Polo-Like Kinase 1 Depletion Induces DNA Damage in Early S Prior to Caspase Activation

Polo-like kinase 1 (Plk1) plays several roles in mitosis, and it has been suggested to have a role in tumorigenesis. We have previously reported that Plk1 depletion results in cell death in cancer cells, whereas normal cells survive similar depletion. However, Plk1 depletion together with p53 depletion induces cell death in normal cells as well. This communication presents evidence on the sequence of events that leads to cell death in cancer cells. DNA damage is detected at the first S phase following Plk1 depletion and is more severe in Plk1-depleted p53-null cancer cells. As a consequence of Plk1 depletion using lentivirus-based small interfering RNA techniques, prereplicative complex (pre-RC) formation is disrupted at the $G_1/S$ transition, and DNA synthesis is reduced during S phase of the first cycle after depletion. The levels of geminin, an inhibitor of DNA pre-RC, and Emi1, an inhibitor of anaphase-promoting complex/cyclosome, are elevated in Plk1-depleted cells. The rate of cell cycling is slower in Plk1-depleted cells than in control cells when synchronized by serum starvation. Plk1 depletion results in disrupted DNA pre-RC formation, reduced DNA synthesis, and DNA damage before cells display severe mitotic catastrophe or apoptosis. Our data suggest that Plk1 is required for cell cycle progression not only in mitosis but also for DNA synthesis, maintenance of DNA integrity, and prevention of cell death.Molecular and Cellular Biolog

A Self-Propelled Biological Process Plk1-Dependent Product-Activated, Feed-Forward Mechanism

Comment on: Park JE, et al. Proc Natl Acad Sci USA 2011; 108:8200-05.Molecular and Cellular Biolog

Crystal structure of xenotropic murine leukaemia virus-related virus (XMRV) ribonuclease H

RNase H (retroviral ribonuclease H) cleaves the phosphate backbone of the RNA template within an RNA/DNA hybrid to complete the synthesis of double-stranded viral DNA. In the present study we have determined the complete structure of the RNase H domain from XMRV (xenotropic murine leukaemia virus-related virus) RT (reverse transcriptase). The basic protrusion motif of the XMRV RNase H domain is folded as a short helix and an adjacent highly bent loop. Structural superposition and subsequent mutagenesis experiments suggest that the basic protrusion motif plays a role in direct binding to the major groove in RNA/DNA hybrid, as well as in establishing the co-ordination among modules in RT necessary for proper function.Molecular and Cellular Biolog

STK295900, a Dual Inhibitor of Topoisomerase 1 and 2, Induces G<inf>2</inf> Arrest in the Absence of DNA Damage

STK295900, a small synthetic molecule belonging to a class of symmetric bibenzimidazoles, exhibits antiproliferative activity against various human cancer cell lines from different origins. Examining the effect of STK295900 in HeLa cells indicates that it induces G2 phase arrest without invoking DNA damage. Further analysis shows that STK295900 inhibits DNA relaxation that is mediated by topoisomerase 1 (Top 1) and topoisomerase 2 (Top 2) in vitro. In addition, STK295900 also exhibits protective effect against DNA damage induced by camptothecin. However, STK295900 does not affect etoposide-induced DNA damage. Moreover, STK295900 preferentially exerts cytotoxic effect on cancer cell lines while camptothecin, etoposide, and Hoechst 33342 affected both cancer and normal cells. Therefore, STK295900 has a potential to be developed as an anticancer chemotherapeutic agent. © 2013 Kim et al

STK295900 inhibits topoisomerases activities.

<p>Supercoiled DNA relaxation assay for (A) topoisomerase 1 (Top 1) and (B) topoisomerase 2 (Top 2). Supercoiled pBR322 plasmid DNA was incubated at 37°C for 30 min with Top 1 enzyme (A) or Top 2α (B) in the presence of various concentrations of indicated compounds. DNA samples were separated by electrophoresis on a 1% agarose gel, stained with ethidium bromide, and visualized by UV light. (−), supercoiled DNA alone; oc, open circular; sc, supercoiled. (C) Antagonistic effect of STK295900 in camptothecin-induced DNA damage. HeLa cells were pretreated with 10 µM of STK295900 for 30 min and then incubated with the indicated concentrations of camptothecin for 1 h. Treated cells were lysed and subjected to immunoblot analyses with antibody against γ-H2A.X. β-actin was used as a loading control. (D) Antagonistic effect of STK295900 on etoposide-induced DNA damage. HeLa cells were pretreated with STK295900 at 10, 20, 30, or 50 µM or ICRF-193 at 10 µM for 30 min. Cells were incubated with 10 µM of etoposide for another 1 h. Treated cells were then lysed and subjected to immunoblot analyses with antibody against γ-H2A.X. β-actin was used as a loading control.</p