RESUMEN
The stress-inducible transcription complex NF-κB induces the transcription of genes that regulate proliferation and apoptosis. Constitutively activated NF-κB is common in breast cancers, and contributes to malignant progression and therapeutic resistance. Ataxia telangiectasia mutated (ATM) is a key regulator of the cellular response to DNA double strand breaks (DSBs), and recent reports have demonstrated that ATM is required for the activation of NF-κB following DNA damage. We investigated the role of ATM in the NF-κB signalling cascade induced by ionising radiation (IR) in breast cancer cell lines using KU55933, a novel and specific inhibitor of ATM. KU55933 suppressed IR-induced IκBα degradation, p50/p65 nuclear translocation and binding to kB consensus sequences. KU55933 also suppressed transcription of an NF-κB dependent reporter gene and inhibited IR-induced DSB repair as assessed by the neutral Comet assay. KU55933 sensitised cells to IR, with a concurrent increase in caspase 3 activity. Importantly, KU55933 sensitised IKKß(+/+) and p65(+/+), but not IKKß(-/-) or p65(-/-), mouse embryonic fibroblasts to IR, despite the equivalent inhibitory effects of KU55933 on DSB repair in both the proficient and the deficient cell lines. P65 siRNA had no effect on DSB repair in either breast cancer cell line. When combined with KU55933, DSB repair was inhibited to the same extent as KU55933 alone in both breast cancer cell lines. P65 siRNA alone sensitised both cell lines to IR. A combination of p65 siRNA and KU55933 resulted in no further sensitisation compared to either one alone. Taken together these data support the hypothesis that KU55933-mediated radio-sensitisation is solely a consequence of its inhibition of NF-κB activation. We conclude that radiotherapy deploying ATM inhibitors may be particularly advantageous in tumours where NF-κB is constitutively activated.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Tolerancia a Radiación/genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Daño del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Femenino , Humanos , Ratones , Morfolinas/farmacología , FN-kappa B/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Pironas/farmacología , ARN Interferente Pequeño/genética , Tolerancia a Radiación/efectos de los fármacos , Radiación Ionizante , Activación Transcripcional/genética , Activación Transcripcional/fisiología , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
DNA-dependent protein kinase (DNA-PK) and poly (ADP-ribose) polymerase-1 (PARP-1) participate in nonhomologous end joining and base excision repair, respectively, and are key determinants of radio- and chemo-resistance. Both PARP-1 and DNA-PK have been identified as therapeutic targets for anticancer drug development. Here we investigate the effects of specific inhibitors on enzyme activities and DNA double-strand break (DSB) repair. The enzyme activities were investigated using purified enzymes and in permeabilized cells. Inhibition, or loss of activity, was compared using potent inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361), and cell lines proficient or deficient for DNA-PK or PARP-1. Inactive DNA-PK suppressed the activity of PARP-1 and vice versa. This was not the consequence of simple substrate competition, since DNA ends were provided in excess. The inhibitory effect of DNA-PK on PARP activity was confirmed in permeabilized cells. Both inhibitors prevented ionizing radiation-induced DSB repair, but only AG14361 prevented single-strand break repair. An increase in DSB levels caused by inhibition of PARP-1 was shown to be caused by a decrease in DSB repair, and not by the formation of additional DSBs. These data point to combined inhibition of PARP-1 and DNA-PK as a powerful strategy for tumor radiosensitization.
Asunto(s)
Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Azulenos , Benzodiazepinas/farmacología , Unión Competitiva , Línea Celular , Línea Celular Tumoral , Cromonas/síntesis química , Cromonas/farmacología , Proteína Quinasa Activada por ADN , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Morfolinas/síntesis química , Morfolinas/farmacología , Proteínas Nucleares , Fosforilación , Poli(ADP-Ribosa) PolimerasasRESUMEN
The DNA repair enzymes, DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase-1 (PARP-1), are key determinants of radio- and chemo-resistance. We have developed and evaluated novel specific inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361) for use in anticancer therapy. PARP-1- and DNA-PK-deficient cell lines were 4-fold more sensitive to ionizing radiation (IR) alone, and showed reduced potentially lethal damage recovery (PLDR) in G(0) cells, compared with their proficient counterparts. NU7026 (10 micro M) potentiated IR cytotoxicity [potentiation factor at 90% cell kill (PF(90)) = 1.51 +/- 0.04] in exponentially growing DNA-PK proficient but not deficient cells. Similarly, AG14361 (0.4 micro M) potentiated IR in PARP-1(+/+) (PF(90) = 1.37 +/- 0.03) but not PARP-1(-/-) cells. When NU7026 and AG14361 were used in combination, their potentiating effects were additive (e.g., PF(90) = 2.81 +/- 0.19 in PARP-1(+/+) cells). Both inhibitors alone reduced PLDR approximately 3-fold in the proficient cell lines. Furthermore, the inhibitor combination completely abolished PLDR. IR-induced DNA double strand break (DNA DSB) repair was inhibited by both NU7026 and AG14361, and use of the inhibitor combination prevented 90% of DNA DSB rejoining, even 24-h postirradiation. Thus, there was a correlation between the ability of the inhibitors to prevent IR-induced DNA DSB repair and their ability to potentiate cytotoxicity. Thus, individually, or in combination, the DNA-PK and PARP-1 inhibitors act as potent radiosensitizers and show potential as tools for anticancer therapeutic intervention.