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International audience; DNA replication checkpoint is activated in response to replication stresses. It maintains the integrity of stalled replication forks and prevents premature segregation of largely unreplicated chromosomes. In budding yeast, Mec1 and Rad53 kinases (homologous to mammalian ATM/ATR and Chk2 kinases, respectively) are the main effectors of this checkpoint control. Using a yeast based screen, we have identified a compound (named here ENA) which inhibits DNA replication and activates Mec1/Rad53 checkpoint. A brief exposure to this compound stops fork progression at or near replication origin and renders the forks incompetent to resume replication despite the presence of a functional checkpoint. ENA also inhibits DNA synthesis in mammalian cells leading to the activation of ATM/ATR pathway and the induction of apoptosis in a p53 independent manner. Interestingly, ENA acts as an effective anti-proliferative agent against a subset of cancer cell lines and as an anti-tumor agent against human xenografts in mice. Thus, ENA is a potent cell cycle inhibitor with conceivable therapeutic potential.

Autophagy is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. PARP-1 has been implicated in different pathways leading to cell death and its inhibition potentiates chemotherapy-induced cell death. Whether PARP-1 participates in the cell's decision to commit to autophagy following DNA damage is still not known. To address this issue PARP-1 wild-type and deficient cells have been treated with a dose of doxorubicin that induces autophagy. Electron microscopy examination and GFP-LC3 transfection revealed autophagic vesicles and increased expression of genes involved in autophagy (bnip-3, cathepsin b and l and beclin-1) in wild-type cells treated with doxo but not in parp-1(-/-) cells or cells treated with a PARP inhibitor. Mechanistically the lack of autophagic features in PARP-1 deficient/PARP inhibited cells is attributed to prevention of ATP and NAD(+) depletion and to the activation of the key autophagy regulator mTOR. Pharmacological or genetical inhibition of autophagy results in increased cell death, suggesting a protective role of autophagy induced by doxorubicin. These results suggest that autophagy might be cytoprotective during the response to DNA damage and suggest that PARP-1 activation is involved in the cell's decision to undergo autophagy.

International audience; This study was designed to analyze the effects of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid) toward the aryl hydrocarbon receptor (AhR) pathway because Ca2+/calmodulin-dependent protein kinase (CaMK) Ialpha, known as a downstream CaMKK effector, has been recently shown to contribute to the AhR cascade. STO-609 failed to alter up-regulation of the AhR target CYP1A1 in response to the potent AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells. STO-609, used at a 25 muM concentration known to fully inhibit CaMKK activity, was surprisingly found to markedly induce CYP1A1 expression and activity by itself in MCF-7 cells; it similarly up-regulated various other AhR target genes in human macrophages. STO-609-related CYP1A1 induction was prevented by chemical inhibition or small interfering RNA-mediated knockdown expression of AhR. Moreover, STO-609 was demonstrated to physically interact with the ligand-binding domain of AhR, as assessed by TCDD binding competition assay, and to induce AhR translocation to the nucleus. As already reported for AhR agonists, STO-609 triggered the increase of [Ca2+](i) and activation of CaMKIalpha, whose inhibition through the use of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester or the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), respectively, prevented STO-609-mediated CYP1A1 activity induction. Taken together, these results demonstrate that the CaMKK inhibitor STO-609 can act as an AhR ligand and, in this way, fully activates the Ca2+/CaMKIalpha/AhR cascade. Such data, therefore, make unlikely any contribution of CaMKK activity to the AhR pathway and, moreover, suggest that caution may be required when using STO-609 as a specific inhibitor of CaMKKs.

p53 deficiency confers resistance to doxo (doxorubicin), a clinically active and widely used antitumour anthracycline antibiotic. The purpose of the present study was to investigate the reversal mechanism of doxo resistance by the potent PARP [poly(ADP-ribose) polymerase] inhibitor ANI (4-amino-1,8-naphthalimide) in the p53-deficient breast cancer cell lines EVSA-T and MDA-MB-231. The effects of ANI, in comparison with doxo alone, on doxo-induced apoptosis, were investigated in matched pairs of EVSA-T or MDA-MB-231 with or without ANI co-treatment. Doxo elicited PARP activation as determined by Western blotting and immunofluorescence of poly(ADP-ribose), and ANI enhanced the cytotoxic activity of doxo 2.3 times and in a caspase-dependent manner. The long-term cytotoxic effect was studied by a colony-forming assay. Using this assay, ANI also significantly potentiates the long-term cytotoxic effect with respect to treatment with doxo alone. Decrease in mitochondrial potential together with an increase in cytochrome c release, association of Bax with the mitochondria and caspase 3 activation were also observed in the presence of ANI. Therefore PARP inhibition may represent a novel way of selectively targeting p53-deficient breast cancer cells. The underlying mechanism is probably a potentiation of unrepaired DNA damage, shifting from DNA repair to apoptosis due to the effective inhibition of PARP activity.