RESUMEN
Reactive oxygen species (ROS) oxidize nucleotide triphosphate pools (e.g., 8-oxodGTP), which may kill cells if incorporated into DNA. Whether cancers avoid poisoning from oxidized nucleotides by preventing incorporation via the oxidized purine diphosphatase MTH1 remains under debate. Also, little is known about DNA polymerases incorporating oxidized nucleotides in cells or how oxidized nucleotides in DNA become toxic. Here we show that replacement of one of the main DNA replicases in human cells, DNA polymerase delta (Pol δ), with an error-prone variant allows increased 8-oxodG accumulation into DNA following treatment with TH588, a dual MTH1 inhibitor and microtubule targeting agent. The resulting elevated genomic 8-oxodG correlated with increased cytotoxicity of TH588. Interestingly, no substantial perturbation of replication fork progression was observed, but rather mitotic progression was impaired and mitotic DNA synthesis triggered. Reducing mitotic arrest by reversin treatment prevented accumulation of genomic 8-oxodG and reduced cytotoxicity of TH588, in line with the notion that mitotic arrest is required for ROS buildup and oxidation of the nucleotide pool. Furthermore, delayed mitosis and increased mitotic cell death was observed following TH588 treatment in cells expressing the error-prone but not wild-type Pol δ variant, which is not observed following treatments with antimitotic agents. Collectively, these results link accumulation of genomic oxidized nucleotides with disturbed mitotic progression. SIGNIFICANCE: These findings uncover a novel link between accumulation of genomic 8-oxodG and perturbed mitotic progression in cancer cells, which can be exploited therapeutically using MTH1 inhibitors.See related commentary by Alnajjar and Sweasy, p. 3459.
Asunto(s)
8-Hidroxi-2'-Desoxicoguanosina , Monoéster Fosfórico Hidrolasas , Enzimas Reparadoras del ADN/genética , Genómica , Humanos , Mitosis/genética , Monoéster Fosfórico Hidrolasas/genética , Pirimidinas/farmacologíaRESUMEN
Mesenchymal stromal cells (MSCs) promote wound healing by expediting the inflammatory phase. Local injection of MSCs into injured vocal folds (VFs) is effective in animal models, suggesting suitability for clinical translation. Despite their therapeutic potential, MSCs do not persist within the VF. This study evaluates whether hyaluronan (HA) hydrogels offer a safe delivery vehicle for local injection of MSCs into VFs, and increase longevity of the cells within the injured tissue. MSCs ± HA hydrogel were exposed to interleukin (IL)1ß, IL8, and chemokine (C-C motif) ligand 4, and evaluated for mRNA expression of matrix remodeling genes and secretion of immunomodulatory/prohealing factors. Chemotaxis/invasion in response to inflammation was evaluated. A lapin model of VF injury evaluated in vivo effects of MSCs ± HA hydrogel on enhancing VF healing. Histological evaluation of inflammation, type I collagen expression, HA hydrogel resorption, and MSC persistence was evaluated at 3 and 25 days after injury. MSCs within HA hydrogel were responsive to their extracellular environment, upregulating immunomodulatory factors when exposed to inflammation. Despite delayed migration out of the gel in vitro, the MSCs did not persist longer within the injured tissue in vivo. MSCs ± HA hydrogel exerted equivalent dampening of inflammation in vivo. The gel was resorbed within 25 days and no edema was evident. HA hydrogels can be safely used in the delivery of MSCs to injured VFs, minimizing leakage of administered cells. MSCs within the HA hydrogel did not persist longer than those in suspension, but did exert comparable therapeutic effects.
Asunto(s)
Ácido Hialurónico/farmacología , Hidrogeles/farmacología , Enfermedades de la Laringe/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/efectos de los fármacos , Pliegues Vocales/lesiones , Animales , Células Cultivadas , Quimiocina CCL4/genética , Quimiocina CCL4/metabolismo , Quimiotaxis , Colágeno/genética , Colágeno/metabolismo , Humanos , Ácido Hialurónico/análogos & derivados , Hidrogeles/química , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Conejos , Andamios del Tejido/efectos adversos , Andamios del Tejido/químicaRESUMEN
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
Treatments with Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors have offered patients carrying cancers with mutated BRCA1 or BRCA2 genes a new and in many cases effective option for disease control. There is potentially a large patient population that may also benefit from PARP inhibitor treatment, either in monotherapy or in combination with chemotherapy. Here, we describe the multifaceted role of PARP inhibitors and discuss which treatment options could potentially be useful to gain disease control without potentiating side effects.
RESUMEN
CK2 phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express an XRCC1 protein (XRCC1(ckm)) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification gives distinct phenotypes in SSB repair and base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the alkylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1(ckm) (CKM cells) or following inhibition with the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1(ckm) protein has a higher affinity for DNA than wild type XRCC1 protein and resides in an immobile fraction on DNA, in particular after damage. We propose a model whereby the increased affinity for DNA sequesters XRCC1(ckm) and the repair enzymes associated with it, at the repair site, which retards kinetics of BER. In conclusion, our results indicate that phosphorylation of XRCC1 by CK2 facilitates the BER incision step, likely by promoting dissociation from DNA.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Roturas del ADN de Cadena Simple/efectos de los fármacos , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Alquilantes/farmacología , Animales , Células CHO , Supervivencia Celular/genética , Cricetinae , Cricetulus , ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Tasa de Mutación , Fosforilación/efectos de los fármacos , Recombinasa Rad51/metabolismo , Ésteres del Ácido Sulfúrico/farmacología , Factores de Tiempo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
The cellular response to double-strand breaks (DSBs) in DNA is a complex signalling network, mobilized by the nuclear protein kinase ataxia-telangiectasia mutated (ATM), which phosphorylates many factors in the various branches of this network. A main question is how ATM regulates DSB repair. Here, we identify the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) as an ATM target. PNKP phosphorylates 5'-OH and dephosphorylates 3'-phosphate DNA ends that are formed at DSB termini caused by DNA-damaging agents, thereby regenerating legitimate ends for further processing. We establish that the ATM phosphorylation targets on human PNKP-Ser 114 and Ser 126-are crucial for cellular survival following DSB induction and for effective DSB repair, being essential for damage-induced enhancement of the activity of PNKP and its proper accumulation at the sites of DNA damage. These findings show a direct functional link between ATM and the DSB-repair machinery.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Citotoxinas/farmacología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Enzimas Reparadoras del ADN/genética , Células HEK293 , Humanos , Ratones , Fosforilación/efectos de los fármacos , Cinostatina/farmacologíaRESUMEN
Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.
Asunto(s)
Roturas del ADN de Cadena Simple , Reparación del ADN , Poli(ADP-Ribosa) Polimerasas/fisiología , Alquilantes/toxicidad , Animales , Línea Celular , ADN Glicosilasas/fisiología , Proteínas de Unión al ADN/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Cinética , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Ésteres del Ácido Sulfúrico/toxicidad , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
XRCC1 is a scaffold protein that interacts with several DNA repair proteins and plays a critical role in DNA base excision repair (BER). XRCC1 protein is in a tight complex with DNA ligase IIIalpha (Lig III) and this complex is involved in the ligation step of both BER and repair of DNA single strand breaks. The majority of XRCC1 has previously been demonstrated to exist in a phosphorylated form and cells containing mutant XRCC1, that is unable to be phosphorylated, display a reduced rate of single strand break repair. Here, in an unbiased assay, we demonstrate that the cytoplasmic form of the casein kinase 2 (CK2) protein is the major protein kinase activity involved in phosphorylation of XRCC1 in human cell extracts and that XRCC1 phosphorylation is required for XRCC1-Lig III complex stability. We demonstrate that XRCC1-Lig III complex containing mutant XRCC1, in which CK2 phosphorylation sites have been mutated, is unstable. We also find that a knockdown of CK2 by siRNA results in both reduced XRCC1 phosphorylation and stability, which also leads to a reduced amount of Lig III and accumulation of DNA strand breaks. We therefore propose that CK2 plays an important role in DNA repair by contributing to the stability of XRCC1-Lig III complex.