RESUMEN
Fascaplysin is a red cytotoxic pigment with anticancer properties isolated from the marine sponge Fascaplysinopsis sp. Recently, structure-activity relationship analysis reported by our group suggested that selective cytotoxicity of fascaplysin derivatives towards tumor cells negatively correlates with their ability to intercalate into DNA. To validate this hypothesis, we synthesized 6- and 7-tert-butylfascaplysins which reveal mitigated DNA-intercalating properties. These derivatives were found to be strongly cytotoxic to drug-resistant human prostate cancer cells, albeit did not demonstrate improved selectivity towards cancer cells when compared to fascaplysin. At the same time, kinome analysis suggested an activation of CHK1/ATR axis in cancer cells shortly after the drug exposure. Further experiments revealed induction of replication stress that is eventually converted to the toxic DNA double-strand breaks, resulting in caspase-independent apoptosis-like cell death. Our observations highlight new DNA-targeting effect of some fascaplysin derivatives and indicate more complex structure-activity relationships within the fascaplysin family, suggesting that cytotoxicity and selectivity of these alkaloids are influenced by multiple factors. Furthermore, combination with clinically-approved inhibitors of ATR/CHK1 as well as testing in tumors particularly sensitive to the DNA damage should be considered in further studies.
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Antineoplásicos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Humanos , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Indoles/farmacología , Indoles/química , Apoptosis/efectos de los fármacos , Relación Estructura-Actividad , Masculino , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , ADN/metabolismo , Animales , Roturas del ADN de Doble Cadena/efectos de los fármacos , Compuestos de Amonio Cuaternario , Carbolinas , IndolizinasRESUMEN
The use of mutation analysis of homologous recombination repair (HRR) genes to estimate PARP-inhibition response may miss a larger proportion of responding patients. Here, we provide preclinical models for castration-resistant prostate cancer (CRPC) that can be used to functionally predict HRR defects. In vitro, CRPC LNCaP sublines revealed an HRR defect and enhanced sensitivity to olaparib and cisplatin due to impaired RAD51 expression and recruitment. Ex vivo-induced castration-resistant tumor slice cultures or tumor slice cultures derived directly from CRPC patients showed increased olaparib- or cisplatin-associated enhancement of residual radiation-induced γH2AX/53BP1 foci. We established patient-derived tumor organoids (PDOs) from CRPC patients. These PDOs are morphologically similar to their primary tumors and genetically clustered with prostate cancer but not with normal prostate or other tumor entities. Using these PDOs, we functionally confirmed the enhanced sensitivity of CRPC patients to olaparib and cisplatin. Moreover, olaparib but not cisplatin significantly decreased the migration rate in CRPC cells. Collectively, we present robust patient-derived preclinical models for CRPC that recapitulate the features of their primary tumors and enable individualized drug screening, allowing translation of treatment sensitivities into tailored clinical therapy recommendations.
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Neoplasias de la Próstata Resistentes a la Castración , Masculino , Humanos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Reparación del ADN por Recombinación , Reparación del ADN/genética , Cisplatino/farmacología , Cisplatino/uso terapéuticoRESUMEN
Background: The oncogene epidermal growth factor receptor variant III (EGFRvIII) is expressed in approximately one-third of all glioblastomas (GBMs). So far it is not clear if EGFRvIII expression induces replication stress in GBM cells, which might serve as a therapeutical target. Methods: Isogenetic EGFRvIII- and EGFRvIII+ cell lines with endogenous EGFRvIII expression were used. Markers of oncogenic and replication stress such as γH2AX, RPA, 53BP1, ATR, and CHK1 were analyzed using western blot, immunofluorescence, and flow cytometry. The DNA fiber assay was performed to analyze replication, transcription was measured by incorporation of EU, and genomic instability was investigated by micronuclei and CGH-Array analysis. Immunohistochemistry staining was used to detect replication stress markers and R-loops in human GBM samples. Results: EGFRvIII+ cells exhibit an activated replication stress response, increased spontaneous DNA damage, elevated levels of single-stranded DNA, and reduced DNA replication velocity, which are all indicative characteristics of replication stress. Furthermore, we show here that EGFRvIII expression is linked to increased genomic instability. EGFRvIII-expressing cells display elevated RNA synthesis and R-loop formation, which could also be confirmed in EGFRvIII-positive GBM patient samples. Targeting replication stress by irinotecan resulted in increased sensitivity of EGFRvIII+ cells. Conclusion: This study demonstrates that EGFRvIII expression is associated with increased replication stress, R-loop accumulation, and genomic instability. This might contribute to intratumoral heterogeneity but may also be exploited for individualized therapy approaches.
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Keratins are the main identification markers of circulating tumor cells (CTCs); however, whether their deregulation is associated with the metastatic process is largely unknown. Previously we have shown by in silico analysis that keratin 16 (KRT16) mRNA upregulation might be associated with more aggressive cancer. Therefore, in this study, we investigated the biological role and the clinical relevance of K16 in metastatic breast cancer. By performing RT-qPCR, western blot, and immunocytochemistry, we investigated the expression patterns of K16 in metastatic breast cancer cell lines and evaluated the clinical relevance of K16 expression in CTCs of 20 metastatic breast cancer patients. High K16 protein expression was associated with an intermediate mesenchymal phenotype. Functional studies showed that K16 has a regulatory effect on EMT and overexpression of K16 significantly enhanced cell motility (p < 0.001). In metastatic breast cancer patients, 64.7% of the detected CTCs expressed K16, which was associated with shorter relapse-free survival (p = 0.0042). Our findings imply that K16 is a metastasis-associated protein that promotes EMT and acts as a positive regulator of cellular motility. Furthermore, determining K16 status in CTCs provides prognostic information that helps to identify patients whose tumors are more prone to metastasize.
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Quantitative cellular in vitro nanoparticle uptake measurements are possible with a large number of different techniques, however, all have their respective restrictions. Here, we demonstrate the application of synchrotron-based X-ray fluorescence imaging (XFI) on prostate tumor cells, which have internalized differently functionalized gold nanoparticles. Total nanoparticle uptake on the order of a few hundred picograms could be conveniently observed with microsamples consisting of only a few hundreds of cells. A comparison with mass spectroscopy quantification is provided, experimental results are both supported and sensitivity limits of this XFI approach extrapolated by Monte-Carlo simulations, yielding a minimum detectable nanoparticle mass of just 5 pg. This study demonstrates the high sensitivity level of XFI, allowing non-destructive uptake measurements with very small microsamples within just seconds of irradiation time.
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Oro , Nanopartículas , Imagen Óptica , Espectrometría por Rayos X , Humanos , Células Tumorales CultivadasRESUMEN
(1) Background: The combination of the first-generation antiandrogens and radiotherapy (RT) has been studied extensively in the clinical setting of prostate cancer (PCa). Here, we evaluated the potential radiosensitizing effect of the second-generation antiandrogens abiraterone acetate, apalutamide and enzalutamide. (2) Methods: Cell proliferation and agarose-colony forming assay were used to measure the effect on survival. Double strand break repair efficiency was monitored using immunofluorescence staining of γH2AX/53BP1. (3) Results: We report retrospectively a minor benefit for PCa patients received first-generation androgen blockers and RT compared to patients treated with RT alone. Combining either of the second-generation antiandrogens and 2Gy suppressed cell growth and increased doubling time significantly more than 2Gy alone, in both hormone-responsive LNCaP and castration-resistant C4-2B cells. These findings were recapitulated in resistant sub-clones to (i) hormone ablation (LNCaP-abl), (ii) abiraterone acetate (LNCaP-abi), (iii) apalutamide (LNCaP-ARN509), (iv) enzalutamide (C4-2B-ENZA), and in castration-resistant 22-RV1 cells. This radiosensitization effect was not observable using the first-generation antiandrogen bicalutamide. Inhibition of DNA DSB repair was found to contribute to the radiosensitization effect of second-generation antiandrogens, as demonstrated by a significant increase in residual γH2AX and 53BP1 foci numbers at 24h post-IR. DSB repair inhibition was further demonstrated in 22 patient-derived tumor slice cultures treated with abiraterone acetate before ex-vivo irradiation with 2Gy. (4) Conclusion: Together, these data show that second-generation antiandrogens can enhance radiosensitivity in PCa through DSB repair inhibition, regardless of their hormonal status. Translated into clinical practice, our results may help to find additional strategies to improve the effectiveness of RT in localized PCa, paving the way for a clinical trial.
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Molecular-targeted therapies and treatment stratification based on molecular biomarkers have rapidly gained momentum in the therapeutic spectrum for patients with prostate cancer, particularly those with aggressive disease. DNA damage repair (DDR) pathways are commonly impaired in prostate cancer. Recent studies have detailed mechanisms interconnecting the DDR with the androgen receptor (AR) signaling pathway as well as its interplay with the immune response. The prominent role of DDR deficiency in prostate cancer development and treatment response encourages innovative strategies for the detection of DDR deficiency in individual tumors. In this review, we describe recent preclinical and early clinical data on the exploitation of DDR defects as predictive biomarkers and also as molecular therapeutic targets.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Biomarcadores de Tumor/genética , Quimioradioterapia/métodos , Reparación del ADN/efectos de los fármacos , Neoplasias de la Próstata/genética , Antagonistas de Andrógenos/farmacología , Antagonistas de Andrógenos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/antagonistas & inhibidores , Reparación del ADN/efectos de la radiación , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Masculino , Terapia Molecular Dirigida/métodos , Mutación , Pronóstico , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Receptores Androgénicos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Transducción de Señal/efectos de la radiación , Resultado del TratamientoRESUMEN
Despite high cure rates, about 20% of patients with advanced germ cell tumors (GCTs) fail cisplatin-based chemotherapy. High levels of DNA methylation have been identified in GCTs and linked to cisplatin resistance. Here, we examined the effects of DNA hypomethylating 5-azacitidine (5-aza) on two embryonal carcinoma cell lines (NCCIT, 2102Ep) and their cisplatin-resistant isogenic derivatives. Effects on cell viability and cisplatin sensitivity were assessed by the trypan blue exclusion method. Western blotting was used to examine induction of apoptosis 5-aza and results were validated by flow cytometry. Single agent treatment with 5-aza strongly impacted viability and induced apoptosis at low nanomolar concentrations, both in cisplatin-sensitive and -resistant cell lines. 5-aza exerted an immediate apoptotic response, followed by a prolonged inhibitory effect on cell viability and cell-cycle progression. Sequential treatment with 5-aza and cisplatin reduced cellular survival of the cisplatin-resistant sublines already at nanomolar concentrations, suggesting a partial restoration of cisplatin sensitivity by the compound. 5-aza demonstrated anti-tumor activity as a single agent at low nanomolar concentrations in GCT cells, irrespective of cisplatin-sensitivity. 5-aza may also have the potential at least to partially restore cisplatin-sensitivity in non-seminoma cells, supporting the hypothesis that combining DNA demethylating agents with cisplatin-based chemotherapy may be a valid therapeutic approach in patients with refractory GCTs.
Asunto(s)
Apoptosis/efectos de los fármacos , Azacitidina/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de Células Germinales y Embrionarias/metabolismo , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Apoptosis/genética , Biomarcadores , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Metilación de ADN , Humanos , Concentración 50 Inhibidora , MasculinoRESUMEN
Here we report that BCL2 blocks DNA double strand break (DSB) repair via nonhomologous end-joining (NHEJ), through sequestration of KU80 protein outside the nucleus. We find that this effect is associated with a repair switch to the error-prone PARP1-dependent end-joining (PARP1-EJ). We present in-vitro proof-of-concept for therapeutic targeting of this switch using PARP inhibitor to specifically enhance the radiosensitivity of BCL2-overexpressing cells. Given its erroneous behavior, PARP1-EJ might allow for the accumulation of genetic alterations and tumor progression. Consistently, we report an inverse correlation between BCL2 expression and biochemical recurrence-free survival of 10.259 prostate cancer (PCa) patients who underwent primary radical-prostatectomy for localized disease. Further, we evaluated retrospectively the impact of BCL2 expression on clinical outcome of 1.426 PCa patients, who had been given salvage radiotherapy at relapse after radical prostatectomy. In line with its role in blocking NHEJ, BCL2 over-expressers showed significantly better response to salvage radiotherapy compared to low-expressers. Collectively, our findings identify BCL2 status in PCa as a putative predictor of (i) radiotherapy response and (ii) response to treatment with PARP inhibitor olaparib as a radiosensitizing agent.
Asunto(s)
Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Neoplasias de la Próstata/terapia , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Regulación hacia Arriba , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Autoantígeno Ku/metabolismo , Masculino , Poli(ADP-Ribosa) Polimerasa-1/genética , Prostatectomía , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Radioterapia , Terapia Recuperativa , Análisis de SupervivenciaRESUMEN
End processing at DNA double strand breaks (DSB) is a decisive step in repair pathway selection. Here, we investigated the role of 53BP1/RIF1 in limiting BRCA1/CtIP-mediated end resection to control DSB repair pathway choice. ATM orchestrates this process through 53BP1 phosphorylation to promote RIF1 recruitment. As cells enter S/G2-phase, end resection is activated, which displaces pATM from DSB sites and diminishes 53BP1 phosphorylation and RIF1 recruitment. Consistently, the kinetics of ATM and 53BP1 phosphorylation in S/G2-phase concur. We show that defective 53BP1/RIF1-mediated DSB end-protection in G1-phase stimulates CtIP/MRE11-dependent end-resection, which requires Polo-like kinase 3. This end resection activity in G1 was shown to produce only short tracks of ssDNA overhangs, as evidenced by the findings that in 53BP1 depleted cells, (i) RPA focus intensity was significantly lower in G1 compared to that in S/G2 phase, and (ii) EXO1 knockdown did not alter either number or intensity of RPA foci in G1 but significantly decreased the RPA focus intensity in S/G2 phase. Importantly, we report that the observed DSB end resection in G1 phase inhibits DNA-PK-dependent nonhomologous end joining but is not sufficient to stimulate HR. Instead, it switches the repair to the alternative PARP1-dependent end joining pathway.
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Proteínas Portadoras/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas Nucleares/genética , Poli(ADP-Ribosa) Polimerasa-1/genética , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína BRCA1/genética , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades , ADN de Cadena Simple/genética , Endodesoxirribonucleasas , Fase G1 , Células HeLa , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Proteínas Supresoras de TumorRESUMEN
Histone demethylases have recently gained interest as potential targets in cancer treatment and several histone demethylases have been implicated in the DNA damage response. We investigated the effects of siRNA-mediated depletion of histone demethylase Jarid1A (KDM5A, RBP2), which demethylates transcription activating tri- and dimethylated lysine 4 at histone H3 (H3K4me3/me2), on growth characteristics and cellular response to radiation in several cancer cell lines. In unirradiated cells Jarid1A depletion lead to histone hyperacetylation while not affecting cell growth. In irradiated cells, depletion of Jarid1A significantly increased cellular radiosensitivity. Unexpectedly, the hyperacetylation phenotype did not lead to disturbed accumulation of DNA damage response and repair factors 53BP1, BRCA1, or Rad51 at damage sites, nor did it influence resolution of radiation-induced foci or rejoining of reporter constructs. We conclude that the radiation sensitivity observed following depletion of Jarid1A is not caused by a deficiency in repair of DNA double-strand breaks.
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Roturas del ADN de Doble Cadena , Reparación del ADN , Histonas/metabolismo , Tolerancia a Radiación , Proteína 2 de Unión a Retinoblastoma/metabolismo , Acetilación , Proliferación Celular/efectos de la radiación , Cromatina/metabolismo , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de la radiación , Regulación hacia Abajo/efectos de la radiación , Técnicas de Silenciamiento del Gen , Genes Reporteros , Células HeLa , Humanos , Lisina/metabolismo , Células MCF-7 , Plásmidos/metabolismo , Tolerancia a Radiación/efectos de la radiación , Radiación IonizanteRESUMEN
Despite the significant contribution of radiotherapy to non-small lung cancer (NSCLC), radioresistance still occurs. One of the major radioresistance mechanisms is the hyperactivation of the PI3K/Akt pathway in which Akt facilitates the repair of DNA double-strand breaks (DSBs) through the stimulation of DNA-PKcs. We investigated if targeting PI3K would be a potential approach for enhancing the radiosensitivity of K-RAS mutated (K-RASmut) NSCLC cell lines A549 and H460. Short-term (1-2 h) pre-treatment of cells with the PI3K inhibitor PI-103 (1 µM) inhibited Akt/DNA-PKcs activity, blocked DSBs repair and induced radiosensitivity, while long-term (24 h) pre-treatment did not. Lack of an effect after 24 h of PI-103 pre-treatment was due to reactivation of K-Ras/MEK/ERK-dependent Akt. However, long-term treatment with the combination of PI-103 and MEK inhibitor PD98059 completely blocked reactivation of Akt and impaired DSBs repair through non-homologous end joining (NHEJ) leading to radiosensitization. The effect of PI3K inhibition on Akt signaling was also tested in A549 mouse xenografts. P-Akt and P-DNA-PKcs were inhibited 30 min post-irradiation in xenografts, which were pretreated by PI-103 30 min before irradiation. However, Akt was reactivated 30 min post-irradiation in tumors, which were pre-treated for 3 h with PI-103 before irradiation. After a 24 h pretreatment with PI-103, a significant reactivation of Akt was achieved 24 h after irradiation. Thus, due to MEK/ERK-dependent reactivation of Akt, targeting PI3K alone is not a suitable approach for radiosensitizing K-RASmut NSCLC cells, indicating that dual targeting of PI3K and MEK is an efficient approach to improve radiotherapy outcome.
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Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genética , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
There is a need to develop new, more efficient therapies for head and neck cancer (HNSCC) patients. It is currently unclear whether defects in DNA repair genes play a role in HNSCCs' resistance to therapy. PARP1 inhibitors (PARPi) were found to be "synthetic lethal" in cancers deficient in BRCA1/2 with impaired homologous recombination. Since tumors rarely have these particular mutations, there is considerable interest in finding alternative determinants of PARPi sensitivity. Effectiveness of combined irradiation and PARPi olaparib was evaluated in ten HNSCC cell lines, subdivided into HR-proficient and HR-deficient cell lines using a GFP-based reporter assay. Both groups were equally sensitive to PARPi alone. Combined treatment revealed stronger synergistic interactions in the HR-deficient group. Because HR is mainly active in S-Phase, replication processes were analyzed. A stronger impact of treatment on replication processes (p = 0.04) and an increased number of radial chromosomes (p = 0.003) were observed in the HR-deficient group. We could show that radiosensitization by inhibition of PARP1 strongly correlates with HR competence in a replication-dependent manner. Our observations indicate that PARP1 inhibitors are promising candidates for enhancing the therapeutic ratio achieved by radiotherapy via disabling DNA replication processes in HR-deficient HNSCCs.
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Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/terapia , Replicación del ADN/efectos de los fármacos , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/terapia , Recombinación Homóloga/genética , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Línea Celular Tumoral , Reparación del ADN/genética , Humanos , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
PURPOSE: The aim of this study was to elucidate the impact of DNA damage response (DDR) proteins 53BP1 and BRCA1 on the double-strand break (DSB)-repair choice. This is important not only in order to understand the underlying mechanisms of DSB-repair pathway regulation but also to determine the therapeutic implications for BRCA1-associated tumors. MATERIALS AND METHODS: Human tumor cell lines A549 and HeLa were used. Non-homologous end-joining (NHEJ) and homologous recombination (HR) were assessed using NHEJ and HR reporter constructs. Colocalization of HR-proteins RPA and RAD51 with 53BP1 was evaluated by confocal microscopy and 3D-analysis. RESULTS: We demonstrate a specific crosstalk between 53BP1 and BRCA1. While 53BP1 does not colocalize with RPA or RAD51 and prohibits the recruitment of BRCA1 to DSBs to stimulate NHEJ, BRCA1 promotes the 53BP1 displacement specifically in S/G2-phase to allow end-resection, initiating HR. HR-efficiency was restored in BRCA1-depleted cells upon additional 53BP1-knockdown. Further, we found that 53BP1-mediated end protection precedes BRCA1-dependent end-resection. CONCLUSION: These results demonstrate that the interplay between 53BP1/NHEJ and BRCA1/HR is of great relevance for tumor treatment, as the 53BP1 status would be highly important for the treatment response of BRCA1-associated tumors.
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Proteína BRCA1/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteína 1 de Unión al Supresor Tumoral P53/fisiología , Ciclo Celular , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades , Células HeLa , Recombinación Homóloga , HumanosRESUMEN
Malignant tumors of the rectum are treated by neoadjuvant radiochemotherapy. This involves a combination of 5-fluorouracil (5-FU) and double stranded DNA-break (DSB)-inducing radiotherapy. Here we explored how 5-FU cooperates with DSB-induction to achieve sustainable DNA damage in colorectal cancer (CRC) cells. After DSB induction by neocarzinostatin, phosphorylated histone 2AX (γ-H2AX) rapidly accumulated but then largely vanished within a few hours. In contrast, when CRC cells were pre-treated with 5-FU, gammaH2AX remained for at least 24 hours. GFP-reporter assays revealed that 5-FU decreases the efficiency of homologous recombination (HR) repair. However, 5-FU did not prevent the initial steps of HR repair, such as the accumulation of RPA and Rad51 at nuclear foci. Thus, we propose that 5-FU interferes with the continuation of HR repair, e. g. the synthesis of new DNA strands. Two key mediators of HR, Rad51 and BRCA2, were found upregulated in CRC biopsies as compared to normal mucosa. Inhibition of HR by targeting Rad51 enhanced DNA damage upon DSB-inducing treatment, outlining an alternative way of enhancing therapeutic efficacy. Taken together, our results strongly suggest that interfering with HR represents a key mechanism to enhance the efficacy when treating CRC with DNA-damaging therapy.
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Antineoplásicos/farmacología , Neoplasias Colorrectales/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Fluorouracilo/farmacología , Reparación del ADN por Recombinación/efectos de los fármacos , Línea Celular Tumoral , Quimioradioterapia , Neoplasias Colorrectales/terapia , Citometría de Flujo , Humanos , Immunoblotting , Microscopía Confocal , Microscopía Fluorescente , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Poly-ADP-ribose-polymerase inhibitors (PARPi) are considered to be optimal tools for specifically enhancing radiosensitivity. This effect has been shown to be replication-dependent and more profound in HR-deficient tumors. Here, we present a new mode of PARPi-mediated radiosensitization which was observed in four out of six HR-proficient tumor cell lines (responders) investigated, but not in normal cells. This effect is replication-independent, as the radiosensitization remained unaffected following the inhibition of replication using aphidicolin. We showed that responders are radiosensitized by Olaparib because their DSB-repair is switched to PARP1-dependent end-joining (PARP1-EJ), as evident by (i) the significant increase in the number of residual γH2AX foci following irradiation with 3Gy and treatment with Olaparib, (ii) the enhanced enrichment of PARP1 at the chromatin after 3Gy and (iii) the inhibition of end-joining activity measured by a specific reporter substrate upon Olaparib treatment. This is the first study which directly demonstrates the switch to PARP1-EJ in tumor cells and its contribution to the response to Olaparib as a radiosensitizer, findings which could widen the scope of application of PARPi in tumor therapy.
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Inhibidores Enzimáticos/farmacología , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Fármacos Sensibilizantes a Radiaciones/farmacología , Western Blotting , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , HumanosRESUMEN
Classical-non-homologous end-joining (C-NHEJ) is considered the main pathway for repairing DNA double strand breaks (DSB) in mammalian cells. When C-NHEJ is defective, cells may switch DSB repair to an alternative-end-joining, which depends on PARP1 and is more erroneous. This PARP1-EJ is suggested to be active especially in tumor cells contributing to their genomic instability. Here, we define conditions under which cells would switch the repair to PARP1-EJ. Using the end jining repair substrate pEJ, we revealed that PARP1-EJ is solely used when Ku is deficient but not when either DNA-PKcs or Xrcc4 is lacking. In the latter case, DSB repair, however, could be shuttled to PARP1-EJ after additional Ku80 down-regulation, which partly rescued the DSB repair in these mutants. We demonstrate here that PARP-EJ may work on DSB ends at high fidelity manner, as evident from the unchanged efficiency upon blocking end resection by either roscovitin or mirin. Furthermore, we demonstrate for that PARP-EJ is likewise involved in the repair of multiple DSBs (I-PpoI- and IR-induced). Importantly, we identified a chromatin signature associated with the switch to PARP1-EJ which is characterized by a strong enrichment of both PARP1 and LigIII at damaged chromatin. Together, these data indicate that Ku is the main regulator for the hierarchal organization between C-NHEJ and PARP1-EJ.
Asunto(s)
Antígenos Nucleares/metabolismo , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Antígenos Nucleares/inmunología , Células CHO , Cromatina/genética , Cromatina/metabolismo , Cricetulus , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/inmunología , Regulación de la Expresión Génica , Humanos , Autoantígeno Ku , Mutación , Proteínas Nucleares/metabolismo , Purinas/farmacología , Pirimidinonas/farmacología , Roscovitina , Tionas/farmacologíaRESUMEN
BACKGROUND: Cellular and clinical sensitivity to ionizing radiation (IR) is determined by DNA double-strand breaks (DSB) repair. Here, we investigate the molecular mechanism underlying the extreme response of a head and neck tumor case (SKX) to standard radiotherapy. METHODS: Immunofluorescence (IF) was used for the assessment of DSB repair, Western blot and real-time PCR for protein and mRNA expression, respectively. RESULTS: SKX cells exhibited a pronounced radiosensitivity associated with numerous residual γ-H2AX foci after IR. This was not associated with lacking canonical repair proteins. SKX cells did not express any ATM protein. Accordingly, immunoblotting revealed no ATM kinase activity toward substrates such as p-SMC1, p-CHK2 and p-KAP1. Sequencing of all 66 exons of ATM showed no mutation. ATM mRNA level was moderately reduced, which could be reverted by 5'-Aza-C treatment but without restoring protein levels. Importantly, we demonstrated a post-transcriptional regulation in SKX cells via 6-fold enhanced levels of miR-421, which targets the 3'-UTR of ATM mRNA. Transfection of SKX cells with either anti-miR-421 inhibitor or a microRNA-insensitive ATM vector recovered ATM expression and abrogated the hyper-radiosensitivity. CONCLUSION: This is the first report describing microRNA-mediated down-regulation of ATM leading to clinically manifest tumor radiosensitivity.
Asunto(s)
Carcinoma de Células Escamosas/radioterapia , Proteínas de Ciclo Celular/antagonistas & inhibidores , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Neoplasias de Cabeza y Cuello/radioterapia , MicroARNs/fisiología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Anciano , Anciano de 80 o más Años , Proteínas de la Ataxia Telangiectasia Mutada , Carcinoma de Células Escamosas/genética , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/fisiología , Línea Celular Tumoral , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/fisiología , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Humanos , MicroARNs/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/fisiología , Tolerancia a Radiación , Carcinoma de Células Escamosas de Cabeza y Cuello , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Double-strand breaks (DSBs) are repaired by two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The endonuclease Artemis and the PIK kinase Ataxia-Telangiectasia Mutated (ATM), mutated in prominent human radiosensitivity syndromes, are essential for repairing a subset of DSBs via NHEJ in G1 and HR in G2. Both proteins have been implicated in DNA end resection, a mandatory step preceding homology search and strand pairing in HR. Here, we show that during S-phase Artemis but not ATM is dispensable for HR of radiation-induced DSBs. In replicating AT cells, numerous Rad51 foci form gradually, indicating a Rad51 recruitment process that is independent of ATM-mediated end resection. Those DSBs decorated with Rad51 persisted through S- and G2-phase indicating incomplete HR resulting in unrepaired DSBs and a pronounced G2 arrest. We demonstrate that in AT cells loading of Rad51 depends on functional ATR/Chk1. The ATR-dependent checkpoint response is most likely activated when the replication fork encounters radiation-induced single-strand breaks leading to generation of long stretches of single-stranded DNA. Together, these results provide new insight into the role of ATM for initiation and completion of HR during S- and G2-phase. The DSB repair defect during S-phase significantly contributes to the radiosensitivity of AT cells.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Reparación del ADN por Recombinación , Fase S/genética , Proteínas Supresoras de Tumor/fisiología , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Línea Celular , Endonucleasas , Humanos , Recombinasa Rad51/análisis , Tolerancia a Radiación , Fase S/efectos de la radiaciónRESUMEN
Non-homologous end-joining (NHEJ), the major repair pathway for DNA double-strand breaks (DSB) in mammalian cells, employs a repertoire of core proteins, the recruitment of which to DSB-ends is Ku-dependent. Lack of either of the core components invariably leads to a repair deficiency. There has been evidence that an alternative end-joining operates in the absence of the core components. We used chromosomal reporter substrates to specifically monitor NHEJ of single I-SceI-induced-DSB for detailed comparison of classical and alternative end-joining. We show that rapid repair of both compatible and non-compatible ends require Ku-protein. In the absence of Ku, cells use a slow but efficient repair mode which experiences increasing sequence-loss with time after DSB induction. Chemical inhibition and PARP1-depletion demonstrated that the alternative end-joining in vivo is completely dependent upon functional PARP1. Furthermore, we show that the requirement for PARP1 depends on the absence of Ku but not on DNA-dependent protein kinase (DNA-PKcs). Extensive sequencing of repair junctions revealed that the alternative rejoining does not require long microhomologies. Together, we show that mammalian cells need Ku for rapid and conservative NHEJ. PARP1-dependent alternative route may partially rescue the deficient repair phenotype presumably at the expense of an enhanced mutation rate.