RESUMEN
Homologous recombination deficiency conferred by alterations in BRCA1 or BRCA2 are common in breast tumors and can drive sensitivity to platinum chemotherapy and PARP inhibitors. Alterations in nucleotide excision repair (NER) activity can also impact sensitivity to DNA damaging agents, but NER activity in breast cancer has been poorly characterized. Here, we apply a novel immunofluorescence-based cellular NER assay to screen a large panel of breast epithelial and cancer cell lines. Although the majority of breast cancer models are NER proficient, we identify an example of a breast cancer cell line with profound NER deficiency. We show that NER deficiency in this model is driven by epigenetic silencing of the ERCC4 gene, leading to lack of expression of the NER nuclease XPF, and that ERCC4 methylation is also strongly correlated with ERCC4 mRNA and XPF protein expression in primary breast tumors. Re-expression of XPF in the ERCC4-deficient breast cancer rescues NER deficiency and cisplatin sensitivity, but does not impact PARP inhibitor sensitivity. These findings demonstrate the potential to use functional assays to identify novel mechanisms of DNA repair deficiency and nominate NER deficiency as a platinum sensitivity biomarker in breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Reparación del ADN , Línea Celular Tumoral , Cisplatino/farmacología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Metilación de ADN/efectos de los fármacos , Metilación de ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Regiones Promotoras Genéticas/genética , Rayos UltravioletaRESUMEN
Because cells are constantly subjected to DNA damaging insults, DNA repair pathways are critical for genome integrity [1]. DNA damage recognition protein complexes (DRCs) recognize DNA damage and initiate DNA repair. The DNA-Damage Binding protein 2 (DDB2) complex is a DRC that initiates nucleotide excision repair (NER) of DNA damage caused by ultraviolet light (UV) [2]-[4]. Using a purified DDB2 DRC, we created a probe ("DDB2 proteo-probe") that hybridizes to nuclei of cells irradiated with UV and not to cells exposed to other genotoxins. The DDB2 proteo-probe recognized UV-irradiated DNA in classical laboratory assays, including cyto- and histo-chemistry, flow cytometry, and slot-blotting. When immobilized, the proteo-probe also bound soluble UV-irradiated DNA in ELISA-like and DNA pull-down assays. In vitro, the DDB2 proteo-probe preferentially bound 6-4-photoproducts [(6-4)PPs] rather than cyclobutane pyrimidine dimers (CPDs). We followed UV-damage repair by cyto-chemistry in cells fixed at different time after UV irradiation, using either the DDB2 proteo-probe or antibodies against CPDs, or (6-4)PPs. The signals obtained with the DDB2 proteo-probe and with the antibody against (6-4)PPs decreased in a nearly identical manner. Since (6-4)PPs are repaired only by nucleotide excision repair (NER), our results strongly suggest the DDB2 proteo-probe hybridizes to DNA containing (6-4)PPs and allows monitoring of their removal during NER. We discuss the general use of purified DRCs as probes, in lieu of antibodies, to recognize and monitor DNA damage and repair.
Asunto(s)
Daño del ADN/fisiología , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Rayos Ultravioleta , Línea Celular , Daño del ADN/genética , Reparación del ADN/genética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Dímeros de Pirimidina/metabolismoRESUMEN
RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.
Asunto(s)
Daño del ADN , Proteína Quinasa Activada por ADN/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Ribosómico/biosíntesis , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Cisplatino/farmacología , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteína Quinasa Activada por ADN/genética , Activación Enzimática/efectos de los fármacos , Genoma Humano , Humanos , Autoantígeno Ku , Proteínas Nucleares/genética , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , ARN Ribosómico/genética , Ribosomas/genética , Ribosomas/metabolismo , Fase S/efectos de los fármacos , Puntos de Control de la Fase S del Ciclo Celular , Rayos UltravioletaRESUMEN
The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.
Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN Helicasas/metabolismo , ADN/metabolismo , Proteómica/métodos , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Western Blotting , Línea Celular Tumoral , Nucléolo Celular/metabolismo , ADN/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Células HeLa , Humanos , Autoantígeno Ku , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Unión Proteica/genética , Transporte de Proteínas/efectos de la radiación , Proteoma/clasificación , Proteoma/genética , Proteoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Factores de Tiempo , Rayos UltravioletaRESUMEN
BACKGROUND: Platinum-containing chemotherapy produces specific DNA damage and is used to treat several human solid tumors. Tumors initially sensitive to platinum-based drugs frequently become resistant. Inhibition of DNA repair is a potential strategy to enhance cisplatin effectiveness. After cisplatin treatment, a balance between repair and apoptosis determines whether cancer cells proliferate or die. DNA-dependent protein kinase (DNA-PK) binds to DNA double strand breaks (DSBs) through its Ku subunits and initiates non-homologous end joining. Inhibition of DNA-PK sensitizes cancer cells to cisplatin killing. The goal of this study is to elucidate the mechanism underlying the effects of DNA-PK on cisplatin sensitivity. RESULTS: Silencing the expression of the catalytic subunit of DNA-PK (DNA-PKcs) increased sensitivity to cisplatin and decreased the appearance of γH2AX after cisplatin treatment. We purified DNA-PK by its Ku86 subunit and identified interactors by tandem mass spectrometry before and after cisplatin treatment. The structure specific recognition protein 1 (SSRP1), Spt16 and γH2AX appeared in the Ku86 complex 5 hours after cisplatin treatment. SSRP1 and Spt16 form the facilitator of chromatin transcription (FACT). The cisplatin-induced association of FACT with Ku86 and γH2AX was abrogated by DNase treatment. In living cells, SSRP1 and Ku86 were recruited at sites of DSBs induced by laser beams. Silencing SSRP1 expression increased sensitivity to cisplatin and decreased γH2AX appearance. However, while silencing SSRP1 in cisplatin-treated cells increased both apoptosis and necrosis, DNA-PKcs silencing, in contrast, favored necrosis over apoptosis. CONCLUSIONS: DNA-PK and FACT both play roles in DNA repair. Therefore both are putative targets for therapeutic inhibition. Since DNA-PK regulates apoptosis, silencing DNA-PKcs redirects cells treated with cisplatin toward necrosis. Silencing FACT however, allows both apoptosis and necrosis. Targeting DNA repair in cancer patients may have different therapeutic effects depending upon the roles played by factors targeted.