Traditional methods to assay enzymatic cleavage of DNA are discontinuous and time consuming. In contrast, recently developed fluorescence methods are continuous and convenient. However, no fluorescence method has been developed for singlestranded DNA digestion. Here we introduce a novel method, based on molecular beacons, to assay single-stranded DNA cleavage by single strandspecific nucleases. A molecular beacon, a hairpinshaped DNA probe labeled with a fluorophore and a quencher, is used as the substrate and enzymatic cleavage leads to fluorescence enhancement in the molecular beacon. This method permits real time detection of DNA cleavage and makes it easy to characterize the activity of DNA nucleases and to study the steady-state cleavage reaction kinetics. The excellent sensitivity, reproducibility and convenience will enable molecular beacons to be widely useful for the study of single-stranded DNA cleaving reactions.DNA cleavage reactions are important for cellular events, such as DNA replication, recombination and repair, as well as for molecular cloning and DNA analysis. These cleavage reactions are usually catalyzed by various enzymes, including the most commonly used restriction nucleases and non-specific nucleases (1,2). To assay the cleavage efficiency of these enzymes, several traditional methods have been used, such as gel electrophoresis, filter binding and high performance liquid chromatography (HPLC) (3-5). All these methods, however, are discontinuous, time consuming and laborious. Furthermore, detecting cleavage at low substrate concentrations necessitates radiolabeling. A continuous UV assay based on hyperchromic effects has been reported, but its application is limited by its narrow dynamic range of substrate concentration (6). Enzyme-linked immunosorbent assay (ELISA) has been used for enzymatic cleavage studies, but also suffers from being discontinuous (7). There have been recent efforts towards the development of fluorescence assays for DNA cleavage (8). These assays are based on fluorescence resonance energy transfer (FRET) or non-FRET quenching mechanisms. Usually, a fluorescence signal enhancement is observed after the cleavage reaction (8-11). These assays are continuous and convenient. However, up to now all of them have been designed only for restriction endonucleases and double-stranded DNA substrates. Furthermore, the potential for achieving high sensitivity through FRET has not been fully realized for the study of DNA cleavage (the signal to background ratio is only~2 or less).In this report, we introduce a fluorescence assay to monitor the cleavage of single-stranded DNA by single-strand-specific nucleases using recently developed molecular beacons. So far, many single-strand-specific nucleases that hydrolyze nucleic acids have been isolated from various sources, including fungi, bacteria, yeast, plants and animals. These enzymes exhibit high selectivity for single-stranded nucleic acids and produce mono-and oligonucleotides terminating in 5′-phosphoryl and 3′-phosphor...

Chromosomal Instability (CIN) is regarded as a unifying feature of heterogeneous tumor populations, driving intratumoral heterogeneity. Polo-Like Kinase 1 (PLK1), a serine-threonine kinase that is often overexpressed across multiple tumor types, is one of the key regulators of CIN and is considered as a potential therapeutic target. However, targeting PLK1 has remained a challenge due to the off-target effects caused by the inhibition of other members of the polo-like family. Here we use synthetic dosage lethality (SDL), where the overexpression of PLK1 is lethal only when another, normally non-lethal, mutation or deletion is present. Rather than directly inhibiting PLK1, we found that inhibition of PP2A causes selective lethality to PLK1-overexpressing breast, pancreatic, ovarian, glioblastoma, and prostate cancer cells. As PP2A is widely regarded as a tumor suppressor, we resorted to gene expression datasets from cancer patients to functionally dissect its therapeutic relevance. We identified two major classes of PP2A subunits that negatively correlated with each other. Interestingly, most mitotic regulators, including PLK1, exhibited SDL interactions with only one class of PP2A subunits (PPP2R1A, PPP2R2D, PPP2R3B, PPP2R5B and PPP2R5D). Validation studies and other functional cell-based assays showed that inhibition of PPP2R5D affects both levels of phospho-Rb as well as sister chromatid cohesion in PLK1-overexpressing cells. Finally, analysis of clinical data revealed that patients with high expression of mitotic regulators and low expression of Class I subunits of PP2A improved survival. Overall, these observations point to a context-dependent role of PP2A that warrants further exploration for therapeutic benefits.

Proteins of the ephrin-B group operate in nonlymphoid cells through the control of their migration and attachment, and are crucial for the development of the vascular, lymphatic, and nervous systems. Ephrin-B activity is deregulated in various nonlymphoid malignancies; however, their precise role in cancer has only started to be addressed. We show here that ephrin-B1, a member of the ephrin-B group, is expressed in pediatric T-cell leukemias, including leukemia cell line Jurkat. Treatment of Jurkat cells with ephrin-Bstimulating EphB3 enhances ephrin-B1 phosphorylation and induces its relocalization into lipid rafts. These events are mediated by the T lineage -specific kinase, Lck, as ephrin-B1 phosphorylation and lipid raft association are blocked in the Lck-deficient clone of Jurkat, JCAM1.6. Ephrin-B1 also induces colocalization of the CrkL and Rac1 cytoskeleton regulators and initiates in leukemic cells a strong repulsive response. The absence of Lck blocks ephrin-B1 -induced signaling and repulsion, confirming the essential role for Lck in ephrin-B1 -mediated responses. This shows a new role for ephrin-B1 in the regulation of leukemic cells through the Lck-dependent Rac1 colocalization with its signaling partner, CrkL, in lipid rafts. In agreement with its repulsive action, ephrin-B1 seems to support metastatic properties of leukemic cells, as suppression of ephrin-B1 signaling inhibits their invasiveness. Because ephrin-B1 -activating EphB proteins are ubiquitously expressed, our findings suggest that ephrin-B1 is likely to play an important role in the regulation of malignant T lymphocytes through the control of lipid-raftassociated signaling, adhesion, and invasive activity, and therefore may represent a novel target for cancer treatment. (Mol Cancer Res 2008;6(2):291 -305)

e15112 Background: Activation of NF- B leads to enhanced proliferation and the expression of anti-apoptotic proteins, which are cancer phenotypes. RIZ1 inactivation through various molecular events was linked to increased proliferation, migration induction and apoptosis inhibition in human cancer. RIZ1 frameshift mutations were recently reported to be confined to MSI-H colorectal tumors and proximal tumor origin, while its hypermethylation was not limited to MSI-H tumors. However, RIZ1 protein expression has not been evaluated in colorectal carcinoma. Methods: TMAs included 28 left-sided and 12 right-sided primary colorectal adenocarcinomas, their matched normal mucosa and their respective distant metastases. Left-sided tumors were compared to right-sided tumors for expression of RIZ1 protein and NF- B activation. RIZ1 immunostaining was scored semiquantitatively (0–3+). NF- B activation was determined by IHC detecting nuclear translocation of its p65 subunit in more than 30% tumor nuclei. Discrepant results were defined as score difference of 2. Results: RIZ1 was less expressed in tumors than in benign mucosa (p<0.0001, r=- 0.377, Chi-Square). The difference between primary vs. metastatic carcinoma was not significant. Low RIZ1 was associated with NF- B activation (p<0.0001, Linear-by-Linear). Left-sided primary tumors showed less RIZ1 protein expression than right-sided (p=0.03, Chi-Square). NF- B activation was more frequent in left-sided primary tumors and their respective metastases (35% in right vs. 67% in left; p=0.002, Chi-Square Test). RIZ1 expression and NF- B activation were almost identical in primary and their respective metastatic tumors with only 3 discrepant results for NF- B status and 2 discrepant results in RIZ1 expression. Conclusions: While RIZ1 downregulation in colorectal adenocarcinoma due to RIZ1 mutations appears to be associated with MSI-H and proximal origin, its protein expression appears to be downregulated more often in distal tumors. NF- B activation is strongly associated with lower RIZ1 protein expression in colorectal adenocarcinoma. No significant financial relationships to disclose.