RESUMEN
Cells respond to double-strand breaks (DSBs) by activating DNA damage response pathways, including cell cycle arrest. We have previously shown that a single double-strand break generated via CRISPR/Cas9 is sufficient to delay cell cycle progression and compromise cell viability. However, we also found that the cellular response to DSBs can vary, independent of the number of lesions. This implies that not all DSBs are equally toxic, and raises the question if the location of a single double-strand break could influence its toxicity. To systematically investigate if DSB-location is a determinant of toxicity we performed a CRISPR/Cas9 screen targeting 6237 single sites in the human genome. Next, we developed a data-driven framework to design CRISPR/Cas9 sgRNA (crRNA) pools targeting specific chromatin features. The chromatin context was defined using ChromHMM states, Lamin-B1 DAM-iD, DNAseI hypersensitivity, and RNA-sequencing data. We computationally designed 6 distinct crRNA pools, each containing 10 crRNAs targeting the same chromatin state. We show that the toxicity of a DSB is highly similar across the different ChromHMM states. Rather, we find that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects. Thus, chromatin features have little to no effect on the toxicity of a single CRISPR/Cas9-induced DSB.
Asunto(s)
Roturas del ADN de Doble Cadena , Sistemas CRISPR-Cas , Cromatina/genética , Reparación del ADN , Humanos , Laminas , ARNRESUMEN
The precise regulation of RNA Polymerase II (Pol II) transcription after genotoxic stress is crucial for proper execution of the DNA damage-induced stress response. While stalling of Pol II on transcription-blocking lesions (TBLs) blocks transcript elongation and initiates DNA repair in cis, TBLs additionally elicit a response in trans that regulates transcription genome-wide. Here we uncover that, after an initial elongation block in cis, TBLs trigger the genome-wide VCP-mediated proteasomal degradation of promoter-bound, P-Ser5-modified Pol II in trans. This degradation is mechanistically distinct from processing of TBL-stalled Pol II, is signaled via GSK3, and contributes to the TBL-induced transcription block, even in transcription-coupled repair-deficient cells. Thus, our data reveal the targeted degradation of promoter-bound Pol II as a critical pathway that allows cells to cope with DNA damage-induced transcription stress and enables the genome-wide adaptation of transcription to genotoxic stress.
Asunto(s)
Glucógeno Sintasa Quinasa 3 , Transcripción Genética , Daño del ADN/genética , Reparación del ADN/genética , Glucógeno Sintasa Quinasa 3/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismoRESUMEN
Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR-Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.
Asunto(s)
Daño del ADN , Reparación del ADN , Inestabilidad Genómica , Factor 1 de Elongación Peptídica/metabolismo , Elongación de la Transcripción Genética , Sistemas CRISPR-Cas , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Evolución Molecular , Células HCT116 , Humanos , Factor 1 de Elongación Peptídica/genética , ARN Polimerasa II/metabolismo , Factor de Transcripción TFIIH/genética , Factor de Transcripción TFIIH/metabolismo , UbiquitinaciónRESUMEN
Liver cancer remains difficult to treat, owing to a paucity of drugs that target critical dependencies1,2; broad-spectrum kinase inhibitors such as sorafenib provide only a modest benefit to patients with hepatocellular carcinoma3. The induction of senescence may represent a strategy for the treatment of cancer, especially when combined with a second drug that selectively eliminates senescent cancer cells (senolysis)4,5. Here, using a kinome-focused genetic screen, we show that pharmacological inhibition of the DNA-replication kinase CDC7 induces senescence selectively in liver cancer cells with mutations in TP53. A follow-up chemical screen identified the antidepressant sertraline as an agent that kills hepatocellular carcinoma cells that have been rendered senescent by inhibition of CDC7. Sertraline suppressed mTOR signalling, and selective drugs that target this pathway were highly effective in causing the apoptotic cell death of hepatocellular carcinoma cells treated with a CDC7 inhibitor. The feedback reactivation of mTOR signalling after its inhibition6 is blocked in cells that have been treated with a CDC7 inhibitor, which leads to the sustained inhibition of mTOR and cell death. Using multiple in vivo mouse models of liver cancer, we show that treatment with combined inhibition of of CDC7 and mTOR results in a marked reduction of tumour growth. Our data indicate that exploiting an induced vulnerability could be an effective treatment for liver cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Terapia Molecular Dirigida , Sertralina/farmacología , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Mutación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Sertralina/uso terapéutico , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND & AIMS: Treatment of liver cancer remains challenging because of a paucity of drugs that target critical dependencies. Sorafenib is a multikinase inhibitor that is approved as the standard therapy for patients with advanced hepatocellular carcinoma, but it only provides limited survival benefit. In this study we aimed to identify potential combination therapies to improve the clinical response to sorafenib. METHODS: To investigate the cause of the limited therapeutic effect of sorafenib, we performed a CRISPR-Cas9 based synthetic lethality screen to search for kinases whose knockout synergizes with sorafenib. Synergistic effects of sorafenib and selumetinib on cell apoptosis and phospho-ERK (p-ERK) were analyzed by caspase-3/7 apoptosis assay and western blot, respectively. p-ERK was measured by immunochemical analysis using a tissue microarray containing 78 liver cancer specimens. The in vivo effects of the combination were also measured in two xenograft models. RESULT: We found that suppression of ERK2 (MAPK1) sensitizes several liver cancer cell lines to sorafenib. Drugs inhibiting the MEK (MEK1/2 [MAP2K1/2]) or ERK (ERK1/2 [MAPK1/3]) kinases reverse unresponsiveness to sorafenib in vitro and in vivo in a subset of liver cancer cell lines characterized by high levels of active p-ERK, through synergistic inhibition of ERK kinase activity. CONCLUSION: Our data provide a combination strategy for treating liver cancer and suggest that tumors with high basal p-ERK levels, which are seen in approximately 30% of liver cancers, are most likely to benefit from such combinatorial treatment. LAY SUMMARY: Sorafenib is approved as the standard therapy for patients with advanced hepatocellular carcinoma, but only provides limited survival benefit. Herein, we found that inhibition of the kinase ERK2 increases the response to sorafenib in liver cancer. Our data indicate that a combination of sorafenib and a MEK inhibitor is most likely to be effective in tumors with high basal phospho-ERK levels.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Sorafenib/administración & dosificación , Biomarcadores , Sinergismo Farmacológico , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Humanos , FosforilaciónRESUMEN
Selective elimination of BRCA1-deficient cells by inhibitors of poly(ADP-ribose) polymerase (PARP) is a prime example of the concept of synthetic lethality in cancer therapy. This interaction is counteracted by the restoration of BRCA1-independent homologous recombination through loss of factors such as 53BP1, RIF1, and REV7/MAD2L2, which inhibit end resection of DNA double-strand breaks (DSBs). To identify additional factors involved in this process, we performed CRISPR/SpCas9-based loss-of-function screens and selected for factors that confer PARP inhibitor (PARPi) resistance in BRCA1-deficient cells. Loss of members of the CTC1-STN1-TEN1 (CST) complex were found to cause PARPi resistance in BRCA1-deficient cells in vitro and in vivo. We show that CTC1 depletion results in the restoration of end resection and that the CST complex may act downstream of 53BP1/RIF1. These data suggest that, in addition to its role in protecting telomeres, the CST complex also contributes to protecting DSBs from end resection.
Asunto(s)
Proteína BRCA1/deficiencia , Roturas del ADN de Doble Cadena , Complejos Multiproteicos/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Animales , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de los fármacos , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Ratones , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Telómero/metabolismoRESUMEN
Current treatment for advanced stage ovarian clear cell cancer is severely hampered by a lack of effective systemic therapy options, leading to a poor outlook for these patients. Sequencing studies revealed that ARID1A is mutated in over 50% of ovarian clear cell carcinomas. To search for a rational approach to target ovarian clear cell cancers with ARID1A mutations, we performed kinome-centered lethality screens in a large panel of ovarian clear cell carcinoma cell lines. Using the largest OCCC cell line panel established to date, we show here that BRD2 inhibition is predominantly lethal in ARID1A mutated ovarian clear cell cancer cells. Importantly, small molecule inhibitors of the BET (bromodomain and extra terminal domain) family of proteins, to which BRD2 belongs, specifically inhibit proliferation of ARID1A mutated cell lines, both in vitro and in ovarian clear cell cancer xenografts and patient-derived xenograft models. BET inhibitors cause a reduction in the expression of multiple SWI/SNF members including ARID1B, providing a potential explanation for the observed lethal interaction with ARID1A loss. Our data indicate that BET inhibition may represent a novel treatment strategy for a subset of ARID1A mutated ovarian clear cell carcinomas.
Asunto(s)
Adenocarcinoma de Células Claras/tratamiento farmacológico , Adenocarcinoma de Células Claras/genética , Mutación/genética , Proteínas Nucleares/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Proteínas/antagonistas & inhibidores , Factores de Transcripción/genética , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN , Femenino , Humanos , Ratones , Ovario/efectos de los fármacosAsunto(s)
Carcinoma Hepatocelular/genética , Quinasas Ciclina-Dependientes/genética , Neoplasias Hepáticas/genética , Sistemas CRISPR-Cas , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/patología , Proteínas Proto-Oncogénicas c-myc/genética , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Senescence is a proliferation arrest that can result from a variety of stresses. Cancer cells can also undergo senescence, but the stresses that provoke cancer cells to undergo senescence are unclear. Here, we use both functional genetic and compound screens in cancer cells harboring a reporter that is activated during senescence to find targets that induce senescence. We show that suppression of the SWI/SNF component SMARCB1 induces senescence in melanoma through strong activation of the MAP kinase pathway. From the compound screen, we identified multiple aurora kinase inhibitors as potent inducers of senescence in RAS mutant lung cancer. Senescent melanoma and lung cancer cells acquire sensitivity to the BCL2 family inhibitor ABT263. We propose a one-two punch approach for the treatment of cancer in which a drug is first used to induce senescence in cancer cells and a second drug is then used to kill senescent cancer cells.
Asunto(s)
Senescencia Celular/genética , Pruebas Genéticas , Ensayos Analíticos de Alto Rendimiento , Neoplasias/genética , Neoplasias/patología , Aurora Quinasas/antagonistas & inhibidores , Aurora Quinasas/metabolismo , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Regulación hacia Abajo/genética , Receptores ErbB/metabolismo , Técnicas de Inactivación de Genes , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Melanoma/genética , Melanoma/patología , Oncogenes , Inhibidores de Proteínas Quinasas/farmacología , Proteína SMARCB1/genética , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismoRESUMEN
Functional genomic screens using shRNA technology are a great tool in biomedical research. As more labs gain access to the necessary reagents and technology to perform such screens, some may lack in-depth knowledge on the difficulties often encountered. With this protocol, we aim to point out the most important caveats of performing shRNA based screens and provide a streamlined workflow that can be easily adapted to meet the specific needs of any particular screening project.
Asunto(s)
Genómica/métodos , ARN Interferente Pequeño/genética , Animales , Línea Celular , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Lentivirus/genética , Mamíferos/genética , Reacción en Cadena de la Polimerasa/métodos , ARN Interferente Pequeño/análisis , Transducción Genética , Flujo de TrabajoRESUMEN
The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, which suggests that AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared with rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-damaging agents and inhibitors of the DNA damage response. Finally, structural modeling of the PARP3-active site with different PARP inhibitors also highlights the potential to develop compounds with different PARP family member specificity profiles for optimal antitumor activity and tolerability. Cancer Res; 76(20); 6084-94. ©2016 AACR.
Asunto(s)
Neoplasias Experimentales/tratamiento farmacológico , Ftalazinas/farmacología , Piperidinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Animales , Médula Ósea/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN , Reparación del ADN , Dacarbazina/administración & dosificación , Dacarbazina/análogos & derivados , Descubrimiento de Drogas , Genes BRCA1 , Humanos , Ratones , Ftalazinas/administración & dosificación , Ftalazinas/toxicidad , Piperazinas/administración & dosificación , Piperidinas/toxicidad , Poli(ADP-Ribosa) Polimerasas/química , Ratas , Temozolomida , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Despite the substantial progress in the development of targeted anticancer drugs, treatment failure due to primary or acquired resistance is still a major hurdle in the effective treatment of most advanced human cancers. Understanding these resistance mechanisms will be instrumental to improve personalized cancer treatment. EXPERIMENTAL DESIGN: Genome-wide loss-of-function genetic screens were performed to identify genes implicated in resistance to HER2/PI3K/mTOR targeting agents in HER2+ breast cancer cell lines. Expression and adjuvant trastuzumab response data from the HER2+ breast cancer trials FinHer and Responsify were used to validate our findings in patient series. RESULTS: We find that reduced ARID1A expression confers resistance to several drugs that inhibit the HER2/PI3K/mTOR signaling cascade at different levels. We demonstrate that ARID1A loss activates annexin A1 (ANXA1) expression, which is required for drug resistance through its activation of AKT. We find that the AKT inhibitor MK2206 restores sensitivity of ARID1A knockdown breast cancer cells to both the mTOR kinase inhibitor AZD8055 and trastuzumab. Consistent with these in vitro data, we find in two independent HER2+ breast cancer patient series that high ANXA1 expression is associated with resistance to adjuvant trastuzumab-based therapy. CONCLUSIONS: Our findings provide a rationale for why tumors accumulate ARID1A mutations and identify high ANXA1 expression as a predictive biomarker for trastuzumab-based treatment. Our findings also suggest strategies to treat breast cancers with elevated ANXA1 expression. Clin Cancer Res; 22(21); 5238-48. ©2016 AACR.
Asunto(s)
Anexina A1/metabolismo , Antineoplásicos Inmunológicos/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Trastuzumab/farmacología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN , Femenino , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Células MCF-7 , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
High-throughput genetic screens have become essential tools for studying a wide variety of biological processes. Here we experimentally compare systems based on clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) or its transcriptionally repressive variant, CRISPR-interference (CRISPRi), with a traditional short hairpin RNA (shRNA)-based system for performing lethality screens. We find that the CRISPR technology performed best, with low noise, minimal off-target effects and consistent activity across reagents.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Perfilación de la Expresión Génica/métodos , Técnicas de Inactivación de Genes/métodos , Marcadores Genéticos/genética , Pruebas Genéticas/métodos , ARN Interferente Pequeño/genéticaRESUMEN
Most BRAF (V600E) mutant melanomas are sensitive to selective BRAF inhibitors, but BRAF mutant colon cancers are intrinsically resistant to these drugs because of feedback activation of EGFR. We performed an RNA-interference-based genetic screen in BRAF mutant colon cancer cells to search for phosphatases whose knockdown induces sensitivity to BRAF inhibition. We found that suppression of protein tyrosine phosphatase non-receptor type 11 (PTPN11) confers sensitivity to BRAF inhibitors in colon cancer. Mechanistically, we found that inhibition of PTPN11 blocks signaling from receptor tyrosine kinases (RTKs) to the RAS-MEK-ERK pathway. PTPN11 suppression is lethal to cells that are driven by activated RTKs and prevents acquired resistance to targeted cancer drugs that results from RTK activation. Our findings identify PTPN11 as a drug target to combat both intrinsic and acquired resistance to several targeted cancer drugs. Moreover, activated PTPN11 can serve as a biomarker of drug resistance resulting from RTK activation.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Melanoma/tratamiento farmacológico , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Animales , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Resistencia a Antineoplásicos/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Vectores Genéticos , Biblioteca Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Indoles/farmacología , Lentivirus/genética , Sistema de Señalización de MAP Quinasas , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Endogámicos NOD , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Sulfonamidas/farmacología , Transducción Genética , Vemurafenib , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
To identify new regulators of homologous recombination repair, we carried out a genome-wide short-interfering RNA screen combined with ionizing irradiation using RAD51 foci formation as readout. All candidates were confirmed by independent short-interfering RNAs and validated in secondary assays like recombination repair activity and RPA foci formation. Network analysis of the top modifiers identified gene clusters involved in recombination repair as well as components of the ribosome, the proteasome and the spliceosome, which are known to be required for effective DNA repair. We identified and characterized the RNA polymerase II-associated protein CDC73/Parafibromin as a new player in recombination repair and show that it is critical for genomic stability. CDC73 interacts with components of the SCF/Cullin and INO80/NuA4 chromatin-remodeling complexes to promote Histone ubiquitination. Our findings indicate that CDC73 is involved in local chromatin decondensation at sites of DNA damage to promote DNA repair. This function of CDC73 is related to but independent of its role in transcriptional elongation.
RESUMEN
Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.
Asunto(s)
Enzimas Reparadoras del ADN/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Nucleótidos/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Animales , Dominio Catalítico , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalización , Daño del ADN , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Masculino , Ratones , Modelos Moleculares , Conformación Molecular , Terapia Molecular Dirigida , Neoplasias/patología , Oxidación-Reducción/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Pirofosfatasas/antagonistas & inhibidores , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de Xenoinjerto , Hidrolasas NudixRESUMEN
The risks of non-cancerous diseases associated with exposure to low doses of radiation are at present not validated by epidemiological data, and pose a great challenge to the scientific community of radiation protection research. Here, we show that premature senescence is induced in human fibroblasts when exposed to chronic low dose rate (LDR) exposure (5 or 15 mGy/h) of gamma rays from a 137Cs source. Using a proteomic approach we determined differentially expressed proteins in cells after chronic LDR radiation compared to control cells. We identified numerous proteins involved in protection against oxidative stress, suggesting that these pathways protect against premature senescence. In order to further study the role of oxidative stress for radiation induced premature senescence, we also used human fibroblasts, isolated from a patient with a congenital deficiency in glutathione synthetase (GS). We found that these GS deficient cells entered premature senescence after a significantly shorter time of chronic LDR exposure as compared to the GS proficient cells. In conclusion, we show that chronic LDR exposure induces premature senescence in human fibroblasts, and propose that a stress induced increase in reactive oxygen species (ROS) is mechanistically involved.