Tumor suppressor ARF regulates the activity of

Tumor suppressor ARF regulates the activity of p53 by inhibiting the functions of Mdm2. ARF is induced by hyperproliferative signals emanating from oncogenic Ras, overexpressed c-myc, and from deregulated E2F . ARF prevents p53 degradation and leads to increased p53 function by sequestering Mdm2 to nucleoli , , and blocking nucleo-cytoplasmic shuttling of Mdm2 , . In addition to regulation of p53, ARF can function as a tumor suppressor independently of p53 , for instance by inhibiting the processing of rRNA or inducing apoptosis through Bax . The stabilization and activation of p53 in response to genotoxic stress is mediated by ATM and ATR kinases and their substrate kinases Chk2 and Chk1 that phosphorylate the N-terminal serine residues of p53 , , , . Phosphorylation favors the association of p53 with co-activators and blocks its degradation, resulting in its protein accumulation and p53-dependent transcription , , . Recently it has been suggested that genotoxic stress and hyperproliferative signals are more tightly intertwined than initially thought. For instance, E2F1 and c-myc are able to induce p53 activation through its phosphorylation by ATM/ATR kinases . Although ARF can induce the phosphorylation of p53 as well , S-phase-promoting E2F1 causes the phosphorylation of p53 independently of ARF , , , . Despite this, it is still unclear, which signals determine an individual cell’s response to oncogene expression.
Introduction Polyploidy is the condition of cells exhibiting the presence of more than two homologous sets of chromosomes. Polyploidy is observed in plants and in some types of animal cells, liver cells, trophoblasts, and megakaryocytes [1]. Abrogation of cell division accompanying over-replication of DNA is thought to result in polyploidization. Some polyploid cell types do not express mitotic regulators, CDK1, Cyclin B, Cyclin A, and Cdc25C, and bypass mitosis, suggesting that decreasing levels of mitotic regulators activate over-replication through abrogation of mitosis in these cell types [1]. A variety of agents, such as microtubule poison, silymarin depolymerizing agents, membrane traffic inhibitors, and topoisomerase inhibitors, have been reported to induce over-replication by disrupting cytokinesis or karyokinesis [2]. These agents are thought to directly act on cytokinesis machineries or chromosome segregation machineries. DNA-damaging agents have also been reported to induce over-replication by disrupting cytokinesis [3]. However, DNA damage is unlikely to be linked directly with disruption of cytokinesis. DNA damage is known to inhibit CDKs, retarding cell cycle progression at G1 or at G2, dependent on cell types [4]. DNA damage activates the ATM/ATR pathway, inhibiting CDK1 activity through the stabilization of phosphorylation at Thr14/Tyr15 of CDK1, resulting in abrogation of cell division [5]. While the ATM/ATR pathway plays a crucial role in abrogation of mitosis in response to DNA damage, whether the ATM/ATR pathway triggered by DNA damage is responsible for DNA-damage-induced over-replication has not been investigated. The DNA-damaging anticancer drugs known as bleomycins, a family of glycopeptides, are an important component in a number of combination chemotherapies [6]. The cytotoxicity of bleomycin is related to its ability to induce single- and double-strand DNA breaks [7]. The extent of these DNA breaks depends on the concentration and time of incubation [8]. Low concentrations of bleomycin caused G2 arrest, and produced enlarged and binucleated cells [8], suggesting that the DNA damage caused by low concentrations of bleomycin may induce over-replication. We wished, therefore, to explore the mechanisms that induce over-replication upon bleomycin treatment.
Materials and methods
Results
Discussion In the present study, we show that low concentrations of bleomycin induce over-replication in a manner dependent on the ATM/ATR pathway. Upon treatment with bleomycin, cells are over-replicated following G2 arrest induced through phosphorylation of CDK1 and degradation of cyclin B1. Abrogation of bleomycin-induced G2 arrest by inhibition of the ATM/ATR pathway promotes cell death instead of over-replication. Our results suggest that in response to bleomycin-induced DNA damage, the ATM/ATR pathway acts as a molecular switch in regulating cell fates between cell death via progress into mitosis and over-replication via sustained G2 arrest (Fig. 8).