The analysis of protein recruitment to laser microirradiation-induced DNA damage in live cells: Best practices for data analysis.

Laser microirradiation coupled with live-cell fluorescence microscopy is a powerful technique that has been used widely in studying the recruitment and retention of proteins at sites of DNA damage. Results obtained from this technique can be found in published works by both seasoned and infrequent users of microscopy. However, like many other microscopy-based techniques, the presentation of data from laser microirradiation experiments is inconsistent; papers report a wide assortment of analytic techniques, not all of which result in accurate and/or appropriate representation of the data.

In vitro screen of a small molecule inhibitor drug library identifies multiple compounds that synergize with oncolytic myxoma virus against human brain tumor-initiating cells.

Background: Brain tumor-initiating cells (BTICs) are stem-like cells hypothesized to form a disease reservoir that mediates tumor recurrence in high-grade gliomas. Oncolytic virotherapy uses replication-competent viruses to target and kill malignant cells and has been evaluated in clinic for glioma therapy with limited results. Myxoma virus (MyxV) is a safe and highly effective oncolytic virus (OV) in conventional glioma models but, as seen with other OVs, is only modestly effective for patient-derived BTICs.

Structural determinants of the catalytic inhibition of human topoisomerase IIα by salicylate analogs and salicylate-based drugs.

We previously identified salicylate as a novel catalytic inhibitor of human DNA topoisomerase II (topo II; EC 5.99.1.

Sodium salicylate is a novel catalytic inhibitor of human DNA topoisomerase II alpha.

We have previously reported that pretreatment of human lymphoblastoid cells with the hydroxyl radical scavenger, N-acetyl cysteine, attenuates doxorubicin-induced DNA damage signalling through the ATM protein kinase. We sought to extend these studies to examine the effects of other hydroxyl radical scavengers in human breast cancer cells. Using MCF-7 cells, we observed that doxorubicin treatment triggered autophosphorylation of ATM on serine 1981 and the ATM-dependent activation of its downstream effectors p53, Chk2, and SMC1.

Altered chloride homeostasis removes synaptic inhibitory constraint of the stress axis.

In mammals, stress elicits a stereotyped endocrine response that requires an increase in the activity of hypothalamic parvocellular neuroendocrine neurons. The output of these cells is normally constrained by powerful GABA-mediated synaptic inhibition. We found that acute restraint stress in rats released the system from inhibitory synaptic drive in vivo by down-regulating the transmembrane anion transporter KCC2.

Doxorubicin activates ATM-dependent phosphorylation of multiple downstream targets in part through the generation of reactive oxygen species.

The requirement for the serine/threonine protein kinase ATM in coordinating the cellular response to DNA damage induced by ionizing radiation has been studied extensively. Many of the anti-tumor chemotherapeutics in clinical use today cause DNA double strand breaks; however, few have been evaluated for their ability to modulate ATM-mediated pathways. We have investigated the requirement for ATM in the cellular response to doxorubicin, a topoisomerase II-stabilizing drug.

DNA damage-induced activation of ATM and ATM-dependent signaling pathways.

Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to ionizing radiation. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage response pathways, most notably cell cycle checkpoints. In this review, we describe recent developments in our understanding of the mechanism of activation of ATM and its downstream signaling pathways, and explore whether DNA double-strand breaks are the sole activators of ATM and ATM-dependent signaling pathways.

The isoflavonoids genistein and quercetin activate different stress signaling pathways as shown by analysis of site-specific phosphorylation of ATM, p53 and histone H2AX.

The ataxia-telangiectasia mutated (ATM) protein kinase is activated in response to ionizing radiation (IR) and activates downstream DNA-damage signaling pathways. Although the role of ATM in the cellular response to ionizing radiation has been well characterized, its role in response to other DNA-damaging agents is less well defined. We previously showed that genistein, a naturally occurring isoflavonoid, induced increased ATM protein kinase activity, ATM-dependent phosphorylation of p53 on serine 15 and activation of the DNA-binding properties of p53.

Cloning and Characterization of the Murine and Rat mrp1 Promoter Regions.

The ATP-binding cassette transporter multidrug resistance protein 1 (MRP1) confers resistance to a number of clinically important chemotherapeutic agents. The proximal promoter region of MRP1 is GC-rich and contains binding sites for members of the Sp1 family of trans-acting factors that seem to be important for basal expression. As an approach to searching for other elements that may contribute to expression, we have sequenced and functionally compared the promoters of the murine and rat mrp1 genes with that of the human gene.