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Molecular Mechanisms of Polyamine Metabolism Affecting Oncogenic SignalingPaz, Edwin Alfredo January 2013 (has links)
Eukaryotic cells tightly regulate metabolism in order to sustain normal processes. Dysregulation of cellular metabolism is associated with multiple diseases including cancer. Polyamine metabolism is a tightly regulated process that is co-opted by multiple cancers for selective growth advantages. Polyamines are small organic molecules with two or more amino groups attached, whose biosynthesis is initiated by ornithine decarboxylase (ODC). Although much is known regarding the effects of polyamine metabolism and ODC on cellular processes, little is known regarding the intracellular signaling events that are regulated by polyamines. Clinical studies demonstrated that the ODC inhibitor difluromethylornithine (DFMO) was an effective chemopreventative strategy causing a reduction of colon adenomas in patients with prior colon polyps. However, the molecular mechanisms leading to this reduction are unknown. This dissertation provides mechanistic insight into the biological roles of the polyamines and show that these amines are regulators of multiple non-coding RNAs involved in cellular responses including effects on the let-7 microRNA family. Moreover, the polyamine modified translation factor eIF5A is demonstrated to regulate the oncofetal factor LIN28. This work also indicates that polyamines regulate the mTOR pathway and suggests alternative signaling nodes for polyamine-mediated regulation of cellular processes. Overall, these findings support the notion that polyamines are oncometabolites that are targetable and serve as a promising approach to manipulate oncogenic signaling for cancer therapy.
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R-2-hydroxyglutarate modulates DNA Replication via Integrated Stress ResponseSharma, Jyoti 06 1900 (has links)
Les gènes de l'isocitrate déshydrogénase (IDH) sont mutés dans 70 à 80 % des gliomes de bas grade. Les enzymes mutantes IDH qui en résultent présentent une activité de gain de fonction, produisant du R-2-hydroxyglutarate (R-2-HG), appelé oncométabolite en raison de son accumulation anormale dans les tumeurs et de ses activités oncogéniques potentielles. Parmi les caractéristiques du cancer telles que la reprogrammation métabolique et épigénétique, le stress réplicatif et la stabilité du génome ont été peu caractérisés dans les cancers IDH-mutants. Par conséquent, cette étude vise à étudier l'impact de l'accumulation de R-2-HG sur la réplication de l'ADN et sa contribution au stress réplicatif dans les cancers IDH-mutants.
Nous avons étudié la dynamique de la fourche de réplication dans des astrocytes humains normaux et confirmé les résultats dans d'autres lignées cellulaires normales et cancéreuses. Nous avons constaté que le traitement exogène par l'octyl-R-2-HG entravait la progression de la fourche de réplication et retardait par conséquent l'achèvement de la phase S. L'évaluation des niveaux de phosphorylation des protéines RPA, CHK1 et H2AX a révélé que la réponse classique au stress réplicatif (RSR) n'était pas activée. Un état cellulaire dans lequel la réplication de l'ADN est altérée sans activation de la RSR a notamment été décrit dans la littérature comme résultant de l'activation de la réponse au stress intégré (ISR). Cependant, l'activation de la RSI dans les cancers mutants IDH n'est pas bien étudiée. En évaluant les marqueurs d'activation de la RSI, tels que la phosphorylation de l'eIF2α et les niveaux de protéines ATF4, nous avons montré que l'octyl-R-2-HG activait la RSI. De plus, le blocage de l'ISR a partiellement sauvé la fourche de réplication et la progression de la phase S. Nous avons répliqué cette étude oncométrique. Nous avons reproduit ce défaut de réplication de l'ADN lié à l'oncométabolite ainsi que l'effet de sauvetage partiel de l'ISRIB lors de l'induction de la surexpression du gène IDH mutant. Nos résultats indiquent que la production de R-2-HG associée à la mIDH peut inhiber la dynamique normale de réplication de l'ADN via la signalisation ISR. / The isocitrate dehydrogenase (IDH) genes are mutated in 70-80% of low-grade gliomas. The resulting IDH mutant enzymes exhibit gain-of-function activity, producing R-2-hydroxyglutarate (R-2-HG), which is referred to as an oncometabolite due to its abnormal accumulation in tumours and potential oncogenic activities. Among the hallmarks of cancer such as metabolic and epigenetic reprogramming, replicative stress and genome stability have been poorly characterized in IDH-mutant cancer. Therefore, this study aims to investigate the impact of R-2-HG accumulation on DNA replication and its contribution to replicative stress in IDH-mutant cancers.
We investigated replication fork dynamics in normal human astrocytes and confirmed the results in other normal and cancer cell lines. We found that exogenous treatment with octyl-R-2-HG impaired replication fork progression and consequently delayed S-phase completion. Assessment of RPA, CHK1 and H2AX protein phosphorylation levels revealed that the classical Replicative Stress Response (RSR) was not activated. Among others, a cell state in which DNA replication was impaired without activation of the RSR has been described in the literature as a result of activation of the Integrated Stress Response (ISR). However, ISR activation in IDH-mutant cancers is not well studied. Hence, by assessing ISR activation markers such as eIF2α phosphorylation and ATF4 protein levels, we showed that octyl-R-2-HG activated ISR. Moreover, blocking ISR partially rescued the replication fork and S-phase progression. We replicated this oncometabolite-related DNA replication defect as well as ISRIB’s partial rescue effect upon induction of mutant IDH gene overexpression. Our results indicate that mIDH-associated R-2-HG production possibly inhibits normal DNA replication dynamics via ISR signalling.
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