Expression et fonctions des gènes nm23 au cours de la différenciation cellulaire chez la souris

Les gènes nm23 appartiennent à une famille génique codant pour différentes isoformes des Nucléoside Di-Phosphate Kinase (NDPK). Ces enzymes catalysent la phosphorylation des nucléosides diphosphates en nucléosides triphosphates autre que l'ATP. Chaque isoforme participerait en outre à des processus tels que la différenciation et la prolifération cellulaires. Chez la souris, le taux d'expression de ces gènes varie d'un organe à l'autre. Les gènes nm23-M1 et M3 s'expriment préférentiellement dans le système nerveux et les organes sensoriels, les ARNm de nm23-M2 présentent une distribution ubiquitaire. Dans certains organes embryonnaires, nm23-M1, -M2 et -M3 sont exprimés dans des zones de différenciation neuronale alors que les ARNm nm23-M4 occupent des zones de prolifération cellulaire. L'expression différentielle des messagers nm23 suggère un rôle spécifique de chaque isoforme, suggérant leur implication dans les phénomènes de différenciation cellulaire. Dans une lignée de cellules PC12, un traitement au NGF stimule l'expression des protéines NDPK avec des modifications de la distribution subcellulaire des différentes isoformes. La suppression des formes A et B sauvages ou mutées provoque des effets complexes mais distincts sur la différenciation des PC12. Ces effets traduisent des rôles différentiels dans leur détermination neurochimique. L'implication dela NDPK A dans les voies de signalisation aboutissant à la différenciation cellulaire et étudié électrophysiologiquement dans une lignée de souris invalidées pour le gène nm23-M1. Des modèles de culture primaire de cellules granulaires du cervelet associé et de cellules cardiaques dissociées nous ont permis de mettre en évidence un rôle direct de la NDPK A dans le phénomène de récupération de la désensibilisation des récepteurs membranaires couplés à des canaux potassiques rectifiant rentrant (GIRK), colocalisés in vivo avec les NDPK.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Small Molecules, Inhibitors of DNA-PK, Targeting DNA Repair, and Beyond

Many current chemotherapies function by damaging genomic DNA in rapidly dividing cells ultimately leading to cell death. This therapeutic approach differentially targets cancer cells that generally display rapid cell division compared to normal tissue cells. However, although these treatments are initially effective in arresting tumor growth and reducing tumor burden, resistance and disease progression eventually occur. A major mechanism underlying this resistance is increased levels of cellular DNA repair. Most cells have complex mechanisms in place to repair DNA damage that occurs due to environmental exposures or normal metabolic processes. These systems, initially overwhelmed when faced with chemotherapy induced DNA damage, become more efficient under constant selective pressure and as a result chemotherapies become less effective. Thus, inhibiting DNA repair pathways using target specific small molecule inhibitors may overcome cellular resistance to DNA damaging chemotherapies. Non-homologous end joining a major mechanism for the repair of double-strand breaks (DSB) in DNA is regulated in part by the serine/threonine kinase, DNA dependent protein kinase (DNA-PK). The DNA-PK holoenzyme acts as a scaffold protein tethering broken DNA ends and recruiting other repair molecules. It also has enzymatic activity that may be involved in DNA damage signaling. Because of its’ central role in repair of DSBs, DNA-PK has been the focus of a number of small molecule studies. In these studies specific DNA-PK inhibitors have shown efficacy in synergizing chemotherapies in vitro. However, compounds currently known to specifically inhibit DNA-PK are limited by poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives. Future improvement in DNA-PK inhibition will likely be achieved by designing new molecules based on the recently reported crystallographic structure of DNA-PK. Computer based drug design will not only assist in identifying novel functional moieties to replace the metabolically labile morpholino group but will also facilitate the design of molecules to target the DNA-PKcs/Ku80 interface or one of the autophosphorylation sites

Simultaneous inhibition of ATR and PARP sensitizes colon cancer cell lines to irinotecan

Enhanced DNA damage repair is one mechanism involved in colon cancer drug resistance. Thus, targeting molecular components of repair pathways with specific small molecule inhibitors may improve the efficacy of chemotherapy. ABT-888 and VE-821, inhibitors of poly-ADP-ribose-polymerase (PARP) and the serine/threonine-kinase Ataxia telangiectasia related (ATR), respectively, were used to treat colon cancer cell lines in combination with the topoisomerase-I inhibitor irinotecan (SN38). Our findings show that each of these DNA repair inhibitors utilized alone at nontoxic single agent concentrations resulted in sensitization to SN38 producing a 1.4-3 fold reduction in the 50% inhibitory concentration (IC50) of SN38 in three colon cancer cell lines. When combined together, nontoxic concentrations of ABT-888 and VE-821 produced a 4.5-27 fold reduction in the IC50 of SN38 with the HCT-116 colon cancer cells demonstrating the highest sensitization as compared to LoVo and HT-29 colon cancer cells. Furthermore, the combination of all three agents was associated with maximal G(2) -M arrest and enhanced DNA-damage (gamma H2AX) in all three colon cancer cell lines. The mechanism of this enhanced sensitization was associated with: (a) maximal suppression of SN38 induced PARP activity in the presence of both inhibitors and (b) ABT-888 producing partial abrogation of the VE-821 enhancement of SN38 induced DNA-PK phosphorylation, resulting in more unrepaired DNA damage; these alterations were only present in the HCT-116 cells which have reduced levels of ATM. This novel combination of DNA repair inhibitors may be useful to enhance the activity of DNA damaging chemotherapies such as irinotecan and help produce sensitization to this drug in colon cancer