RESUMO
Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress.
Assuntos
Replicação do DNA , Proteína Quinase Ativada por DNA/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Recombinação Homóloga , Proteínas Nucleares/metabolismo , Proteína de Replicação A/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Células CHO , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Cricetinae , Cricetulus , Proteína Quinase Ativada por DNA/genética , Humanos , Mutação , Proteínas Nucleares/genética , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína de Replicação A/genética , Serina/genética , Serina/metabolismoRESUMO
BACKGROUND: Resistance to chemotherapy is a major limitation in the treatment of head and neck squamous cell carcinomas (HNSCCs), accounting for high mortality rates in patients. Here, we investigated the role of replication protein A (RPA) in cisplatin and etoposide resistance. METHODS: We used 6 parental HNSCC cell lines. We also generated 1 cisplatin-resistant progeny subline from a parental cisplatin-sensitive cell line, to examine cisplatin resistance and sensitivity with respect to RPA2 hyperphosphorylation and cell-cycle response. RESULTS: Cisplatin-resistant HNSCC cell levels of hyperphosphorylated RPA2 in response to cisplatin were 80% to 90% greater compared with cisplatin-sensitive cell lines. RPA2 hyperphosphorylation could be induced in the cisplatin-resistant HNSCC subline. The absence of RPA2 hyperphosphorylation correlated with a defect in cell-cycle progression and cell survival. CONCLUSION: Loss of RPA2 hyperphosphorylation occurs in HNSCC cells and may be a marker of cellular sensitivities to cisplatin and etoposide in HNSCC.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/farmacologia , Proteína de Replicação A/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Ciclo Celular , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , FosforilaçãoRESUMO
Human replication protein A (RPA), the primary single-stranded DNA-binding protein, was previously found to be inhibited after heat shock by complex formation with nucleolin. Here we show that nucleolin-RPA complex formation is stimulated after genotoxic stresses such as treatment with camptothecin or exposure to ionizing radiation. Complex formation in vitro and in vivo requires a 63-residue glycine-arginine-rich (GAR) domain located at the extreme C terminus of nucleolin, with this domain sufficient to inhibit DNA replication in vitro. Fluorescence resonance energy transfer studies demonstrate that the nucleolin-RPA interaction after stress occurs both in the nucleoplasm and in the nucleolus. Expression of the GAR domain or a nucleolin mutant (TM) with a constitutive interaction with RPA is sufficient to inhibit entry into S phase. Increasing cellular RPA levels by overexpression of the RPA2 subunit minimizes the inhibitory effects of nucleolin GAR or TM expression on chromosomal DNA replication. The arrest is independent of p53 activation by ATM or ATR and does not involve heightened expression of p21. Our data reveal a novel cellular mechanism that represses genomic replication in response to genotoxic stress by inhibition of an essential DNA replication factor.
Assuntos
Dano ao DNA , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Camptotecina/toxicidade , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Replicação do DNA/genética , DNA Viral/genética , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Mutação/genética , Fosfoproteínas/genética , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Subunidades Proteicas/análise , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/genética , Radiação Ionizante , Proteína de Replicação A , Vírus 40 dos Símios/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/fisiologia , NucleolinaRESUMO
We recently discovered that heat shock causes nucleolin to relocalize from the nucleolus to the nucleoplasm, whereupon it binds replication protein A and inhibits DNA replication initiation. We report that nucleolin mobilization also occurs following exposure to ionizing radiation (IR) and treatment with camptothecin. Mobilization was selective in that another nucleolar marker, upstream binding factor, did not relocalize in response to IR. Nucleolin relocalization was dependent on p53 and stress, the latter initially stimulating nucleolin-p53 complex formation. Nucleolin relocalization and complex formation in vivo were independent of p53 transactivation but required the p53 C-terminal regulatory domain. Nucleolin and p53 also interact directly in vitro, with a similar requirement for p53 domains. These data indicate a novel p53-dependent mechanism in which cell stress mobilizes nucleolin for transient replication inhibition and DNA repair.
