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.