In higher eukaryotes, cell cycle progression is controlled by cyclin dependent kinases (Cdks) complexed with cyclins. A-type cyclins are involved at both G1/S and G2/M transitions of the cell cycle. Cyclin A2 activates cdc2 (Cdk1) on passage into mitosis and Cdk2 at the G1/S transition. Antisense constructs, or antibodies directed against cyclin A2 block cultured mammalian cells at both of these transitions. In contrast, overexpression of cyclin A2 appears to advance S phase entry and confer anchorage-independent growth, and can lead to apoptosis. A second A-type cyclin, cyclin A1 has been described recently which, in the mouse, is expressed in germ cells but not somatic tissues. To address the possible redundancy between different cyclins in vivo and also the control of early embryonic cell cycles, we undertook the targeted deletion of the murine cyclin A2 gene. The homozygous null mutant is embryonically lethal, demonstrating that the cyclin A2 gene is essential. Surprisingly, homozygous null mutant embryos develop normally until post-implantation, around day 5.5 p.c. This observation may be explained by the persistence of a maternal pool of cyclin A2 protein until at least the blastocyst stage, or an unexpected role for cyclin A1 during early embryo development.

Germline mutations in the RB1 gene confer hereditary predisposition to retinoblastoma. We have performed a mutation survey of the RB1 gene in 232 patients with hereditary or non hereditary retinoblastoma. We systematically explored all 27 exons and flanking sequences as well as the promotor. All types of point mutations are represented and are found unequally distributed along the RB1 gene sequence. In the population we studied, exons 3, 8, 18 and 19 are preferentially altered. The range of frequency of detection of germline mutations is about 20%, indicating that other mechanisms of inactivation of RB1 should be involved. The spectrum of mutations presented here should help to improve the clinical management of retinoblastoma and to understand the molecular mechanisms leading to tumorigenesis.

DNA methylation is involved in the regulation of gene expression and plays an important role in normal developmental processes and diseases, such as cancer. DNA methyltransferases are the enzymes responsible for DNA methylation on the position 5 of cytidine in a CpG context. In order to identify and characterize novel inhibitors of these enzymes, we developed a fluorescence-based throughput screening by using a short DNA duplex immobilized on 96-well plates. We have screened 114 flavones and flavanones for the inhibition of the murine catalytic Dnmt3a/3L complex and found 36 hits with IC(50) values in the lower micromolar and high nanomolar ranges. The assay, together with inhibition tests on two other methyltransferases, structure-activity relationships and docking studies, gave insights on the mechanism of inhibition. Finally, two derivatives effected zebrafish embryo development, and induced a global demethylation of the genome, at doses lower than the control drug, 5-azacytidine.

In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)—the enzymes responsible for DNA methylation—are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(β-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First, we observe an increase in the DNA binding when zebularine is incorporated into the DNA, compared to deoxycytidine and 5-fluorodeoxycytidine, together with a strong decrease in the dissociation rate. Second, we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible, differing thus from 5-fluorodeoxycytidine. Third, no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA, hindering the methylation and decreasing the dissociation, thereby trapping the enzyme and preventing turnover even at other sites.

DNA methyltransferases (DNMT) are promising drug targets in cancer provided that new, more specific, and chemically stable inhibitors are discovered. Among the non-nucleoside DNMT inhibitors, N-phthaloyl-l-tryptophan 1 (RG108) was first identified as inhibitor of DNMT1. Here, 1 analogues were synthesized to understand its interaction with DNMT. The indole, carboxylate, and phthalimide moieties were modified. Homologated and conformationally constrained analogues were prepared. The latter were synthesized from prolinohomotryptophan derivatives through a methodology based amino-zinc-ene-enolate cyclization. All compounds were tested for their ability to inhibit DNMT1 in vitro. Among them, constrained compounds 16-18 and NPys derivatives 10-11 were found to be at least 10-fold more potent than the reference compound. The cytotoxicity on the tumor DU145 cell line of the most potent inhibitors was correlated to their inhibitory potency. Finally, docking studies were conducted in order to understand their binding mode. This study provides insights for the design of the next-generation of DNMT inhibitors.

DNA methyltransferases (DNMTs) are responsible for DNA methylation, an epigenetic modification involved in gene regulation. Families of conjugates of procainamide, an inhibitor of DNMT1, were conceived and produced by rapid synthetic pathways. Six compounds resulted in potent inhibitors of the murine catalytic Dnmt3A/3L complex and of human DNMT1, at least 50 times greater than that of the parent compounds. The inhibitors showed selectivity for C5 DNA methyltransferases. The cytotoxicity of the inhibitors was validated on two tumour cell lines (DU145 and HCT116) and correlated with the DNMT inhibitory potency. The inhibition potency of procainamide conjugated to phthalimide through alkyl linkers depended on the length of the linker; the dodecane linker was the best.

Progression through the mammalian cell cycle is regulated by the sequential activation and inactivation of the cyclin-dependent kinases. In adult cells, cyclin A2-dependent kinases are required for entry into S and M phases, completion of S phase, and centrosome duplication. However, mouse embryos lacking the cyclin A2 gene nonetheless complete preimplantation development, but die soon after implantation. In this report, we investigated whether a contribution of maternal cyclin A2 mRNA and protein to early embryonic cell cycles might explain these conflicting observations. Our data show that a maternal stock of cyclin A2 mRNA is present in the oocyte and persists after fertilization until the second mitotic cell cycle, when it is degraded to undetectable levels coincident with transcriptional activation of the zygotic genome. A portion of maternally derived cyclin A2 protein is stable during the first mitosis and persists in the cytoplasm, but is completely degraded at the second mitosis. The ability of cyclin A2-null mutants to develop normally from the four-cell to the postimplantation stage in the absence of detectable cyclin A2 gene product indicates therefore that cyclin A2 is dispensable for cellular progression during the preimplantation nongrowth period of mouse embryo development.

In order to discover new inhibitors of the DNA methyltransferase 3A/3L complex, we used a medium-throughput nonradioactive screen on a random collection of 1120 small organic compounds. After a primary hit detection against DNA methylation activity of the murine Dnmt3A/3L catalytic complex, we further evaluated the EC50 of the 12 most potent hits as well as their cytotoxicity on DU145 prostate cancer cultured cells. Interestingly, most of the inhibitors showed low micromolar activities and little cytotoxicity. Dichlone, a small halogenated naphthoquinone, classically used as pesticide and fungicide, showed the lowest EC50 at 460 nM. We briefly assessed the selectivity of a subset of our new inhibitors against hDNMT1 and bacterial Dnmts, including M. SssI and EcoDam, and the protein lysine methyltransferase PKMT G9a and the mode of inhibition. Globally, the tested molecules showed a clear preference for the DNA methyltransferases, but poor selectivity among them. Two molecules including Dichlone efficiently reactivated YFP gene expression in a stable HEK293 cell line by promoter demethylation. Their efficacy was comparable to the DNMT inhibitor of reference 5-azacytidine.