These samples were normalized by blotting with an antibody against -tubulin. genotoxic therapies. Keywords:microRNAs, p53, Wee1, Chk1, checkpoint kinases, genotoxic stress TP53 is a universal tumour suppressor whose major function is to control the stability of genome in response to various forms of genotoxic stress. Acting mostly as a transcription factor, p53 regulates the gene expression programme to prevent damaged cells from propagation. Products of p53-dependent genes induce DNA repair, cell cycle arrest in G1/S and G2/M phases, and apoptosis.1G1/S arrest is mediated mostly by the p21 protein whose expression is induced by p53.1,2,3,4The former binds to and inhibits the kinase activity of cdk2/cyclin E and cdk4/cyclin D complexes, which are essential for G1/S transition. The p53-mediated G2 arrest involves, in addition to the p21 protein, other p53 targets such as members of the 14-3-3 family of proteins5,6and GADD45.7The 14-3-3 protein binds to and inhibits the phosphatase activity of Cdc25, whereas GADD45 attenuates the activity of mitotic cdc2 (cdk1) kinase by sequestering its cyclin partner, cyclin B.8Activity of cdk1 is also regulated by two checkpoint kinases Chek1 and Wee1. On DNA damage, Chk1 and Wee1 phosphorylate and inhibit cdk1, thus preventing entry into mitosis.9,10,11CHK1 also phosphorylates p53 and together with ATM/ATR stabilizes and activates p53, further enhancing the G2-M checkpoint in the cell cycle.12 Recently, it has been demonstrated that microRNAs (miRNAs) are the integral part of the p53 network.13,14miRNAs are a group of small (2025 nucleotides) non-coding RNA molecules, which are expressed endogenously in cells and that regulate gene expression at the post-transcriptional level.15To date, there is more than a dozen of different families of microRNA genes controlled by p53.16,17Importantly, p53 has been shown to regulate both transcription and maturation of miRNA genes, whose products aid in the processes of cell cycle arrest and apoptosis.16 Here, we report an identification of two abundantly expressed microRNAs, miR-16 and miR-26a, whose expression is regulated by p53 during the genotoxic stress. Importantly, among the targets of these miRs are two critical checkpoint kinases, Chk1 and Wee1. The p53-dependent augmentation of miR-16 and miR-26a expression levels led to the cell cycle arrest of tumour cells in G1/S and increased apoptosis. Strikingly, the bioinformatics analysis of survival times for patients with breast and prostate cancers has revealed that co-expression of miR-16 and miR-26a correlated with a better survival outcome. Collectively, our data provide a novel mechanism whereby p53 represses Chk1 and Wee1 expression, at least in part, via upregulation of miR-16 and miR-26a and thus sensitizes tumour cells to genotoxic therapies. == Results == == p53 and genotoxic stress control the expression of miR-16 and miR-26a == To define the spectrum of microRNA regulated by p53 on genotoxic stress, we employed the microarray expression analysis of microRNAs in a matching pair of cell lines that differ in their p53 Isotretinoin expression status. Control U2-OS cells stably expressing non-specific scrambled shRNA (U2-OS scr) showed low levels of p53, as these cells overexpress p53-specific E3 ubiquitin ligase, Mdm2, which targets p53 for the 26S proteasome-dependent degradation.18,19,20,21,22A matching cell line (U2-OS KDp53) with suppressed expression of p53 due to the presence Isotretinoin of p53-specific shRNA also showed very Isotretinoin low level of p53 expression. However, doxorubicin treatment stabilized p53 in U2-OS scr cells (Figure Sema3d 1, left), whereas in U2-OS KDp53 cells the level of p53 was still low (Figure 1a). Isotretinoin == Figure 1. == Expression of miR-16 and miR-26a is induced in the presence of p53 by doxorubicin. (a) Comparison of the p53 levels in U2-OS cells stably expressing scrambled shRNAversusp53-specific shRNA (U2-OS KDp53) on treatment with doxorubicin. U2-OS cells with wild type and knocked-down expression of p53 were continuously treated with 0.5M of doxorubicin for 0, 6, and 12 h before harvesting. Cell lysates were then prepared and analysed by western blotting using p53-speicific antibody. Anti-tubulin serum was used as loading control. (b) Cell cycle distribution of U2-OS cells with.