RESUMEN
Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo IIα depletion accelerated origin cluster activation, and topo IIα add-back negated overinitiation. Therefore, topo IIα is not required for DNA replication, but topo IIα clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks. Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo IIα restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters.
Asunto(s)
Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Óvulo/enzimología , Origen de Réplica , Xenopus/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Puntos de Control del Ciclo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dicetopiperazinas , Masculino , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Óvulo/citología , Piperazinas/farmacología , Replicón , Fase S , Espermatozoides/citología , Espermatozoides/metabolismo , Factores de Tiempo , Xenopus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
Midkine (MDK) is a member of a new family of neurotrophic factors considered as rate-limiting growth and angiogenic factors implicated in the onset, invasion, and metastatic process of neuronal tumors, including neuroblastoma. We showed that all neuroblastoma cell lines highly expressed MDK, indicating that it is a critical player in tumor development, which may henceforth represent an attractive therapeutic target. We showed that the knockdown of MDK expression by siRNA led to a marked and significant decrease in neuroblastoma (IGR-N91 and SH-SY5Y) cell proliferation in vitro. Using a new strategy, we then evaluated the antitumor effect of a truncated MDK protein, lacking the C-terminal 81-121 portion of the molecule (MDKΔ81-121), which may act as a dominant-negative effector for its mitogenic, angiogenic, and tumorigenic activities by heterodimerizing with the wild-type protein. In vitro studies showed that MDKΔ81-121 selectively inhibited MDK-dependent tumor cells and was able to strongly reduce endothelial cell proliferation and migration and to induce ER stress-mediated apoptosis. We then investigated the effects of MDKΔ81-121 in vivo using electrotransfer of a plasmid encoding a secretable form of MDKΔ81-121 into tibialis cranialis muscles of nude mice. We showed that MDKΔ81-121 dramatically inhibited (up to 91%) tumor development and growth. This inhibition was correlated with the detection of the MDKΔ81-121 molecule in plasma and the suppression of intratumor neovascularization. Our findings demonstrate that MDK inhibition is a tractable therapeutic target for this lethal pediatric malignancy.