos c/análise , Citoplasma/química , Expressão Gênica , Histonas/metabolismo , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína X Associada a bcl-2/biossínteseRESUMO
The G2/M checkpoint inhibits mitotic entry upon DNA damage, thereby preventing segregation of broken chromosomes and preserving genome stability. The tumor suppressor proteins BRCA1, PALB2 and BRCA2 constitute a BRCA1-PALB2-BRCA2 axis that is essential for homologous recombination (HR)-based DNA doublestrand break repair. Besides HR, BRCA1 has been implicated in both the initial activation and the maintenance of the G2/M checkpoint, while BRCA2 and PALB2 have been shown to be critical for its maintenance. Here we show that all three proteins can play a significant role in both checkpoint activation and checkpoint maintenance, depending on cell type and context, and that PALB2 links BRCA1 and BRCA2 in the checkpoint response. The BRCA1-PALB2 interaction can be important for checkpoint activation, whereas the PALB2-BRCA2 complex formation appears to be more critical for checkpoint maintenance. Interestingly, the function of PALB2 in checkpoint response appears to be independent of CHK1 and CHK2 phosphorylation. Following ionizing radiation, cells with disengaged BRCA1-PALB2 interaction show greatly increased chromosomal abnormalities due apparently to combined defects in HR and checkpoint control. These findings provide new insights into DNA damage checkpoint control and further underscore the critical importance of the proper cooperation of the BRCA and PALB2 proteins in genome maintenance.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Células HCT116 , Células HEK293 , Humanos , Camundongos , Fosforilação , Reparo de DNA por RecombinaçãoRESUMO
The specific function of PP2A, a major serine/threonine phosphatase, is mediated by regulatory targeting subunits, such as members of the B55 family. Although implicated in cell division and other pathways, the specific substrates and functions of B55 targeting subunits are largely undefined. In this study we identified over 100 binding proteins of B55α and B55ß in Xenopus egg extracts that are involved in metabolism, mitochondria function, molecular trafficking, cell division, cytoskeleton, DNA replication, DNA repair, and cell signaling. Among the B55α and B55ß-associated proteins were numerous mitotic regulators, including many substrates of CDK1. Consistently, upregulation of B55α accelerated M-phase exit and inhibited M-phase entry. Moreover, specific substrates of CDK2, including factors of DNA replication and chromatin remodeling were identified within the interactomes of B55α and B55ß, suggesting a role for these phosphatase subunits in DNA replication. In particular, we confirmed in human cells that B55α binds RPA and mediates the dephosphorylation of RPA2. The B55-RPA association is disrupted after replication stress, consistent with the induction of RPA2 phosphorylation. Thus, we report here a new mechanism that accounts for both how RPA phosphorylation is modulated by PP2A and how the phosphorylation of RPA2 is abruptly induced after replication stress.
Assuntos
Proteína Fosfatase 2/metabolismo , Proteína de Replicação A/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Ciclo Celular/fisiologia , Estruturas Cromossômicas , Mitose/fisiologia , Fosforilação , Mapas de Interação de Proteínas , Subunidades Proteicas/metabolismo , Proteólise , Proteína de Replicação A/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
Signaling from arrested replication forks plays a role in maintaining genome stability. We have investigated this process in xeroderma pigmentosum variant cells that carry a mutation in the POLH gene and lack functional DNA polymerase eta (poleta). Poleta is required for error-free bypass of UV-induced cyclobutane pyrimidine dimers; in the absence of poleta in XPV cells, DNA replication is arrested at sites of UV-induced DNA damage, and mutagenic bypass of lesions is ultimately carried out by other, error-prone, DNA polymerases. The present study investigates whether poleta expression influences the activation of a number of UV-induced DNA damage responses. In a stably transfected XPV cell line (TR30-9) in which active poleta can be induced by addition of tetracycline, expression of poleta determines the extent of DNA double-strand break formation following UV-irradiation. UV-induced phosphorylation of replication protein A (RPA), a key DNA-binding protein involved in DNA replication, repair and recombination, is increased in cells lacking poleta compared to when poleta is expressed in the same cell line. To identify the protein kinase responsible for increased UV-induced hyperphosphorylation of the p34 subunit of RPA, we have used NU7441, a specific small molecule inhibitor of DNA-PK. DNA-PK is necessary for RPA p34 hyperphosphorylation, but DNA-PK-mediated phosphorylation is not required for recruitment of RPA p34 into nuclear foci in response to UV-irradiation. The results demonstrate that activation of a UV-induced DNA damage response pathway, involving phosphorylation of RPA p34 by DNA-PK, is enhanced in cells lacking poleta.
Assuntos
Proteína Quinase Ativada por DNA/metabolismo , DNA Polimerase Dirigida por DNA/deficiência , Proteína de Replicação A/metabolismo , Raios Ultravioleta , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Quinase 1 do Ponto de Checagem , DNA/biossíntese , DNA/efeitos da radiação , Dano ao DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Relação Dose-Resposta à Radiação , Fibroblastos/citologia , Regulação Enzimológica da Expressão Gênica , Histonas/metabolismo , Humanos , Cinética , Proteínas Nucleares/metabolismo , Fosforilação/efeitos da radiação , Proteínas Quinases/metabolismo , Tetraciclina/metabolismoRESUMO
Activation of the cellular DNA damage response (DDR) is an important determinant of cell sensitivity to cisplatin and other chemotherapeutic drugs that eliminate tumor cells through induction of DNA damage. It is therefore important to investigate whether alterations of the DNA damage-signaling pathway confer chemoresistance in cancer cells and whether pharmacologic manipulation of the DDR pathway can resensitize these cells to cancer therapy. In a panel of oral/laryngeal squamous cell carcinoma (SCC) cell lines, we observed deficiencies in DNA damage signaling in correlation with cisplatin resistance, but not with DNA repair. These deficiencies are consistent with reduced expression of components of the ataxia telangiectasia mutated (ATM)-dependent signaling pathway and, in particular, strong upregulation of Wip1, a negative regulator of the ATM pathway. Wip1 knockdown or inhibition enhanced DNA damage signaling and resensitized oral SCC cells to cisplatin. In contrast to the previously reported involvement of Wip1 in cancer, Wip1 upregulation and function in these SCC cells is independent of p53. Finally, using xenograft tumor models, we showed that Wip1 upregulation promotes tumorigenesis and its inhibition improves the tumor response to cisplatin. Thus, this study reveals that chemoresistance in oral SCCs is partially attributed to deficiencies in DNA damage signaling, and Wip1 is an effective drug target for enhanced cancer therapy.
Assuntos
Cisplatino/farmacologia , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Bucais/patologia , Transdução de Sinais/efeitos dos fármacos , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/uso terapêutico , Reparo do DNA/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Nus , Neoplasias Bucais/tratamento farmacológico , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2C , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Post-translational phosphorylation of proteins provides a mechanism for cells to switch on or off many diverse processes, including responses to replication stress. Replication-stress-induced phosphorylation enables the rapid activation of numerous proteins involved in DNA replication, DNA repair and cell cycle checkpoints, including replication protein A (RPA). Here, we report that hydroxyurea (HU)-induced RPA phosphorylation requires both NBS1 (NBN) and NBS1 phosphorylation. Transfection of both phosphospecific and nonphosphospecific anti-NBS1 antibodies blocked hyperphosphorylation of RPA in HeLa cells. Nijmegen breakage syndrome (NBS) cells stably transfected with an empty vector or with S343A-NBS1 or S278A/S343A phospho-mutants were unable to hyperphosphorylate RPA in DNA-damage-associated foci following HU treatment. The stable transfection of fully functional NBS1 in NBS cells restored RPA hyperphosphorylation. Retention of ATR on chromatin in both NBS cells and in NBS cells expressing S278A/S343A NBS1 mutants decreased after DNA damage, suggesting that ATR is the kinase responsible for RPA phosphorylation. The importance of RPA hyperphosphorylation is demonstrated by the ability of cells expressing a phospho-mutant form of RPA32 (RPA2) to suppress and delay HU-induced apoptosis. Our findings suggest that RPA hyperphosphorylation requires NBS1 and is important for the cellular response to DNA damage.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Replicação A/metabolismo , Anexina A5/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular Transformada , Transformação Celular Viral , Fibroblastos/metabolismo , Vetores Genéticos , Células HeLa , Humanos , Fosforilação , Retroviridae/genética , Vírus 40 dos Símios/fisiologia , Frações Subcelulares/metabolismo , TransfecçãoRESUMO
NAD(P)H:quinone oxidoreductase (NQO1) catalyzes the two- or four-electron reduction of numerous endogenous and environmental quinones (e.g., the vitamin E alpha-tocopherol quinone, menadione, benzene quinones). In laboratory animals treated with various environmental chemicals, inhibition of NQO1 metabolism has long been known to increase the risk of toxicity or cancer. Currently, there are 22 reported single-nucleotide polymorphisms (SNPs) in the NQO1 gene. Compared with the human consensus (reference, "wild-type") NQO1*1 allele coding for normal NQO1 enzyme and activity, the NQO1*2 allele encodes a nonsynonymous mutation (P187S) that has negligible NQO1 activity. The NQO1*2 allelic frequency ranges between 0.22 (Caucasian) and 0.45 (Asian) in various ethnic populations. A large epidemiologic investigation of a benzene-exposed population has shown that NQO1*2 homozygotes exhibit as much as a 7-fold greater risk of bone marrow toxicity, leading to diseases such as aplastic anemia and leukemia. The extent of the contribution of polymorphisms in other genes involved in the metabolism of benzene and related compounds-such as the P450 2E1 (CYP2E1), myeloperoxidase (MPO), glutathione-S-transferase (GSTM1, GSTT1), microsomal epoxide hydrolase (EPHX1), and other genes-should also be considered. However, it now seems clear that a lowered or absent NQO1 activity can increase one's risk of bone marrow toxicity, after environmental exposure to benzene and benzene-like compounds. In cancer patients, the NQO1*2 allele appears to be associated with increased risk of chemotherapy-related myeloid leukemia. Many other epidemiological studies, attempting to find an association between the NQO1 polymorphism and one or another human disease, have now begun to appear in the medical literature.
Assuntos
Benzeno/efeitos adversos , Doenças da Medula Óssea/genética , Exposição Ambiental/efeitos adversos , Predisposição Genética para Doença , NAD(P)H Desidrogenase (Quinona)/genética , Polimorfismo Genético , Alelos , Animais , Doenças da Medula Óssea/induzido quimicamente , Doenças da Medula Óssea/enzimologia , Frequência do Gene , Genética Populacional , Humanos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Mutação Puntual , Polimorfismo de Fragmento de RestriçãoRESUMO
In response to replicative stress, cells relocate and activate DNA repair and cell cycle arrest proteins such as replication protein A (RPA, a three subunit protein complex required for DNA replication and DNA repair) and the MRN complex (consisting of Mre11, Rad50, and Nbs1; involved in DNA double-strand break repair). There is increasing evidence that both of these complexes play a central role in DNA damage recognition, activation of cell cycle checkpoints, and DNA repair pathways. Here we demonstrate that RPA and the MRN complex co-localize to discrete foci and interact in response to DNA replication fork blockage induced by hydroxyurea (HU) or ultraviolet light (UV). Members of both RPA and the MRN complexes become phosphorylated during S-phase and in response to replication fork blockage. Analysis of RPA and Mre11 in fractionated lysates (cytoplasmic/nucleoplasmic, chromatin-bound, and nuclear matrix fractions) showed increased hyperphosphorylated-RPA and phosphorylated-Mre11 in the chromatin-bound fractions. HU and UV treatment also led to co-localization of hyperphosphorylated RPA and Mre11 to discrete detergent-resistant nuclear foci. An interaction between RPA and Mre11 was demonstrated by co-immunoprecipitation of both protein complexes with anti-Mre11, anti-Rad50, anti-NBS1, or anti-RPA antibodies. Phosphatase treatment with calf intestinal phosphatase or lambda-phosphatase not only de-phosphorylated RPA and Mre11 but also abrogated the ability of RPA and the MRN complex to co-immunoprecipitate. Together, these data demonstrate that RPA and the MRN complex co-localize and interact after HU- or UV-induced replication stress and suggest that protein phosphorylation may play a role in this interaction.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Hidrolases Anidrido Ácido , Western Blotting , Ciclo Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Citoplasma/metabolismo , Dano ao DNA , Reparo do DNA , Fase G1 , Células HeLa , Humanos , Hidroxiureia/farmacologia , Proteína Homóloga a MRE11 , Microscopia de Fluorescência , Modelos Biológicos , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Testes de Precipitina , Ligação Proteica , Proteína de Replicação A , Fase S , Frações Subcelulares , Fatores de Tempo , Raios UltravioletaRESUMO
The heterotrimeric DNA-binding protein, replication protein A (RPA), consists of 70-, 34-, and 14-kDa subunits and is involved in maintaining genomic stability by playing key roles in DNA replication, repair, and recombination. RPA participates in these processes through its interaction with other proteins and its strong affinity for single-stranded DNA (ssDNA). RPA-p34 is phosphorylated in a cell-cycle-dependent fashion primarily at Ser-29 and Ser-23, which are consensus sites for Cdc2 cyclin-dependent kinase. By systematically examining RPA-p34 phosphorylation throughout the cell cycle, we have found there are distinct phosphorylated forms of RPA-p34 in different cell-cycle stages. We have isolated and purified a unique phosphorylated form of RPA that is specifically associated with the mitotic phase of the cell cycle. The mitotic form of RPA (m-hRPA) shows no difference in ssDNA binding activity as compared with recombinant RPA (r-hRPA), yet binds less efficiently to double-stranded DNA (dsDNA). These data suggest that mitotic phosphorylation of RPA-p34 inhibits the destabilization of dsDNA by RPA complex, thereby decreasing the binding affinity for dsDNA. The m-hRPA also exhibits altered interactions with certain DNA replication and repair proteins. Using highly purified proteins, m-hRPA exhibited decreased binding to ATM, DNA pol alpha, and DNA-PK as compared to unphosphorylated recombinant RPA (r-hRPA). Dephosphorylation of m-hRPA was able to restore the interaction with each of these proteins. Interestingly, the interaction of RPA with XPA was not altered by RPA phosphorylation. These data suggest that phosphorylation of RPA-p34 plays an important role in regulating RPA functions in DNA metabolism by altering specific protein-protein interactions.